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2024 – Triplet–Triplet Annihilation Upconverting Liposomes: Mechanistic Insights into the Role of Membranes in Two-Dimensional TTA-UC
Vesicle Prep Pro Publication in Appl. Mater. Interfaces (2024) Authors: Prabhakaran A., Jha K.K., Sia R.C., Arellano Reyes R.A., Sarangi N.K, Kogut M., Guthmuller J., Czub J., Dietzek-Ivanšić B., Keyes T.E.

Triplet–triplet annihilation upconversion (TTA-UC) implemented in nanoparticle assemblies is of emerging interest in biomedical applications, including in drug delivery and imaging. As it is a bimolecular process, ensuring sufficient mobility of the sensitizer and annihilator to facilitate effective collision in the nanoparticle is key. Liposomes can provide the benefits of two-dimensional confinement and condensed concentration of the sensitizer and annihilator along with superior fluidity compared to other nanoparticle assemblies. They are also biocompatible and widely applied across drug delivery modalities. However, there are relatively few liposomal TTA-UC systems reported to date, so systematic studies of the influence of the liposomal environment on TTA-UC are currently lacking. Here, we report the first example of a BODIPY-based sensitizer TTA-UC system within liposomes and use this system to study TTA-UC generation and compare the relative intensity of the anti-Stokes signal for this system as a function of liposome composition and membrane fluidity. We report for the first time on time-resolved spectroscopic studies of TTA-UC in membranes. Nanosecond transient absorption data reveal the BODIPY-perylene dyad sensitizer has a long triplet lifetime in liposome with contributions from three triplet excited states, whose lifetimes are reduced upon coinclusion of the annihilator due to triplet–triplet energy transfer, to a greater extent than in solution. This indicates triplet energy transfer between the sensitizer and the annihilator is enhanced in the membrane system. Molecular dynamics simulations of the sensitizer and annihilator TTA collision complex are modeled in the membrane and confirm the co-orientation of the pair within the membrane structure and that the persistence time of the bound complex exceeds the TTA kinetics. Modeling also reliably predicted the diffusion coefficient for the sensitizer which matches closely with the experimental values from fluorescence correlation spectroscopy. The relative intensity of the TTA-UC output across nine liposomal systems of different lipid compositions was explored to examine the influence of membrane viscosity on upconversion (UC). UC showed the highest relative intensity for the most fluidic membranes and the weakest intensity for highly viscous membrane compositions, including a phase separation membrane. Overall, our study reveals that the co-orientation of the UC pair within the membrane is crucial for effective TTA-UC within a biomembrane and that the intensity of the TTA-UC output can be tuned in liposomal nanoparticles by modifying the phase and fluidity of the liposome. These new insights will aid in the design of liposomal TTA-UC systems for biomedical applications.

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2024 – Transporter function characterization via continuous-exchange cell-free synthesis and solid supported membrane-based electrophysiology
SURFE2R N1 Publication in Bioelectrochemistry (2024) Authors: Dong F., Lojko P., Bazzone A., Bernhard F., Borodina I.

Functional characterization of transporters is impeded by the high cost and technical challenges of current transporter assays. Thus, in this work, we developed a new characterization workflow that combines cell-free protein synthesis (CFPS) and solid supported membrane-based electrophysiology (SSME). For this, membrane protein synthesis was accomplished in a continuous exchange cell-free system (CECF) in the presence of nanodiscs. The resulting transporters expressed in nanodiscs were incorporated into proteoliposomes and assayed in the presence of different substrates using the surface electrogenic event reader. As a proof of concept, we validated this workflow to express and characterize five diverse transporters: the drug/H+-coupled antiporters EmrE and SugE, the lactose permease LacY, the Na+/H+ antiporter NhaA from Escherichia coli, and the mitochondrial carrier AAC2 from Saccharomyces cerevisiae. For all transporters kinetic parameters, such as KM, IMAX, and pH dependency, were evaluated. This robust and expedite workflow (e.g., can be executed within only five workdays) offers a convenient direct functional assessment of transporter protein activity and has the ability to facilitate applications of transporters in medical and biotechnological research.

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2024 – Susceptibility to innate immune activation in genetically-mediated myocarditis
CardioExcyte 96 Publication in J Clin Invest. (2024) Authors: Selgrade D.F., Fullenkamp D.E., Chychula I.A., Li B., Dellefave-Castillo L., Dubash A., Ohiri J., Monroe T.O., Blancard M., Tomar G., Holgren C., Burridge P., George A.L., Demonbreun A., Puckelwartz M., George S.A., Efimov I.R., Green K.J., McNally E.M.,

Myocarditis is clinically characterized by chest pain, arrhythmias, and heart failure, and treatment is often supportive. Mutations in DSP, a gene encoding the desmosomal protein
desmoplakin, have been increasingly implicated in myocarditis. To model DSP-associated myocarditis and assess the role of innate immunity, we generated engineered heart tissues
(EHTs) using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from patients with heterozygous DSP truncations (DSPtv) and a gene-edited homozygous deletion
cell line (DSP-/-). At baseline, DSP-/- EHTs displayed a transcriptomic signature of innate immune activation which was mirrored by cytokine release. Importantly, DSP-/- EHTs were
hypersensitive to Toll-like receptor (TLR) stimulation demonstrating more contractile dysfunction compared to isogenic controls. Relative to DSP-/- EHTs, heterozygous DSPtv EHTs had less functional impairment. DSPtv EHTs displayed heightened sensitivity to TLR stimulation, and when subjected to strain, DSPtv EHTs developed functional deficits indicating reduced
contractile reserve compared to healthy control. Colchicine or NFκB inhibitors improved strain-induced force deficits in DSPtv EHTs. Genomic correction of DSP p.R1951X using adenine
base editing reduced inflammatory biomarker release from EHTs. Thus, EHTs replicate electrical and contractile phenotypes seen in human myocarditis implicating cytokine release as
a key part of the myogenic susceptibility to inflammation. This heightened innate immune activation and sensitivity is a target for clinical intervention.

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2024 – Phosphoproteomics reveals a novel mechanism underlying the proarrhythmic effects of nilotinib, vandetanib, and mobocertinib
CardioExcyte 96 Publication in Toxicology (2024) Authors: Wu W., Sun J., Zhang J., Zhao H., Qiu S., Li C., Shi C., Xu Y.

The use of tyrosine kinase inhibitors (TKIs) has resulted in significant occurrence of arrhythmias. However, the precise mechanism of the proarrhythmic effect is not fully understood. In this study, we found that nilotinib (NIL), vandetanib (VAN), and mobocertinib (MOB) induced the development of “cellrhythmia” (arrhythmia-like events) in a concentration-dependent manner in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Continuous administration of NIL, VAN, or MOB in animals significantly prolonged the action potential durations (APD) and increased susceptibility to arrhythmias. Using phosphoproteomic analysis, we identified proteins with altered phosphorylation levels after treatment with 3 μM NIL, VAN, and MOB for 1.5 h. Using these identified proteins as substrates, we performed kinase–substrate enrichment analysis to identify the kinases driving the changes in phosphorylation levels of these proteins. MAPK and WNK were both inhibited by NIL, VAN, and MOB. A selective inhibitor of WNK1, WNK-IN-11, induced concentration- and time-dependent cellrhythmias and prolonged field potential duration (FPD) in hiPSC-CMs in vitro; furthermore, administration in guinea pigs confirmed that WNK-IN-11 prolonged ventricular repolarization and increased susceptibility to arrhythmias. Findings indicated that WNK1 inhibition had an in vivo and in vitro arrhythmogenic phenotype similar to TKIs. Additionally, three of TKIs reduced hERG and KCNQ1 expression at protein level, not at transcription level. Similarly, the knockdown of WNK1 decreased hERG and KCNQ1 protein expression in hiPSC-CMs.

Collectively, our data suggest that the proarrhythmic effects of NIL, VAN, and MOB occur through a kinase inhibition mechanism. NIL, VAN, and MOB inhibit WNK1 kinase, leading to a decrease in hERG and KCNQ1 protein expression, thereby prolonging action potential repolarization and consequently cause arrhythmias.

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2024 – Single-cell ionic current phenotyping elucidates non-canonical features and predictive potential of cardiomyocytes during automated drug experiments
Patchliner Publication in J Physiol. (2024) Authors: Clark A.P., Wei S., Christini D.J., Krogh-Madsen T.

All new drugs must go through preclinical screening tests to determine their proarrhythmic potential. While these assays effectively filter out dangerous drugs, they are too
conservative, often misclassifying safe compounds as proarrhythmic. In this study, we attempt to address this shortcoming with a novel, medium-throughput drug-screening approach: we use an automated patch-clamp system to acquire optimized voltage clamp (VC) and action potential (AP) data from human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) at several drug concentrations (baseline, 3×, 10× and 20× the effective free plasma concentrations). With our novel method, we show correlations between INa block and upstroke slowing after treatment with flecainide or quinine. Additionally, after quinine treatment, we identify significant reductions in current during voltage steps designed to isolate If and IKs. However, we do not detect any IKr block by either drug, and upon further investigation, do not see any IKr present in the iPSC-CMs when prepared for automated patch experiments (i.e. in suspension) – this is in contrast to similar experiments we have conducted with these cells using the manual patch setup. In this study, we: (1) present a proof-of-concept demonstration of a single-cell medium-throughput drug study, and (2) characterize the non-canonical electrophysiology of iPSC-CMs when prepared for experiments in a medium-throughput setting.

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2024 – An experimental investigation of rundown of the L-type calcium current
SyncroPatch 384PE (a predecessor model of the SyncroPatch 384) Publication in Wellcome Open Res (2024) Authors: Agrawal A., Clerx M., Wang K., Gissinger E., Gavaghan D., Polonchuk L., Mirams G.

Background
L-type calcium channels (LCCs) are multi-protein macro-molecular ion channel complexes that are involved in several critical functions in cardiac, skeletal, neuronal, smooth muscle, and endocrine cells. Like other ion channels, LCCs can be selectively over-expressed in a host cell line and studied using voltage-clamp patch-clamp experiments. However, L-type calcium current (ICaL) recordings commonly exhibit a reduction in current magnitude over time, commonly termed ‘rundown’. Previous studies have shown the effect of phosphorylation on rundown, here we provide evidence that accumulation of Ca2+ inside the cell also contributes towards ICaL rundown.

Methods
We generated experimental conditions that should promote the accumulation of sub-membrane Ca2+ in a CHO expression system, by increasing calcium import or decreasing export. These interventions took the form of: a decrease in inter-pulse duration between sweeps, block of the sodium-calcium exchanger, and increased temperature.

Results
On average, we found that current reduced to 63% of its initial value within 325 seconds. This reduction of current with time was found to follow two main patterns: linear or saturating decay. Additionally, current magnitude in some cells increased before stabilising or decaying.

Conclusions
This study shows that the rundown of ICaL in patch-clamp experiments can be reduced by modifying the experimental conditions, and implies that reduced accumulation of Ca2+ inside the cell membrane reduces calcium-dependent inactivation of ICaL.

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2024 – Receptor-recognition and antiviral mechanisms of retrovirus-derived human proteins
SURFE2R N1 Publication in Nature structural & molecular biology (2024) Authors: Khare S., Villalba M., Canul-Tec J., Cajiao A.B., Kumar A., Backovic M., Rey F., Pardon E., Steyaert J., Perez C., Reyes N.

Human syncytin-1 and suppressyn are cellular proteins of retroviral origin involved in cell–cell fusion events to establish the maternal–fetal interface in the placenta. In cell culture, they restrict infections from members of the largest interference group of vertebrate retroviruses, and are regarded as host immunity factors expressed during development. At the core of the syncytin-1 and suppressyn functions are poorly understood mechanisms to recognize a common cellular receptor, the membrane transporter ASCT2. Here, we present cryo-electron microscopy structures of human ASCT2 in complexes with the receptor-binding domains of syncytin-1 and suppressyn. Despite their evolutionary divergence, the two placental proteins occupy similar positions in ASCT2, and are stabilized by the formation of a hybrid β-sheet or ‘clamp’ with the receptor. Structural predictions of the receptor-binding domains of extant retroviruses indicate overlapping binding interfaces and clamping sites with ASCT2, revealing a competition mechanism between the placental proteins and the retroviruses. Our work uncovers a common ASCT2 recognition mechanism by a large group of endogenous and disease-causing retroviruses, and provides high-resolution views on how placental human proteins exert morphological and immunological functions.li

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2024 – The antimicrobial fibupeptide lugdunin forms water-filled channel structures in lipid membranes
Orbit 16 (a predecessor model of the Orbit 16 TC) Publication in Nature communications (2024) Authors: Ruppelt D., Trollmann M., Dema T., Wirtz S., Flegel H., Mönnikes S., Grond S., Böckmann R., Steinem C.

Recently, a novel cyclo-heptapeptide composed of alternating D,L-amino acids and a unique thiazolidine heterocycle, called lugdunin, was discovered, which is produced by the nasal and skin commensal Staphylococcus lugdunensis. Lugdunin displays potent antimicrobial activity against a broad spectrum of Gram-positive bacteria, including challenging-to-treat methicillin-resistant Staphylococcus aureus (MRSA). Lugdunin specifically inhibits target bacteria by dissipating their membrane potential. However, the precise mode of action of this new class of fibupeptides remains largely elusive. Here, we disclose the mechanism by which lugdunin rapidly destabilizes the bacterial membrane potential using an in vitro approach. The peptide strongly partitions into lipid compositions resembling Gram-positive bacterial membranes but less in those harboring the eukaryotic membrane component cholesterol. Upon insertion, lugdunin forms hydrogen-bonded antiparallel β-sheets by the formation of peptide nanotubes, as demonstrated by ATR-FTIR spectroscopy and molecular dynamics simulations. These hydrophilic nanotubes filled with a water wire facilitate not only the translocation of protons but also of monovalent cations as demonstrated by voltage-clamp experiments on black lipid membranes. Collectively, our results provide evidence that the natural fibupeptide lugdunin acts as a peptidic channel that is spontaneously formed by an intricate stacking mechanism, leading to the dissipation of a bacterial cell’s membrane potential.

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2024 – Conformational Flexibility Driving Charge-Selective Substrate Translocation Across Bacterial Transporter
Orbit 16 TC Publication in Chemical Science (2024) Authors: Vikraman D., Majumdar B.B., Sharavanakkumar S.K., Weichbrodt C., Fertig N., Winterhalter M., Mondal J., Kozhinjampara M.

Bacterial membrane porins facilitate the translocation of small molecules while restricting large molecules, and this mechanism remains elusive at the molecular level. Here, we investigate the selective uptake of large cyclic sugars across an unusual passive membrane transporter CymA, comprising a charged zone and a constricting N terminus segment. Using a combination of electrical recordings, protein mutagenesis and molecular dynamics simulations, we establish substrate translocation across CymA governed by the electrostatic pore properties and conformational dynamics of the constriction segment. Notably, we show that the variation in pH of the environment resulted in reversible modulation of the substrate binding site in the pore, thereby regulating charge-selective transport of cationic, anionic and neutral cyclic sugars. The quantitative kinetics of cyclic sugar translocation across CymA obtained in electrical recordings at different pHs are comparable with molecular dynamics simulations that revealed transport pathway, energetics and favorable affinity sites in the pore for substrate binding. We further define the molecular basis of cyclic sugar translocation and establish that the constriction segment is flexible and can reside inside or outside the pore, regulating substrate translocation distinct from the ligand-gated transport mechanism. Our study contributes novel insights into energy-independent large molecular membrane transport for targeted drug design strategies.

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2024 – The interaction of plant flavones with amphotericin B: Consequences for its pore-forming ability
Vesicle Prep Pro Publication in Biomedicine & Pharmacotherapy (2024) Authors: Malykhina A., Efimova S, Andriianov V., Ostroumova O.

The growth of antibiotic resistance to antifungal drugs contributes to the search for new ways to enhance their effectiveness and reduce toxicity. The undeniable advantage of polyene macrolide antibiotic amphotericin B (AmB) which ensures low pathogen resistance is its mechanism of action related to the formation of transmembrane pores in target lipid membranes. Here, we investigated the effects of plant flavones, chrysin, wogonin, baicalein, apigenin, scutellarein, luteolin, morin and fisetin on the pore-forming activity of AmB in the sterol-enriched membranes by electrophysiological assays. Сhrysin, wogonin, baicalein, apigenin, scutellarein, and luteolin were shown to decrease the AmB pore-forming activity in the bilayers composed of palmitoyloleylphosphocholine independently of their sterol composition. Morin and fisetin led to the increase and decrease in the AmB pore-forming activity in the ergosterol- and cholesterol-containing bilayers respectively. Differential scanning microcalorimetry of the gel-to-liquid crystalline phase transition of membrane forming lipids, molecular dynamics simulations, and absorbance spectroscopy revealed the possibility of direct interactions between AmB and some flavones in the water and/or in the lipid bilayer. The influence of these interactions on the antibiotic partitioning between aqueous solution and membrane and/or its transition between different states in the bilayer was discussed.

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2024 – Unveiling the crucial role of betaine: Modulation of GABA homeostasis via SLC6A1 transporter (GAT1)
Patchliner Pre-print Publication in bioRxiv (2024) Authors: Bhatt M., Lazzarin E., Alberto-Silva A.S., Domingo G., Zerlotti R., Gradisch R., Bazzone A., Sitte H., Stockner T., Bossi E.

Betaine is an endogenous osmolyte that exhibits therapeutic potential by mitigating various neurological disorders. However, the underlying cellular and molecular mechanisms responsible for its neuroprotective effects remain puzzling. In this study, we describe a possible mechanism behind the positive impact of betaine in preserving neurons from excitotoxicity. Using electrophysiology, mass spectroscopy, radiolabelled cellular assay, and molecular dynamics simulation we demonstrate that betaine at mM concentration acts as a slow substrate of GAT1 (slc6a1), the predominant GABA transporter in the central nervous system. Intriguingly, when betaine is present at low concentration (0.01-3 mM) with GABA (at concentration <K0.5), it blocks the GABA reuptake. This GAT1 modulation occurs through the temporal  inhibition of the transporter, i.e., the prolonged occupancy by betaine impedes the rapid transition of the transporter to the inward conformation. The temporal inhibition results in a crucial regulatory mechanism contributing to the maintenance of GABA homeostasis, preserving neurons from excitotoxicity.

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2024 – Independent compartmentalization of functional, metabolic, and transcriptional maturation of hiPSC-derived cardiomyocytes
CardioExcyte 96 and SyncroPatch 384 Publication in Cell Reports (2024) Authors: Fetterman A., Blancard M., Lyra-Leite D., Vanoye C., Fonoudi H., Jouni M., DeKeyser J.M., Lenny B., Sapkota Y., George A., Burridge P.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) recapitulate numerous disease and drug response phenotypes, but cell immaturity may limit their accuracy and fidelity as a model system. Cell culture medium modification is a common method for enhancing maturation, yet prior studies have used complex media with little understanding of individual component contribution, which may compromise long-term hiPSC-CM viability. Here, we developed high-throughput methods to measure hiPSC-CM maturation, determined factors that enhanced viability, and then systematically assessed the contribution of individual maturation medium components. We developed a medium that is compatible with extended culture. We discovered that hiPSC-CM maturation can be sub-specified into electrophysiological/EC coupling, metabolism, and gene expression and that induction of these attributes is largely independent. In this work, we establish a defined baseline for future studies of cardiomyocyte maturation. Furthermore, we provide a selection of medium formulae, optimized for distinct applications and priorities, that promote measurable attributes of maturation.

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2024 – YeeD is an essential partner for YeeE-mediated thiosulfate uptake in bacteria and regulates thiosulfate ion decomposition
SURFE2R N1 Publication in PLoS Biol (2024) Authors: Ikei M., Miyazaki R., Monden K., Naito Y., Takeuchi A., Takahashi Y.S., Tanaka Y., Murata K., Mori T., Ichikawa M., Tsukazaki T.

Uptake of thiosulfate ions as an inorganic sulfur source from the environment is important for bacterial sulfur assimilation. Recently, a selective thiosulfate uptake pathway involving a membrane protein YeeE (TsuA) in Escherichia coli was characterized. YeeE-like proteins are conserved in some bacteria, archaea, and eukaryotes. However, the precise function of YeeE, along with its potential partner protein in the thiosulfate ion uptake pathway, remained unclear. Here, we assessed selective thiosulfate transport via Spirochaeta thermophila YeeE in vitro and characterized Ecoli YeeD (TsuB) as an adjacent and essential protein for YeeE-mediated thiosulfate uptake in vivo. We further showed that Sthermophila YeeD possesses thiosulfate decomposition activity and that a conserved cysteine in YeeD was modified to several forms in the presence of thiosulfate. Finally, the crystal structures of Sthermophila YeeE-YeeD fusion proteins at 3.34-Å and 2.60-Å resolutions revealed their interactions. The association was evaluated by a binding assay using purified Sthermophila YeeE and YeeD. Based on these results, a model of the sophisticated uptake of thiosulfate ions by YeeE and YeeD is proposed.

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2024 – A sustainable chemiosmotic strategy for driving solute transport in synthetic cells
SURFE2R N1 Pre-Print Publication in Research Square (2024) Authors: Poolman B., Ruiz M.P., Anshari Z., Gaastra B., Slotboom D.

Cellular homeostasis requires sustained provision of metabolic energy in the form of ATP and electrochemical ion gradients. Primary and secondary active transporters are prominent consumers of cellular energy, and couple ATP hydrolysis and ion gradient dissipation, respectively, to translocation of molecules across biological membranes. Active transport is essential for the translocation of most charged and/or large hydrophilic molecules, both for nutrient uptake into and waste export from living cells. Endeavours to build synthetic cells crucially depend on simulating real cell behaviour by supplying stable and sustained energy sources and deploying them for membrane transport. Here, we provide synthetic cells with long-lasting metabolic energy supply in the form of an electrochemical proton gradient. Leveraging the L-malate decarboxylation pathway from Lactococcus lactis we generate a stable proton gradient and electrical potential in lipid vesicles by electrogenic L-malate/L-lactate exchange coupled to L-malate decarboxylation. By co-reconstitution of the pathway with the Escherichia coli transporters GltP and LacY, the synthetic cells maintain accumulation of L-glutamate and lactose over periods of hours, mimicking nutrient feeding in living cells. This study underscores the potential of harnessing a proton motive force via a simple metabolic network, involving electrogenic substrate/product exchange and substrate decarboxylation, paving the way for the development of more complex synthetic systems.

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2024 – Structural and mechanistic insights into human choline and ethanolamine transport
SURFE2R N1 Pre-Print Publication in bioRxiv (2024) Authors: Ri K., Weng T.H., Claveras Cabezudo A., Jösting W., Yu Z., Bazzone A., Leong N., Welsch S., Doty R., Gursu G., Ying Lim T.J., Schmidt S.L., Abkowitz J.L., Hummer G., Wu D., Nguyen L., Safarian S.

Human feline leukemia virus subgroup C receptor-related proteins 1 and 2 (FLVCR1 and 2) are members of the major facilitator superfamily. Their dysfunction is linked to several clinical disorders, including PCARP, HSAN, and Fowler syndrome. Earlier studies concluded that FLVCR1 may function as a putative heme exporter, while FLVCR2 was suggested to act as a heme importer, yet conclusive biochemical and detailed molecular evidence remained elusive for the function of both transporters. Here, we show that FLVCR1 and FLVCR2 facilitate the transport of choline and ethanolamine across human plasma membranes, utilizing a concentration-driven substrate translocation process. Through structural and computational analyses, we have identified distinct conformational states of FLVCRs and unraveled the coordination chemistry underlying their substrate interactions. Within the binding pocket of both transporters, we identify fully conserved tryptophan and tyrosine residues holding a central role in the formation of cation-π interactions, essential for choline and ethanolamine selectivity. Our findings not only clarify the mechanisms of choline and ethanolamine transport by FLVCR1 and FLVCR2, enhancing our comprehension of disease-associated mutations that interfere with these vital processes, but also shed light on the conformational dynamics of these MFS-type proteins during the transport cycle.

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2024 – Analgesic effect of Botulinum toxin in neuropathic pain is sodium channel independent
SyncroPatch 384i (a predecessor model of the SyncroPatch 384) Publication in Neuropharmacology (2024) Authors: Kesdoğan A., Neureiter A., Gaebler A., Kalia A., Körner J., Lampert A.

Botulinum neurotoxin type A BoNT/A is used off-label as a third line therapy for neuropathic pain. However, the mechanism of action remains unclear. In recent years, the role of voltage-gated sodium channels (Nav) in neuropathic pain became evident and it was suggested that block of sodium channels by BoNT/A would contribute to its analgesic effect.

We assessed sodium channel function in the presence of BoNT/A in heterologously expressed Nav1.7, Nav1.3, and the neuronal cell line ND7/23 by high throughput automated and manual patch-clamp. We used both the full protein and the isolated catalytic light chain LC/A for acute or long-term extracellular or intracellular exposure. To assess the toxin’s effect in a human cellular system, we differentiated induced pluripotent stem cells (iPSC) into sensory neurons from a healthy control and a patient suffering from a hereditary neuropathic pain syndrome (inherited erythromelalgia) carrying the Nav1.7/p.Q875E-mutation and carried out multi electrode array measurements.

Both BoNT/A and the isolated catalytic light chain LC/A showed limited effects in heterologous expression systems and the neuronal cell line ND7/23. Spontaneous activity in iPSC derived sensory neurons remained unaltered upon BoNT/A exposure both in neurons from the healthy control and the mutation carrying patient.

BoNT/A may not specifically be beneficial in pain syndromes linked to sodium channel variants. The favorable effects of BoNT/A in neuropathic pain are likely based on mechanisms other than sodium channel blockage and new approaches to understand BoNT/A’s therapeutic effects are necessary.

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Immuno-oncology research – AltaZ
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Virology research – AtlaZ
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2024 – Interaction of selected alkoxy naringenin oximes with model and bacterial membranes
Vesicle Prep Pro Publication in Biomedicine & Pharmacotherapy (2024) Authors: Wesołowska O., Duda-Madej A., Błaszczyk M., Środa-Pomianek K., Kozłowska J., Anioł M.

Naringenin is a flavonoid found in many fruits and herbs, most notably in grapefruits. In recent years, this compound and its derivatives have been of great interest due to their high biological activity, including fungicidal and bactericidal effects, also in relation to multidrug-resistant bacteria. Membrane interactions of naringenin oxime (NO) and its 7-O-alkyl (7-alkoxy) derivatives, such as methyl (7MENO), ethyl (7ETNO), isopropyl (7IPNO), n-butyl (7BUNO) and n-pentyl (7PENO) were studied. Thermotropic properties of model membranes were investigated via differential scanning calorimetry (DSC), the influence on lipid raft mimicking giant unilamellar vesicles (GUVs) via fluorescence microscopy, and membrane permeability via measuring calcein leakage from liposomes. Molecular calculations supplemented the study. The influence of naringenin oximes on two strains of multidrug resistant bacteria: Staphylococcus aureus KJ and Enterococcus faecalis 37VRE was also investigated. In DSC studies all compounds reduced the temperature and enthalpy of main phase transition and caused disappearing of the pretransition. NO was the least active. The reduction in the area of surface domains in GUVs was observed for NO. Compounds NO and 7BUNO resulted in very low secretion of calcein from liposomes (permeability < 3 %). The highest results were observed for 7MENO (88.4 %) and 7IPNO (78.5 %). When bacterial membrane permeability was investigated all compounds caused significant release of propidium iodide from S. aureus (31.6–87.0 % for concentration 128 μg/mL). In the case of E. faecalis, 7ETNO (75.7 %) and NO (28.8 %) were the most active. The rest of the tested compounds showed less activity (permeability < 13.9 %). The strong evidence was observed that antibacterial activity of the tested compounds may be associated with their interaction with bacterial membrane.

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2024 – Proximal telomeric decompaction due to telomere shortening drives FOXC1-dependent myocardial senescence
CardioExcyte 96 Publication in Nucleic Acids Research (2024) Authors: Li B., Xiong W., Zuo W., Shi Y., Wang T., Chang L., Wu Y., Ma H., Bian Q., Chang A.

Telomeres, TTAGGGn DNA repeat sequences located at the ends of eukaryotic chromosomes, play a pivotal role in aging and are targets of DNA damage response. Although we and others have demonstrated presence of short telomeres in genetic cardiomyopathic and heart failure cardiomyocytes, little is known about the role of telomere lengths in cardiomyocyte. Here, we demonstrate that in heart failure patient cardiomyocytes, telomeres are shortened compared to healthy controls. We generated isogenic human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) with short telomeres (sTL-CMs) and normal telomeres (nTL-CMs) as model. Compared to nTL-CMs, short telomeres result in cardiac dysfunction and expression of senescent markers. Using Hi-C and RNASeq, we observe that short telomeres induced TAD insulation decrease near telomeric ends and this correlated with a transcription upregulation in sTL-CMs. FOXC1, a key transcription factor involved in early cardiogenesis, was upregulated in sTL-CMs and its protein levels were negatively correlated with telomere lengths in heart failure patients. Overexpression of FOXC1 induced hiPSC-CM aging, mitochondrial and contractile dysfunction; knockdown of FOXC1 rescued these phenotypes. Overall, the work presented demonstrate that increased chromatin accessibility due to telomere shortening resulted in the induction of FOXC1-dependent expression network responsible for contractile dysfunction and myocardial senescence.

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2024 – Exploring new nanopore sensors from the aerolysin family
Orbit mini Pre-print in bioRxiv (2024) Authors: Cirauqui N., Bada Juarez J.F., Meireles F.A., Barry J., Bokori-Brown M., Marcaida M., Cao C., Dal Peraro M.

Aerolysin-like proteins are a family of β-pore-forming toxins which are widely present in all kingdoms of life. Recently, this family of proteins is gaining attention because of their biotechnological application as nanopore sensors for sensing and sequencing of biomolecules. Here, we explore the possibilities of using the knowledge of the sequence and structure of proteins to screen and explore new potential nanopore candidates. However, in spite of the conserved structural fold, the sequence identity in this family is very low. This complicates their sequence alignment, hindering the understanding of their pore structure and properties, therefore limiting further biotechnological applications. In an attempt to further understand the properties of aerolysin-like pores, we analyzed the pore structure of three family members, Clostridium perfringens epsilon toxin (ETX), Laetiporus sulphureus lectin (LSL) and Bacillus thuringiensis parasporin-2, comparing it to aerolysin. Their structure and sensing capabilities for ssDNA were first assessed by in silico methods. Moreover, ETX was characterized experimentally in planar lipid membranes for the detection of biomolecules. We found that ETX can form three distinct pore conformations, each presenting a specific open pore current, and only one of them being able to translocate ssDNA. When the ssDNA translocate through ETX, the depth of current blockage is higher compared to aerolysin which indicates a higher sensitivity for molecular sensing. Our findings open a new venue for improving and diversifying nanopore capabilities for molecular sensing.

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2024 – Single-molecule evidence of Entropic Pulling by Hsp70 chaperones
Orbit mini Pre-print in bioRxiv (2024) Authors: Rukes V., Rebeaud M., Perrin L., De Los Rios P., Cao C.

Hsp70 chaperones are central components of the cellular network that ensure the structural quality of proteins. Despite their crucial roles in processes as diverse as the prevention of protein aggregation and protein translocation into organelles, their molecular mechanism of action has remained a hotly debated issue. Due to a lack of suitable methods, no experimental data has directly proven any of the models that have been proposed (Power Stroke, Brownian Ratchet, and Entropic Pulling). Recently, nanopores have emerged as a powerful tool to analyze the function of motor enzymes, as well as protein-protein interactions. Here, we used an in vitro single-molecule nanopore to mimic in vivo translocation of proteins, and to investigate the molecular mechanism of Hsp70. Our experiments demonstrate that Hsp70s forcefully extract polypeptide substrates that are trapped inside the pore. The forces they exert are strong at the molecular level, being equivalent to 46 pN over distances of 1 nm, and depend on the size of Hsp70. These findings provide unambiguous evidence supporting the Entropic Pulling mechanism of action of Hsp70s, thus solving a long-standing debate, and proposing a potentially universal principle governing diverse cellular processes. In addition, these results emphasize the utility of biological nanopores for studying protein function at the single-molecule level.

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2024 – Discovery of a Novel Potent Tetrazole Antifungal Candidate with High Selectivity and Broad Spectrum
SyncroPatch 384 Publication in J. Med. Chem. (2024) Authors: Ni T., Hao Y., Ding Z., Chi X., Xie F., Wang R., Bao J., Yan L., Li L., Wang T., Zhang D., Jiang Y.

Thirty-one novel albaconazole derivatives were designed and synthesized based on our previous work. All compounds exhibited potent in vitro antifungal activities against seven pathogenic fungi. Among them, tetrazole compound D2 was the most potent antifungal with MIC values of <0.008, <0.008, and 2 μg/mL against Candida albicansCryptococcus neoformans, and Aspergillus fumigatus, respectively, the three most common and critical priority pathogenic fungi. In addition, compound D2 also exhibited potent activity against fluconazole-resistant C. auris isolates. Notably, compound D2 showed a lower inhibitory activity in vitro against human CYP450 enzymes as well as a lower inhibitory effect on the hERG K+ channel, indicating a low risk of drug–drug interactions and QT prolongation. Moreover, with improved pharmacokinetic profiles, compound D2 showed better in vivo efficacy than albaconazole at reducing fungal burden and extending the survival of C. albicans-infected mice. Taken together, compound D2 will be further investigated as a promising candidate.

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2024 – Discovery of TNG908: A Selective, Brain Penetrant, MTA-Cooperative PRMT5 Inhibitor That Is Synthetically Lethal with MTAP-Deleted Cancers
SyncroPatch 384 PE (a predecessor model of the SyncroPatch 384) Publication in J. Med. Chem. (2024) Authors: Cottrell K.M., Briggs K.J., Whittington D.A., Jahic H., Ali J.A., Davis C.B., Gong S., Gotur D., Gu L., McCarren P., Tonini M., Tsai A., Wilker E., Yuan H., Zhang M., Zhang W., Huang A., Maxwell J.P.

It has been shown that PRMT5 inhibition by small molecules can selectively kill cancer cells with homozygous deletion of the MTAP gene if the inhibitors can leverage the consequence of MTAP deletion, namely, accumulation of the MTAP substrate MTA. Herein, we describe the discovery of TNG908, a potent inhibitor that binds the PRMT5·MTA complex, leading to 15-fold-selective killing of MTAP-deleted (MTAP-null) cells compared to MTAPintact (MTAP WT) cells. TNG908 shows selective antitumor activity when dosed orally in mouse xenograft models, and its physicochemical properties are amenable for crossing the blood–brain barrier (BBB), supporting clinical study for the treatment of both CNS and non-CNS tumors with MTAP loss.

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2024 – Structural basis of human Nav1.5 gating mechanisms
SyncroPatch 384i (a predecessor model of the SyncroPatch 384) Pre-Print Publication in Research Square (2024) Authors: Chinthalapudi K., Biswas R., López-Serrano A., Huang H.L., Ramirez-Navarro A., Grandinetti G., Heissler S., Deschênes I.

Voltage-gated Nav1.5 channels are central to the generation and propagation of cardiac action potentials. Aberrations in their function are associated with a wide spectrum of cardiac diseases including arrhythmias and heart failure. Despite decades of progress in Nav1.5 biology, the lack of structural insights into intracellular regions has hampered our understanding of its gating mechanisms. Here we present three cryo-EM structures of human Nav1.5 in previously unanticipated open states, revealing sequential conformational changes in gating charges of the voltage-sensing domains (VSDs) and several intracellular regions. Despite the channel being in the open state, these structures show the IFM motif repositioned in the receptor site but not dislodged. In particular, our structural findings highlight a dynamic C-terminal domain (CTD) and III-IV linker interaction, which regulates the conformation of VSDs and pore opening. Electrophysiological studies confirm that disrupting this interaction results in the fast inactivation of Nav1.5. Together, our structure-function studies establish a foundation for understanding the gating mechanisms of Nav1.5 and the mechanisms underlying CTD-related channelopathies.

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PatchControlHT Suite – Patchliner
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2024 – Response of heterologously expressed pressure sensor-actuator-modulator macromolecule to external mechanical stress
Port-a-Patch Publication in Molecular Sciences (2024) Authors: Batabyal S., Idigo C., Narcisse D., Dibas A., Mohanty S.

Cells from different organs in the body experience a range of mechanical and osmotic pressures that change in various diseases, including neurological, cardiovascular, ophthalmological, and renal diseases. Here, we demonstrate the use of an engineered Sensor-Actuator-Modulator (SAM) of microbial origin derived from a mechanosensitive channel of large conductance (MscL) for sensing external mechanical stress and modulating activities of mammalian cells. SAM is reliably expressed in the mammalian cell membrane and acts as a tension-activated pressure release valve. Further, the activities of heterologously expressed SAM in mammalian cells could be modulated by osmotic pressure. A comparison of the mechanosensitive activities of SAM-variants from different microbial origins shows differential inward current and dye uptake in response to mechanical stress exerted by hypo-osmotic shock. The use of SAM channels as mechanical stress-activated modulators in mammalian cells could provide new therapeutic approaches for treating disorders related to mechanical or osmotic pressure.

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2024 – Thermosensitive TRP Channels Are Functionally Expressed and Influence the Lipogenesis in Human Meibomian Gland Cells
Port-a-Patch Publication in Molecular Sciences (2024) Authors: Keller M., Mergler S., Li A., Zahn I., Paulsen F., Garreis F.

While the involvement of thermosensitive transient receptor potential channels (TRPs) in dry eye disease (DED) has been known for years, their expression in the meibomian gland (MG) has never been investigated. This study aims to show their expression and involvement in the lipogenesis of the MG, providing a possible new drug target in the treatment of DED. Our RT-PCR, Western blot and immunofluorescence analysis showed the expression of TRPV1TRPV3TRPV4 and TRPM8 in the MG at the gene and the protein level. RT-PCR also showed gene expression of TRPV2 but not TRPA1. Calcium imaging and planar patch-clamping performed on an immortalized human meibomian gland epithelial cell line (hMGECs) demonstrated increasing whole-cell currents after the application of capsaicin (TRPV1) or icilin (TRPM8). Decreasing whole-cell currents could be registered after the application of AMG9810 (TRPV1) or AMTB (TRPM8). Oil red O staining on hMGECs showed an increase in lipid expression after TRPV1 activation and a decrease after TRPM8 activation. We conclude that thermo-TRPs are expressed at the gene and the protein level in MGs. Moreover, TRPV1 and TRPM8’s functional expression and their contribution to their lipid expression could be demonstrated. Therefore, TRPs are potential drug targets and their clinical relevance in the therapy of meibomian gland dysfunction requires further investigation.

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2024 – High-throughput single biomarker identification using droplet nanopore
Orbit 16 (a predecessor model of the Orbit 16 TC) Publication in Chem. Sci. (2024) Authors: Zhang L.L., Zhonga C.B., Huanga T.J., Zhangb L.M., Yanb F., Ying Y.L.

Biomarkers are present in various metabolism processes, demanding precise and meticulous analysis at the single-molecule level for accurate clinical diagnosis. Given the need for high sensitivity, biological nanopore have been applied for single biomarker sensing. However, the detection of low-volume biomarkers poses challenges due to their low concentrations in dilute buffer solutions, as well as difficulty in parallel detection. Here, a droplet nanopore technique is developed for low-volume and high-throughput single biomarker detection at the sub-microliter scale, which shows a 2000-fold volume reduction compared to conventional setups. To prove the concept, this nanopore sensing platform not only enables multichannel recording but also significantly lowers the detection limit for various types of biomarkers such as angiotensin II, to 42 pg. This advancement enables direct biomarker detection at the picogram level. Such a leap forward in detection capability positions this nanopore sensing platform as a promising candidate for point-of-care testing of biomarker at single-molecule level, while substantially minimizing the need for sample dilution.

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2024 – Automated Patch Clamp for the Detection of Tetrodotoxin in Pufferfish Samples
Patchliner Publication in Marine Drugs (2024) Authors: Campàs M., Reverté J., Tudó À., Alkassar M., Diogène J., Sureda FX.

Tetrodotoxin (TTX) is a marine toxin responsible for many intoxications around the world. Its presence in some pufferfish species and, as recently reported, in shellfish, poses a serious health concern. Although TTX is not routinely monitored, there is a need for fast, sensitive, reliable, and simple methods for its detection and quantification. In this work, we describe the use of an automated patch clamp (APC) system with Neuro-2a cells for the determination of TTX contents in pufferfish samples. The cells showed an IC50 of 6.4 nM for TTX and were not affected by the presence of muscle, skin, liver, and gonad tissues of a Sphoeroides pachygaster specimen (TTX-free) when analysed at 10 mg/mL. The LOD achieved with this technique was 0.05 mg TTX equiv./kg, which is far below the Japanese regulatory limit of 2 mg TTX equiv./kg. The APC system was applied to the analysis of extracts of a Lagocephalus sceleratus specimen, showing TTX contents that followed the trend of gonads > liver > skin > muscle. The APC system, providing an in vitro toxicological approach, offers the advantages of being sensitive, rapid, and reliable for the detection of TTX-like compounds in seafood.

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2024 – Comprehensive promotion of iPSC-CM maturation by integrating metabolic medium, nanopatterning, and electrostimulation
Patchliner Pre-print Publication in Research Square (2024) Authors: Li W., Luo X., Strano A., Arun S., Gamm O., Pötsch M., Hasse M., Steiner R., Fischer K., Pöche J., Ulbricht Y., Lesche M.

The immaturity of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) is a major limitation for their use in drug screening to identify pro-arrhythmogenic or cardiotoxic molecules, thus hindering their potential role in guiding personalised drug selection for patients. Here, we demonstrate an approach that combines lipid-enriched maturation medium, nanopatterning of culture surfaces and electrostimulation to generate iPSC-CMs with an advanced electrophysiological, structural and metabolic phenotype. Through a systematic, stepwise parallel testing of the three stimuli, electrostimulation emerged as the pivotal factor to enhance mitochondrial development and to improve the electrophysiological properties of iPSC-CMs. The combined approach brought a substantial modification in their current composition by increasing INaItoIK1 and IKr but decreasing ICa−L, resulting in a significant change in their sensitivity to cardioactive drugs. Transcriptome analysis revealed that activation of HMCES and TFAM targets played a role in mitochondrial development, whereas the downregulation of MAPK/PI3K signalling pathways and SRF targets were associated with polyploidy of iPSC-CMs. Taken together, our study provides mechanistic insights into the maturation of iPSC-CMs with a more adult-like drug response.

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GPCRs – Profiling the pharmacology of GPCRs by time-resolved impedance measurements
AtlaZ Application note

This Application Note demonstrates the in vitro characterization of GPCR pharmacology, covering agonist and antagonist mode of action, dose-response relationships, and the involvement of signal transduction cascades. Experimentally,
cells expressing H1R or Y4R are cultured on planar gold-film electrodes integrated into standard cell culture dishes.

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Replacing radiolabeling techniques with the SURFE2R N1
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2024 – Robust inference of cooperative behaviour of multiple ion channels in voltage-clamp recordings
Orbit e16 (a predecessor model of the Orbit 16 TC) Pre-print Publication in Arxiv (2024) Authors: Requadt R., Fink M., Kubica P., Steinem C., Munk A., Li H.

Recent experimental studies have shed light on the intriguing possibility that ion channels exhibit cooperative behaviour. However, a comprehensive understanding of such cooperativity remains elusive, primarily due to limitations in measuring separately the response of each channel. Rather, only the superimposed channel response can be observed, challenging existing data analysis methods. To address this gap, we propose IDC (Idealisation, Discretisation, and Cooperativity inference), a robust statistical data analysis methodology that requires only voltage-clamp current recordings of an ensemble of ion channels. The framework of IDC enables us to integrate recent advancements in idealisation techniques and coupled Markov models. Further, in the cooperativity inference phase of IDC, we introduce a minimum distance estimator and establish its statistical guarantee in the form of asymptotic consistency. We demonstrate the effectiveness and robustness of IDC through extensive simulation studies. As an application, we investigate gramicidin D channels. Our findings reveal that these channels act independently, even at varying applied voltages during voltage-clamp experiments. An implementation of IDC is available from GitLab.

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2024 – Discovery of a novel natural compound, vitekwangin B, with ANO1 protein reduction properties and anticancer potential
Patchliner Publication in Frontiers in Pharmacology (2024) Authors: Yohan S., Sion L., Minuk K., Dongguk K., Baek J.S., Raju D., Armin S., SeonJu P., Xuan N.N., Thi Thanh H.P., Oh-Bin K., Wan N., Joohan W.

Background: Prostate cancer and non-small cell lung cancer (NSCLC) present significant challenges in the development of effective therapeutic strategies. Hormone therapies for prostate cancer target androgen receptors and prostate-specific antigen markers. However, treatment options for prostatic small-cell neuroendocrine carcinoma are limited. NSCLC, on the other hand, is primarily treated with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors but exhibits resistance. This study explored a novel therapeutic approach by investigating the potential anticancer properties of vitekwangin B, a natural compound derived from Vitex trifolia.

Methods: Vitekwangin B was chromatographically isolated from the fruits of V. trifolia. ANO1 protein levels in prostate cancer and NSCLC cells were verified and evaluated again after vitekwangin B treatment.

Results: Vitekwangin B did not inhibit anoctamin1 (ANO1) channel function but significantly reduced ANO1 protein levels. These results demonstrate that vitekwangin B effectively inhibited cancer cell viability and induced apoptosis in prostate cancer and NSCLC cells. Moreover, it exhibited minimal toxicity to liver cells and did not affect hERG channel activity, making it a promising candidate for further development as an anticancer drug.

Conclusion: Vitekwangin B may offer a new direction for cancer therapy by targeting ANO1 protein, potentially improving treatment outcomes in patients with prostate cancer and NSCLC. Further research is needed to explore its full potential and overcome existing drug resistance challenges.

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2024 – The funnel-web spider venom derived single knot peptide Hc3a modulates acid-sensing ion channel 1a desensitisation
Patchliner Publication in Biochemical Pharmacology (2024) Authors: Budusan E., Payne C., Gonzalez T., Obergrussberger A., Becker N., Clark R., Rosengren K., Rash L., Cristofori-Armstrong B.

Acid-sensing ion channel 1a (ASIC1a) is a proton-gated channel involved in synaptic transmission, pain signalling, and several ischemia-associated pathological conditions. The spider venom-derived peptides PcTx1 and Hi1a are two of the most potent ASIC1a inhibitors known and have been instrumental in furthering our understanding of the structure, function, and biological roles of ASICs. To date, homologous spider peptides with different pharmacological profiles at ASIC1a have yet to be discovered. Here we report the characterisation of Hc3a, a single inhibitor cystine knot peptide from the Australian funnel-web spider Hadronyche cerberea with sequence similarity to PcTx1. We show that Hc3a has complex pharmacology and binds different ASIC1a conformational states (closed, open, and desensitised) with different affinities, with the most prominent effect on desensitisation. Hc3a slows the desensitisation kinetics of proton-activated ASIC1a currents across multiple application pHs, and when bound directly to ASIC1a in the desensitised conformation promotes current inhibition. The solution structure of Hc3a was solved, and the peptide-channel interaction examined via mutagenesis studies to highlight how small differences in sequence between Hc3a and PcTx1 can lead to peptides with distinct pharmacology. The discovery of Hc3a expands the pharmacological diversity of spider venom peptides targeting ASIC1a and adds to the toolbox of compounds to study the intricacies of ASIC1 gating.

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Diabetic cardiomyopathy – “In vitro cardiomyopathy model to evaluate ischemic sensitivity”
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2024 – Using automated patch clamp electrophysiology platforms in ion channel drug discovery: an industry perspective
SyncroPatch 384 Publication (review) in Expert Opinion on Drug Discovery (2024) Authors: Rogers M., Obergrussberger A., Kondratskyi A., Fertig N.

Introduction
Automated patch clamp (APC) is now well established as a mature technology for ion channel drug discovery in academia, biotech and pharma companies, and in contract research organizations (CRO), for a variety of applications including channelopathy research, compound screening, target validation and cardiac safety testing.

Areas covered
Ion channels are an important class of drugged and approved drug targets. The authors present a review of the current state of ion channel drug discovery along with new and exciting developments in ion channel research involving APC. This includes topics such as native and iPSC-derived cells in ion channel drug discovery, channelopathy research, organellar and biologics in ion channel drug discovery.

Expert opinion
It is our belief that APC will continue to play a critical role in ion channel drug discovery, not only in ‘classical’ hit screening, target validation and cardiac safety testing, but extending these applications to include high throughput organellar recordings and optogenetics. In this way, with advancements in APC capabilities and applications, together with high resolution cryo-EM structures, ion channel drug discovery will be re-invigorated, leading to a growing list of ion channel ligands in clinical development.

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2024 – Janus microparticles-based targeted and spatially-controlled piezoelectric neural stimulation via low-intensity focused ultrasound
Buffer solutions Publication in Nature communications (2024) Authors: Han M., Yildiz E., Bozuyuk U., Aydin A., Yu Y., Bhargava A., Karaz S., Sitti M.

Electrical stimulation is a fundamental tool in studying neural circuits, treating neurological diseases, and advancing regenerative medicine. Injectable, free-standing piezoelectric particle systems have emerged as non-genetic and wireless alternatives for electrode-based tethered stimulation systems. However, achieving cell-specific and high-frequency piezoelectric neural stimulation remains challenging due to high-intensity thresholds, non-specific diffusion, and internalization of particles. Here, we develop cell-sized 20 μm-diameter silica-based piezoelectric magnetic Janus microparticles (PEMPs), enabling clinically-relevant high-frequency neural stimulation of primary neurons under low-intensity focused ultrasound. Owing to its functionally anisotropic design, half of the PEMP acts as a piezoelectric electrode via conjugated barium titanate nanoparticles to induce electrical stimulation, while the nickel-gold nanofilm-coated magnetic half provides spatial and orientational control on neural stimulation via external uniform rotating magnetic fields. Furthermore, surface functionalization with targeting antibodies enables cell-specific binding/targeting and stimulation of dopaminergic neurons. Taking advantage of such functionalities, the PEMP design offers unique features towards wireless neural stimulation for minimally invasive treatment of neurological diseases.

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2024 – Nanopore analysis of cis-diols in fruits
Orbit mini Publication in Nature communications (2024) Authors: Fan P., Cao Z., Zhang S., Wang Y., Xiao Y., Jia W:, Zhang P., Huang S.

Natural fruits contain a large variety of cis-diols. However, due to the lack of a high-resolution sensor that can simultaneously identify all cis-diols without a need of complex sample pretreatment, direct and rapid analysis of fruits in a hand-held device has never been previously reported. Nanopore, a versatile single molecule sensor, can be specially engineered to perform this task. A hetero-octameric Mycobacterium smegmatis porin A (MspA) nanopore modified with a sole phenylboronic acid (PBA) adapter is prepared. This engineered MspA accurately recognizes 1,2-diphenols, alditols, α-hydroxy acids and saccharides in prune, grape, lemon, different varieties of kiwifruits and commercial juice products. Assisted with a custom machine learning program, an accuracy of 99.3% is reported and the sample pretreatment is significantly simplified. Enantiomers such as DL-malic acids can also be directly identified, enabling sensing of synthetic food additives. Though demonstrated with fruits, these results suggest wide applications of nanopore in food and drug administration uses.

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Developing new therapies targeting P2X receptors
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Targeting TRP channels for the treatment of eye diseases
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SyncroPatch 384 – NaV with adaptive voltage clamp
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2024 – Nanoscale architecture and dynamics of CaV1.3 channel clusters in cardiac myocytes revealed by single channel nanoscopy
SyncroPatch 384 Pre-Print Publication in bioRxiv (2024) Authors: Schwenzer N., Tsukanov R., Kohl T., Basak S., Seibertz F., Voigt N., Enderlein J., Lehnart S.

The clustering of L-type calcium channels in cardiac myocytes presents an important mechanism for functional regulation of calcium signaling. Here we applied targeted super-resolution imaging techniques for the study of atrial-specific CaV1.3 channel clusters in human iPSC-derived atrial cardiomyocytes (hiPSC-aCM). We thereby clarified cluster localization, dimensions, architecture, and dynamics, which were largely unexplored previously. Live-cell STimulated Emission Depletion (STED) imaging identified that cell surface-localized clusters contained 9 channel molecules within 120 nm diameter on average. DNA Points Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) optimized for molecular mapping revealed an irregular arrangement of channels with significant spacing. Single Particle Tracking (SPT) further evidenced that clustered channels do not associate into rigidly packed structures (oligomers or lattices), but rather co-diffuse in confined and stationary membrane nanodomains. Immunofluorescence showed consistent cell-surface colocalization with Ryanodine Receptor type 2 and Junctophilin-2 forming stable calcium release units, similar to dyadic junctions containing CaV1.2 in ventricular cardiomyocytes. Lastly, novel genetic constructs for live-cell imaging showed that the cytosolic C-terminal tail of CaV1.3 by itself is sufficient for cluster formation. In conclusion, a novel strategy for LTCC clustering studies in atrial cells was established, suitable for a wide range of super-resolution imaging techniques. Based on live-cell STED, DNA-PAINT and SPT data, we propose that CaV1.3 channel clusters consist of mobile individual channels inside defined membrane nanodomains.

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Targeting TRP channels for the treatment of eye diseases
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2024 – Wnt dose escalation during the exit from pluripotency identifies tranilast as a regulator of cardiac mesoderm
CardioExcyte 96 Publication in Developmental Cell (2024) Authors: Wu Z., Shen S., Mizikovsky D., Cao Y., Naval-Sanchez M., Tan S.Z., Alvarez Y., Sun Y., Chen X., Zhao Q., Kim D., Yang P., Hill T., Jones A., Fairlie D., Pébay A., Hewitt A., Tam P., White M., Nefzger C., Palpant N.

Wnt signaling is a critical determinant of cell lineage development. This study used Wnt dose-dependent induction programs to gain insights into molecular regulation of stem cell differentiation. We performed single-cell RNA sequencing of hiPSCs responding to a dose escalation protocol with Wnt agonist CHIR-99021 during the exit from pluripotency to identify cell types and genetic activity driven by Wnt stimulation. Results of activated gene sets and cell types were used to build a multiple regression model that predicts the efficiency of cardiomyocyte differentiation. Cross-referencing Wnt-associated gene expression profiles to the Connectivity Map database, we identified the small-molecule drug, tranilast. We found that tranilast synergistically activates Wnt signaling to promote cardiac lineage differentiation, which we validate by in vitro analysis of hiPSC differentiation and in vivo analysis of developing quail embryos. Our study provides an integrated workflow that links experimental datasets, prediction models, and small-molecule databases to identify drug-like compounds that control cell differentiation.

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2024 – Secretion of the fungal toxin candidalysin is dependent on conserved precursor peptide sequences
Orbit 16 (a predecessor model of the Orbit 16TC) Publication in Nature Microbiology (2024) Authors: Müller R., König A., Groth S., Zarnowski R., Visser C., Handrianz T., Maufrais C., Krüger T., Himmel M., Lee S., Priest E., Yildirim D., Richardson J., Blango M., Bougnoux M.E., Kniemeyer O., d’Enfert C., Brakhage A., Andes D., Trümper V., Nehls C., Kasper L., Mogavero S., Gutsmann T., Naglik J., Allert S., Hube B.

The opportunistic fungal pathogen Candida albicans damages host cells via its peptide toxin, candidalysin. Before secretion, candidalysin is embedded in a precursor protein, Ece1, which consists of a signal peptide, the precursor of candidalysin and seven non-candidalysin Ece1 peptides (NCEPs), and is found to be conserved in clinical isolates. Here we show that the Ece1 polyprotein does not resemble the usual precursor structure of peptide toxins. C. albicans cells are not susceptible to their own toxin, and single NCEPs adjacent to candidalysin are sufficient to prevent host cell toxicity. Using a series of Ece1 mutants, mass spectrometry and anti-candidalysin nanobodies, we show that NCEPs play a role in intracellular Ece1 folding and candidalysin secretion. Removal of single NCEPs or modifications of peptide sequences cause an unfolded protein response (UPR), which in turn inhibits hypha formation and pathogenicity in vitro. Our data indicate that the Ece1 precursor is not required to block premature pore-forming toxicity, but rather to prevent intracellular auto-aggregation of candidalysin sequences.

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2024 – Reprogramming of DNA methylation patterns mediates perfluorooctane sulfonate-induced fetal cardiac dysplasia
CardioExcyte 96 Publication in Science of the Total Environment (2024) Authors: Qiu M., Chen J., Liu M., Shi Y., Nie Z., Dong G., Li X., Chen J., Ou Y., Zhuang J.

Prenatal exposure to perfluorooctane sulfonate (PFOS) is associated with adverse health effects, including congenital heart disease, yet the underlying mechanisms remain elusive. Herein, we aimed to evaluate the embryotoxicity of PFOS using C57BL/6 J mice to characterize fetal heart defects after PFOS exposure, with the induction of human embryonic stem cells (hESC) into cardiomyocytes (CMs) as a model of early-stage heart development. We also performed DNA methylation analysis to clarify potential underlying mechanisms and identify targets of PFOS. Our results revealed that PFOS caused septal defects and excessive ventricular trabeculation cardiomyopathy at 5 mg/kg/day in embryonic mice and inhibited the proliferation and pluripotency of ESCs at concentrations >20 μM. Moreover, it decreased the beating rate and the population of CMs during cardiac differentiation. Decreases were observed in the abundances of NPPA+ trabecular and HEY2+ compact CMs. Additionally, DNA methyl transferases and ten-eleven translocation (TET) dioxygenases were regulated dynamically by PFOS, with TETs inhibitor treatment inducing significant decreases similar as PFOS. 850 K DNA methylation analysis combined with expression analysis revealed several potential targets of PFOS, including SORBS2FHOD1SLIT2SLIT3ADCY9, and HDAC9. In conclusion, PFOS may reprogram DNA methylation, especially demethylation, to induce cardiac toxicity, causing ventricular defects in vivo and abnormal cardiac differentiation in vitro.

 

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Hepatocytes – “Comprehensive impedance-based hepatotoxicity assay for metabolically active iPSC-derived hepatocytes”
CardioExcyte 96, AtlaZ Application Note: Cells were kindly provided by FUJIFILM CellularDynamics, FCDI

Hepatotoxicity and drug-induced liver injury (DILI) are leading reasons for drug failure to market, leading to approximately 18% of market withdrawals of drugs in the last decade.
Additionally, only half of drugs with a potential to induce hepatotoxicity are actually identified during preclinical animal studies. This highlights the importance of generating more
advanced cell-based models and experimental strategies to enhance the predictivity of these assays. Current existing in vitro models employed to predict DILI mostly focus on
hepatocytes, though primary hepatocytes do not maintain their phenotype. iCell® Hepatocytes 2.0 (FUJIFILM Cellular Dynamics, FCDI) are human iPSC-derived cells with a
wide variety of basic and functional characteristics which make them amenable to applications such as compound mediated ADME-T and DILI toxicity. In addition to displaying
characteristic hepatocyte morphology, (i.e., polygonal shape, polynucleation and formation of bile canalicular channels), these cells also express liver cell markers, including albumin,
A1AT, and HNF4a, and exhibit basic and induced P450 functions, as observed in primary human hepatocytes. iCell® Hepatocytes 2.0 maintain morphology, marker expression,
and metabolic function in culture over a longer time frame compared to primary human hepatocytes, rendering these cells useful for investigations of acute and chronic DILI responses in a 2D culture system using impedance. Combining these cells with the planar gold-film electrodes on the impedance systems reveal alterations in confluency, cell contact (morphological shape) and conductivity of adherent cells, thereby providing a measure of toxicity. Dose-dependent harmful effects of drugs could be evaluated over time in a functional 2D cell-based model without the need for 3D spheroid formation.

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2024 – Nanobody-mediated neutralization of candidalysin prevents epithelial damage and inflammatory responses that drive vulvovaginal candidiasis pathogenesis
Orbit 16 (a predecessor model of the Orbit 16TC) Publication in American Society for Microbiology (2024) Authors: Valentine M., Rudolph P., Dietschmann A., Tsavou A., Mogavero S., Lee S., Priest E., Zhurgenbayeva G., Jablonowski N., Timme S., Eggeling C., Allert S., Dolk E., Naglik J., Figge M., Gresnigt M., Hube B.

Candida albicans can cause mucosal infections in humans. This includes oropharyngeal candidiasis, which is commonly observed in human immunodeficiency virus infected patients, and vulvovaginal candidiasis (VVC), which is the most frequent manifestation of candidiasis. Epithelial cell invasion by C. albicans hyphae is accompanied by the secretion of candidalysin, a peptide toxin that causes epithelial cell cytotoxicity. During vaginal infections, candidalysin-driven tissue damage triggers epithelial signaling pathways, leading to hyperinflammatory responses and immunopathology, a hallmark of VVC. Therefore, we proposed blocking candidalysin activity using nanobodies to reduce epithelial damage and inflammation as a therapeutic strategy for VVC. Anti-candidalysin nanobodies were confirmed to localize around epithelial-invading C. albicans hyphae, even within the invasion pocket where candidalysin is secreted. The nanobodies reduced candidalysin-induced damage to epithelial cells and downstream proinflammatory responses. Accordingly, the nanobodies also decreased neutrophil activation and recruitment. In silico mathematical modeling enabled the quantification of epithelial damage caused by candidalysin under various nanobody dosing strategies. Thus, nanobody-mediated neutralization of candidalysin offers a novel therapeutic approach to block immunopathogenic events during VVC and alleviate symptoms.

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Mitochondrial Membrane Research
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SyncroPatch 384 – DataControl 384
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SyncroPatch 384 – Optogenetic Stimulation Tool
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Patchliner – Single Channel Recordings
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TMEM175, TPC2 – “High throughput organellar electrophysiology of TMEM175 and TPC2 from freshly isolated lysosomes recorded on the SyncroPatch 384”
SyncroPatch 384 application note: in collaboration with Axxam

Intracellular ion channels are known to play an essential role in various signaling pathways for health and disease, considering that over 80% of transport processes occur inside
the cells. Among the variety of organellar channels and transporters the proton leak channel transmembrane protein 175 (TMEM175) and the lysosomal two-pore channel (TPC) have received increasing attention in the field given their potential roles in connecting lysosomal homeostasis with pathophysiological conditions such as Parkinson’s disease and cancer. Consequently, the interest to explore intracellular ion channels as therapeutic targets has grown tremendously indicating a need for high-throughput electrophysiology including patch clamp. There has been some progress in alternative approaches such as solid supported membrane electrophysiology (SSME using the SURFE2R 96SE) recently, however, until now, HTS patch clamp has lacked the possibility to collect data from native lysosomes.

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SyncroPatch 384 – Advanced Temperature Control
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2013 – Functional TRPV1 expression in human corneal fibroblasts
Port-a-Patch Publication in Experimental Eye Research (2013) Authors: Yang Y., Yang H., Wang Z., Mergler S., Wolosin JM., Reinach PS.

Corneal wound healing in mice subsequent to an alkali burn results in dysregulated inflammation and opacification. Transient receptor potential vanilloid subtype 1 (TRPV1) channel activation in all tissue layers by endogenous ligands contributes to this sight compromising outcome since in TRPV1 knockout mice wound healing results instead in tissue transparency restoration. However, it is not known if primary human stromal fibroblasts exhibit such expression even though functional TRPV1 expression is evident in an immortalized human corneal epithelial cell line. In primary human corneal fibroblasts (HCF), TRPV1 gene expression and localization were identified based on the results of quantitative RT-PCR and immunocytochemistry, respectively. Western blot analysis identified a 100 kD protein corresponding to TRPV1 protein expression in a positive control. Single-cell fluorescence imaging detected in fura2-AM loaded cells Ca(2+) transients that rose 1.8-fold above the baseline induced by a selective TRPV1 agonist, capsaicin (CAP), which were blocked by a TRPV1 antagonist, capsazepine (CPZ) or exposure to a Ca(2+) free medium. The whole-cell mode of the planar patch-clamp technique identified TRPV1-induced currents that rose 1.76-fold between -60 and +130 mV. CAP-induced time dependent changes in the phosphorylation status of mitogen activated protein kinase (MAPK) signaling mediators that led to a 2.5-fold increase in IL-6 release after 24 h. This rise did not occur either in TRPV1 siRNA gene silenced cells or during exposure to SB203580 (10 μM), a selective p38 MAPK inhibitor. Taken together, identification of functional TRPV1 expression in HCF suggests that in vivo its activation by injury contributes to corneal opacification and inflammation during wound healing. These undesirable effects may result in part from increases in IL-6 expression mediated by p-p38 MAPK signaling.

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2013 – Cannabinoid receptor 1 suppresses transient receptor potential vanilloid 1-induced inflammatory responses to corneal injury
Port-a-Patch Publication in Cellular Signalling (2013) Authors: Yang Y., Yang H., Wang Z., Varadaraj K., Kumari SS., Mergler S., Okada Y., Saika S., Kingsley PJ., Marnett LJ., Reinach PS.

Cannabinoid receptor type 1 (CB1)-induced suppression of transient receptor potential vanilloid type 1 (TRPV1) activation provides a therapeutic option to reduce inflammation and pain in different animal disease models through mechanisms involving dampening of TRPV1 activation and signaling events. As we found in both mouse corneal epithelium and human corneal epithelial cells (HCEC) that there is CB1 and TRPV1 expression colocalization based on overlap of coimmunostaining, we determined in mouse corneal wound healing models and in human corneal epithelial cells (HCEC) if they interact with one another to reduce TRPV1-induced inflammatory and scarring responses. Corneal epithelial debridement elicited in vivo a more rapid wound healing response in wildtype (WT) than in CB1(-/-) mice suggesting functional interaction between CB1 and TRPV1. CB1 activation by injury is tenable based on the identification in mouse corneas of 2-arachidonylglycerol (2-AG) with tandem LC-MS/MS, a selective endocannabinoid CB1 ligand. Suppression of corneal TRPV1 activation by CB1 is indicated since following alkali burning, CB1 activation with WIN55,212-2 (WIN) reduced immune cell stromal infiltration and scarring. Western blot analysis of coimmunoprecipitates identified protein-protein interaction between CB1 and TRPV1. Other immunocomplexes were also identified containing transforming growth factor kinase 1 (TAK1), TRPV1 and CB1. CB1 siRNA gene silencing prevented suppression by WIN of TRPV1-induced TAK1-JNK1 signaling. WIN reduced TRPV1-induced Ca(2+) transients in fura2-loaded HCEC whereas pertussis toxin (PTX) preincubation obviated suppression by WIN of such rises caused by capsaicin (CAP). Whole cell patch clamp analysis of HCEC showed that WIN blocked subsequent CAP-induced increases in nonselective outward currents. Taken together, CB1 activation by injury-induced release of endocannabinoids such as 2-AG downregulates TRPV1 mediated inflammation and corneal opacification. Such suppression occurs through protein-protein interaction between TRPV1 and CB1 leading to declines in TRPV1 phosphorylation status. CB1 activation of the GTP binding protein, G(i/o) contributes to CB1 mediated TRPV1 dephosphorylation leading to TRPV1 desensitization, declines in TRPV1-induced increases in currents and pro-inflammatory signaling events.

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2012 – Altered calcium regulation by thermosensitive transient receptor potential channels in etoposide-resistant WERI-Rb1 retinoblastoma cells
Port-a-Patch Publication in Experimental Eye Research (2012) Authors: Mergler S, Cheng Y, Skosyrski S, Garreis F, Pietrzak P, Kociok N, Dwarakanath A, Reinach PS, Kakkassery V.

Differences in transient receptor potential (TRP) and cannabinoid receptor type 1 (CB1) expression levels can serve as prognostic factors for retinoblastoma (RB) tumor progression. We hypothesized in RB tissue that such differences are also indicators of whether or not they are sensitive to etoposide. Accordingly, we compared in malignant etoposide-sensitive and etoposide-resistant WERI-Rb1 cells TRPV1, TRPM8 and TRPA1 subtype and CB1 gene expression pattern levels and accompanying functional activity using quantitative real-time RT-PCR, immunohistochemistry, immunofluorescence microscopy, calcium imaging as well as patch-clamp technology. Gene expression patterns were evaluated in enucleated human RB tissues (n = 4). Both etoposide-resistant and etoposide-sensitive WERI-Rb1 cells expressed all of the aforementioned channels based on responses to known activators and thermal challenges. However, TRPA1 was absent in the etoposide-resistant counterpart. Even though both types of RB cells express TRPV1 as well as TRPM8 and CB1, the capsaicin (50 μM) (CAP)-induced Ca(2+) rise caused by TRPV1 activation was prompt and transient only in etoposide-resistant RB cells (n = 8). In this cell type, the inability of CB1 activation (10 μM WIN) to suppress Ca(2+) responses to CAP (50 μM; n = 4) may be attributable to the absence of TRPA1 gene expression. Therefore, using genetic approaches to upregulate TRPA1 expression could provide a means to induce etoposide sensitivity and suppress RB cell tumorigenesis.

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2015 – Thyronamine induces TRPM8 channel activation in human conjunctival epithelial cells
Port-a-Patch Publication in Cellular Signalling (2015) Authors: Khajavi N, Reinach PS, Slavi N, Skrzypski M, Lucius A, Strauß O, Köhrle J, Mergler S.

3-Iodothyronamine (T1AM), an endogenous thyroid hormone (TH) metabolite, induces numerous responses including a spontaneously reversible body temperature decline. As such an effect is associated in the eye with increases in basal tear flow and thermosensitive transient receptor potential melastatin 8 (TRPM8) channel activation, we determined in human conjunctival epithelial cells (IOBA-NHC) if T1AM also acts as a cooling agent to directly affect TRPM8 activation at a constant temperature. RT-PCR and quantitative real-time PCR (qPCR) along with immunocytochemistry probed for TRPM8 gene and protein expression whereas functional activity was evaluated by comparing the effects of T1AM with those of TRPM8 mediators on intracellular Ca2 + ([Ca2 +]i) and whole-cell currents. TRPM8 gene and protein expression was evident and icilin (20 μM), a TRPM8 agonist, increased Ca2 + influx as well as whole-cell currents whereas BCTC (10 μM), a TRPM8 antagonist, suppressed these effects. Similarly, either temperature lowering below 23 °C or T1AM (1 μM) induced Ca2 + transients that were blocked by this antagonist. TRPM8 activation by both 1 µM T1AM and 20 μM icilin prevented capsaicin (CAP) (20 μM) from inducing increases in Ca2 + influx through TRP vanilloid 1 (TRPV1) activation, whereas BCTC did not block this response. CAP (20 μM) induced a 2.5-fold increase in IL-6 release whereas during exposure to 20 μM capsazepine this rise was completely blocked. Similarly, T1AM (1 μM) prevented this response. Taken together, T1AM like icilin is a cooling agent since they both directly elicit TRPM8 activation at a constant temperature. Moreover, there is an inverse association between changes in TRPM8 and TRPV1 activity since these cooling agents blocked both CAP-induced TRPV1 activation and downstream rises in IL-6 release.

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2024 – Transport mechanism of DgoT, a bacterial homolog of SLC17 organic anion transporters
SURFE2R N1 Preprint Publication in bioRxiv (2024) Authors: Dmitrieva N., Gholami S., Alleva C., Carloni P., Alfonso-Prieto M., Fahlke C.

The solute carrier 17 (SLC17) family contains anion transporters that accumulate neurotransmitters in secretory vesicles, remove carboxylated monosaccharides from lysosomes, or extrude organic anions from the kidneys and the liver. We combined experimental and computational approaches to describe the transport mechanisms of a model bacterial protein, the D-galactonate transporter DgoT, at atomic resolution. We found that protonation of D46 and E133 precedes galactonate binding and that substrate binding induces closure of the extracellular gate, with the conserved R47 coupling substrate binding to transmembrane helix movement. After isomerization to an inward-facing conformation, deprotonation of E133 and subsequent proton trans-fer from D46 to E133 opens the intracellular gate and permits galactonate dissociation. After release of the second proton, apo DgoT returns to the outward-facing conformation. Our results provide a framework to understand how various SLC17 transport functions with distinct transport stoichiometries can be attained through subtle variations in proton and substrate binding/unbinding.

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2024 – Methoxychalcones as potential anticancer agents for colon cancer: Is membrane perturbing potency relevant?
Vesicle Prep Pro Publication in Biochimica et Biophysica Acta (BBA) - General Subjects (2024) Authors: Palko-Łabuz A., Wesołowska O., Błaszczyk M., Uryga A., Sobieszczańska B., Skonieczna M., Kostrzewa-Susłow E., Janeczko T., Środa-Pomianek K.

Chalcones are naturally produced by many plants, and constitute precursors for the synthesis of flavons and flavanons. They were shown to possess antibacterial, antifungal, anti-cancer, and anti- inflammatory properties. The goal of the study was to assess the suitability of three synthetic methoxychalcones as potential anticancer agents. In a panel of colon cancer cell lines they were demonstrated to be cytotoxic, proapoptotic, causing cell cycle arrest, and increasing intracellular level of reactive oxygen species. Anticancer activity of the compounds was not diminished in the presence of stool extract containing microbial enzymes that could change the structure of chalcones. Moreover, methoxychalcones interacted strongly with model phosphatidylcholine membranes as detected by differential scanning calorimetry. Metohoxychalcones particularly affected the properties of lipid domains in giant unilamellar liposomes formed from raft-mimicking lipid composition. This may be of importance since many molecular targets for therapy of metastatic colon cancer are raft-associated receptors (e.g., receptor tyrosine kinases). The importance of membrane perturbing potency of methoxychalcones for their biological activity was additionally corroborated by the results obtained by molecular modelling.

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2024 – Novel Cocrystal Structures of Peptide Antagonists Bound to the Human Melanocortin Receptor 4 Unveil Unexplored Grounds for Structure-Based Drug Design
SyncroPatch 384 PE (a predecessor model of the SyncroPatch 384) Publication in J. Med. Chem. (2024) Authors: Gimenez L.E., Martin C., Yu J., Hollanders C., Hernandez C.C., Wu Y., Yao D., Han G.W., Dahir N.S., Wu L., Van der Poorten O., Lamouroux A., Mannes M., Zhao S., Tourwé D., Stevens R.C., Cone R.D., Ballet S.

Melanocortin 4 receptor (MC4-R) antagonists are actively sought for treating cancer cachexia. We determined the structures of complexes with PG-934 and SBL-MC-31. These peptides differ from SHU9119 by substituting His6 with Pro6 and inserting Gly10 or Arg10. The structures revealed two subpockets at the TM7-TM1-TM2 domains, separated by N2857.36. Two peptide series based on the complexed peptides led to an antagonist activity and selectivity SAR study. Most ligands retained the SHU9119 potency, but several SBL-MC-31-derived peptides significantly enhanced MC4-R selectivity over MC1-R by 60- to 132-fold. We also investigated MC4-R coupling to the K+ channel, Kir7.1. Some peptides activated the channel, whereas others induced channel closure independently of G protein coupling. In cell culture studies, channel activation correlated with increased feeding, while a peptide with Kir7.1 inhibitory activity reduced eating. These results highlight the potential for targeting the MC4-R:Kir7.1 complex for treating positive and restrictive eating disorders.

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2024 – Prognostic Value of Multiplexed Assays of Variant Effect and Automated Patch-clamping for KCNH2-LQTS Risk Stratification
SyncroPatch 384 PE (a predecessor model of the SyncroPatch 384) Pre-Print Publication in medRxiv (2024) Authors: Liu J., Huang J., Wang K., Li Y., Li C., Zhu Y., He X., Zhang Y., Zhao Y., Hu C., Xi Z., Tong M., Li M., Gong P., Hou Y.

Background: Long QT syndrome (LQTS) is a lethal arrhythmia condition, frequently caused by rare loss-of-function variants in the cardiac potassium channel encoded by KCNH2. Variant-based risk stratification is complicated by heterogenous clinical data, incomplete penetrance, and low-throughput functional data. Objective: To test the utility of variant-specific features, including high-throughput functional data, to predict cardiac events among KCNH2 variant heterozygotes. Methods: We quantified cell-surface trafficking of 18,796 missense variants in KCNH2 and recorded potassium current densities for 506 KCNH2 variants. Next, we deeply phenotyped 1150 KCNH2 missense variant patients, including ECG features, cardiac event history (528 total cardiac events), and mortality. We then assessed variant functional, in silico, structural, and LQTS penetrance data to stratify event-free survival for cardiac events in the study cohort. Results: Traditional risk factors of QT interval adjusted for heart rate (Hazard Ratio 1.09 [1.07-1.12]) and sex (HR 0.60 [0.47-0.76]) were most significant for predicting events; however, variant-specific current density (HR 0.44 [0.26-0.70]) and estimates of LQTS penetrance (HR 1.93; [1.13-3.39]) were independently predictive of severe cardiac events when controlling for patient-specific features. Conclusion: We show that high-throughput functional data, and other variant-specific features, meaningfully contribute to both diagnosis and prognosis of a clinically actionable monogenic disease.

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2024 – Cellular-level analyses of SCN5A mutations in left ventricular noncompaction cardiomyopathy suggest electrophysiological mechanisms for ventricular tachycardia
CardioExcyte 96 Publication in Biochemistry and Biophysics Reports (2024) Authors: Li Y., Liu S., Huang J., Xie Y., Hou A., Wei Y.

Left ventricular noncompaction cardiomyopathy (LVNC) is a cardiovascular disease characterized by arrhythmia and heart failure. In this study, LVNC myocardial samples were collected from patients who underwent heart transplantation and were analyzed using exome sequencing. Approximately half of the LVNC patients carried SCN5A variants, which are associated with clinical symptoms of ventricular tachycardia. To investigate the electrophysiological functions of these SCN5A variants and the underlying mechanism by which they increase arrhythmia susceptibility in LVNC patients, functional evaluations were conducted in CHO–K1 cells and human embryonic stem cell-derived cardiomyocytes (hESC-CMs) using patch-clamp or microelectrode array (MEA) techniques. These findings demonstrated that these SCN5A mutants exhibited gain-of-function properties, leading to increased channel activation and enhanced fast inactivation in CHO–K1 cells. Additionally, these mutants enhanced the excitability and contractility of the cardiomyocyte population in hESC-CMs models. All SCN5A variants induced fibrillation-like arrhythmia and increased the heart rate in cardiomyocytes. However, the administration of Lidocaine, an antiarrhythmic drug that acts on sodium ion channels, was able to rescue or alleviate fibrillation-like arrhythmias and secondary beat phenomenon. Based on these findings, it is speculated that SCN5A variants may contribute to susceptibility to arrhythmia in LVNC patients. Furthermore, the construction of cardiomyocyte models with SCN5A variants and their application in drug screening may facilitate the development of precise therapies for arrhythmia in the future.

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2024 – A dynamically gated triangular DNA nanopore for molecular sensing and cross-membrane transport
Vesicle Prep Pro and Orbit mini Preprint Publication in Research Square (2024) Authors: Liu X., Liu F., Chhabra H., Maffeo C., Huang Q., Aksimentiev A., Arai T.

Synthetic membrane nanopores made of DNA are promising systems to sense and control molecular transport in biosensing, sequencing, and synthetic cells. Dynamically gating cargo transport like the natural ion channels and systematically increasing the lumen size have become long-standing desires in developing nanopores. Here, we design a triangular DNA nanopore with a large dynamically-gated lumen. It can switch between expanded and contracted states without changing its stable triangular shape, whereby specific DNA bindings as stimuli mechanically pinch and release the three corners of the triangular frame. Transmission electron microscopy images and molecular dynamics simulations illustrated the large lumen up to 539 nm2, the stable architectures, and the high shape retention. Single channel current recordings and fluorescence influx studies demonstrated the low-noise repeatable readouts and the controllable cross-membrane macromolecular transport. We envision that the proposed DNA nanopores could offer powerful tools in molecular sensing, drug delivery, and the creation of synthetic cells.

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2024 – 5,6-diHETE lactone (EPA-L) mediates hypertensive microvascular dilation by activating the endothelial GPR-PLC-IP3 signaling pathway
Port-a-Patch Publication in Biochemical and Biophysical Research Communications (2024) Authors: Asulin M., Gorodetzer N., Fridman R., Ben-Shushan R., Cohen Z., Beyer A., Chuyun D., Gutterman D., Szuchman-Sapir A.

Endothelial microvascular dysfunction affects multi-organ pathologic processes that contribute to increased vascular tone and is at the base of impaired metabolic and cardiovascular diseases. The vascular dilation impaired by nitric oxide (NO) deficiency in such dysfunctional endothelium is often balanced by endothelial-derived hyperpolarizing factors (EDHFs), which play a critical role in managing vascular tone. Our latest research has uncovered a new group of lactone oxylipins produced in the polyunsaturated fatty acids (PUFAs) CYP450 epoxygenase pathway, significantly affecting vascular dilation. The lactone oxylipin, derived from arachidonic acid (5,6-diHET lactone, AA-L), has been previously shown to facilitate vasodilation dependent on the endothelium in isolated human microvessels. The administration of the lactone oxylipin derived from eicosapentaenoic acid (5,6-diHETE lactone, EPA-L) to hypertensive rats demonstrated a significant decrease in blood pressure and improvement in the relaxation of microvessels. However, the molecular signaling processes that underlie these observations were not fully understood. The current study delineates the molecular pathways through which EPA-L promotes endothelium-dependent vascular dilation. In microvessels from hypertensive individuals, it was found that EPA-L mediates endothelium-dependent vasodilation while the signaling pathway was not dependent on NO. In vitro studies on human endothelial cells showed that the hyperpolarization mediated by EPA-L relies on G-protein-coupled receptor (GPR)-phospholipase C (PLC)-IP3 signaling that further activates calcium-dependent potassium flux. The pathway was confirmed using a range of inhibitors and cells overexpressing GPR40, where a specific antagonist reduced the calcium levels and outward currents induced by EPA-L. The downstream AKT and endothelial NO synthase (eNOS) phosphorylations were non-significant. These findings show that the GPR-PLC-IP3 pathway is a key mediator in the EPA-L-triggered vasodilation of arterioles. Therefore, EPA-L is identified as a significant lactone-based PUFA metabolite that contributes to endothelial and vascular health.

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2024 – Solid-phase synthesis as a tool to create exactly defined, branched polymer vectors for cell membrane targeting
Vesicle Prep Pro Publication in Macromolecules (2024) Authors: Elter J., Liščáková V., Moravec O., Vragović M., Filipová M., Štěpánek P., Šácha P., Hrubý M.

Modern drug formulations often require, besides the active drug molecule, auxiliaries to enhance their pharmacological properties. Tailor-made, biocompatible polymers covalently connected to the drug molecule can fulfill this function by increasing its solubility, reducing its toxicity, and guiding it to a specific target. If targeting membrane-bound proteins, localization of the drug close to the cell membrane and its target is beneficial to increase drug efficiency and residence time. In this study, we present the synthesis of highly defined, branched polymeric structures with membrane-binding properties. One to three hydrophilic poly(ethylene oxide) or poly(2-ethyloxazoline) side chains were connected via a peptoid backbone using a two-step iterative protocol for solid-phase peptoid synthesis. Additional groups, e.g., a hydrophobic anchor for membrane attachment, were introduced. Due to the nature of solid-phase synthesis, the number and order of the side chains and additional units can be precisely defined. The method proved to be versatile for the generation of multifunctional, branched polymeric structures of molecular weights up to approximately 7000 g mol–1. The behavior of all compounds towards biological membranes and cells was investigated using liposomes as cell membrane models, HEK293 and U251-MG cell lines, and red blood cells, thereby demonstrating their potential value as drug auxiliaries with cell membrane affinity.

Application Note PDF
Cardiomyocytes – Monitoring the effect of Triiodothyronine (T3) and Dexmethasone (Dex) on the calcium handling property of hiPSC-Cardiomyocytes
CardioExcyte 96 Application Note - Cells kindly provided by Bayer AG, and analysis software kindly provided by Hamamatsu

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) provide new avenues for disease modelling, drug discovery and cell therapy. However, structural and functional immaturity of these cells, poses a great challenge. Therefore, there is a highly unmet need for hiPSC-CMs with a mature phenotype. Recently, it has been reported that triiodothyronine (T3) and dexamethasone (Dex) play significant roles in hiPSCCMs maturation, by promoting the structural T-tubule development, enhancing the electrophysiological maturation and improving calcium handling ability.

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2024 – Dissecting the membrane association mechanism of aerolysin pore at femtomolar concentrations using water as a probe
Orbit mini Pre-print in bioRxiv (2024) Authors: Roesel T., Cao C., Bada J., Dal Peraro M., Roke S.

Aerolysin is a bacterial pore-forming toxin able to form transmembrane pores at the host plasma membrane of narrow internal diameter and great stability. These assets make it a highly promising nanopore for the detection of biopolymers such as nucleic acids and
peptides. While much is known about aerolysin from a microbiological and structural side, its membrane association and pore-formation mechanism are not yet fully disclosed. Here, we studied the interaction of femtomolar concentrations of aerolysin and its mutants with
liposomes in aqueous solution using angle-resolved second harmonic scattering (AR-SHS), in combination with single-channel current measurements. The measurements were so sensitive to detect electrostatic changes on the membrane-bound aerolysin induced by pH
variation induced by the changes in the hydration shell of aerolysin. We reported for the first time the membrane binding affinity of aerolysin at different stages of the pore formation mechanism: while wt aerolysin has a binding affinity as high as 20 fM, the quasi-pore state
and the prepore state show gradually decreasing membrane affinities, incomplete insertion and pore opening signature. Moreover, we quantitatively characterized the membrane affinity of mutants relevant for applications to nanopore sensing. This approach opens new
possibilities to efficiently screen biological pores suitable for conducting molecular sensing and sequencing measurements, as well as to probe pore forming processes.

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2024 – Recording ten-fold larger IKr conductances with automated patch clamping using equimolar Cs+ solutions
Patchliner and SyncroPatch 384 Publication in Frontiers in Physiology (2024) Authors: Bloothooft M., Verbruggen B., Seibertz F., van der Heyden M.A.G., Voigt N., de Boer T.P.

Background: The rapid delayed rectifier potassium current (IKr) is important for cardiac repolarization and is most often involved in drug-induced arrhythmias. However, accurately measuring this current can be challenging in human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes because of its small current density. Interestingly, the ion channel conducting IKr, hERG channel, is not only permeable to K+ ions but also to Cs+ ions when present in equimolar concentrations inside and outside of the cell.

Methods: In this study, IhERG was measured from Chinese hamster ovary (CHO)-hERG cells and hiPSC-CM using either Cs+ or K+ as the charge carrier. Equimolar Cs+ has been used in the literature in manual patch-clamp experiments, and here, we apply this approach using automated patch-clamp systems. Four different (pre)clinical drugs were tested to compare their effects on Cs+- and K+-based currents.

Results: Using equimolar Cs+ solutions gave rise to approximately ten-fold larger hERG conductances. Comparison of Cs+- and K+-mediated currents upon application of dofetilide, desipramine, moxifloxacin, or LUF7244 revealed many similarities in inhibition or activation properties of the drugs studied. Using equimolar Cs+ solutions gave rise to approximately ten-fold larger hERG conductances. In hiPSC-CM, the Cs+-based conductance is larger compared to the known K+-based conductance, and the Cs+ hERG conductance can be inhibited similarly to the K+-based conductance.

Conclusion: Using equimolar Cs+ instead of K+ for IhERG measurements in an automated patch-clamp system gives rise to a new method by which, for example, quick scans can be performed on effects of drugs on hERG currents. This application is specifically relevant when such experiments are performed using cells which express small IKr current densities in combination with small membrane capacitances.

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2024 – The Inhibition of the Membrane-Bound Transcription Factor Site-1 Protease (MBTP1) Alleviates the p.Phe508del-Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Defects in Cystic Fibrosis Cells
Port-a-Patch Publication in Cells (2024) Authors: Santinelli R., Benz N., Guellec J., Quinquis F., Kocas E., Thomas J., Montier T., Ka C., Luczka-Majérus E., Sage E., Férec C., Coraux C., Trouvé P.

Cystic Fibrosis (CF) is present due to mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, the most frequent variant being p.phe508del. The CFTR protein is a chloride (Cl-) channel which is defective and almost absent of cell membranes when the p.Phe508del mutation is present. The p.Phe508del-CFTR protein is retained in the endoplasmic reticulum (ER) and together with inflammation and infection triggers the Unfolded Protein Response (UPR). During the UPR, the Activating Transcription Factor 6 (ATF6) is activated with cleavage and then decreases the expression of p.Phe508del-CFTR. We have previously shown that the inhibition of the activation of ATF6 alleviates the p.Phe508del-CFTR defects in cells overexpressing the mutated protein. In the present paper, our aim was to inhibit the cleavage of ATF6, and thus its activation in a human bronchial cell line with endogenous p.Phe508del-CFTR expression and in bronchial cells from patients, to be more relevant to CF. This was achieved by inhibiting the protease MBTP1 which is responsible for the cleavage of ATF6. We show here that this inhibition leads to increased mRNA and p.Phe508del-CFTR expression and, consequently, to increased Cl-efflux. We also explain the mechanisms linked to these increases with the modulation of genes when MBTP1 is inhibited. Indeed, RT-qPCR assays show that genes such as HSPA1B, CEBPB, VIMP, PFND2, MAPK8, XBP1, INSIG1, and CALR are modulated. In conclusion, we show that the inhibition of MBTP1 has a beneficial effect in relevant models to CF and that this is due to the modulation of genes involved in the disease.

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2024 – Dynamic conformational changes of a tardigrade group-3 late embryogenesis abundant protein modulate membrane biophysical properties
Vesicle Prep Pro Publication in PNAS Nexus (2024) Authors: Li X., Yu C., Gomez-Navarro N., Stancheva V., Zhu H., Murthy A., Wozny M., Malhotra K., Johnson C., Blackledge M., Santhanam B., Liu W., Huang J., Freund S., Miller E., Babu M.

A number of Intrinsically disordered proteins (IDPs) encoded in stress-tolerant organisms such as tardigrade, can confer fitness advantage and abiotic stress tolerance when heterologously expressed. Tardigrade-specific disordered proteins including the cytosolic abundant heat soluble (CAHS) proteins are proposed to confer stress tolerance through vitrification or gelation, whereas evolutionarily conserved IDPs in tardigrades may contribute to stress tolerance through other biophysical mechanisms. Here we characterized the mechanism of action of an evolutionarily conserved, tardigrade IDP, HeLEA1, which belongs to the group-3 late embryogenesis abundant (LEA) protein family. HeLEA1 homologs are found across different kingdoms of life. HeLEA1 is intrinsically disordered in solution but shows a propensity for helical structure across its entire sequence. HeLEA1 interacts with negatively charged membranes via dynamic disorder-to-helical transition, mainly driven by electrostatic interactions. Membrane interaction of HeLEA1 is shown to ameliorate excess surface tension and lipid packing defects. HeLEA1 localizes to the mitochondrial matrix when expressed in yeast and interacts with model membranes mimicking inner mitochondrial membrane. Yeast expressing HeLEA1 show enhanced tolerance to hyperosmotic stress under non-fermentative growth and increased mitochondrial membrane potential. Evolutionary analysis suggests that although HeLEA1 homologs have diverged their sequences to localize to different subcellular organelles, all homologs maintain a weak hydrophobic moment that is characteristic of weak and reversible membrane interaction. We suggest that such a dynamic and weak protein-membrane interaction buffering alterations in lipid packing could be a conserved strategy for regulating membrane properties and represents a general biophysical solution for stress tolerance across the domains of life.

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2024 – Engineered cocultures of iPSC-derived atrial cardiomyocytes and atrial fibroblasts for modeling atrial fibrillation
SyncroPatch 384 Publication in Sci. Adv. (2024) Authors: Brown G.E., Han Y.D., Michell A.R., Ly O.T., Vanoye C.G., Spanghero E., George Jr. A.L., Darbar D., Khetani S.R.

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia treatable with antiarrhythmic drugs; however, patient responses remain highly variable. Human induced pluripotent stem cell–derived atrial cardiomyocytes (iPSC-aCMs) are useful for discovering precision therapeutics, but current platforms yield phenotypically immature cells and are not easily scalable for high-throughput screening. Here, primary adult atrial, but not ventricular, fibroblasts induced greater functional iPSC-aCM maturation, partly through connexin-40 and ephrin-B1 signaling. We developed a protein patterning process within multiwell plates to engineer patterned iPSC-aCM and atrial fibroblast coculture (PC) that significantly enhanced iPSC-aCM structural, electrical, contractile, and metabolic maturation for 6+ weeks compared to conventional mono-/coculture. PC displayed greater sensitivity for detecting drug efficacy than monoculture and enabled the modeling and pharmacological or gene editing treatment of an AF-like electrophysiological phenotype due to a mutated sodium channel. Overall, PC is useful for elucidating cell signaling in the atria, drug screening, and modeling AF.

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2024 – A Cost-Effective Pichia pastoris Cell-Free System Driven by Glycolytic Intermediates Enables the Production of Complex Eukaryotic Proteins
Orbit 16 (a predecessor model of the Orbit 16 TC) Publication in Bioengineering (2024) Authors: Schloßhauer J., Dondapati S., Kubick S., Zemella A.

Cell-free systems are particularly attractive for screening applications and the production of difficult-to-express proteins. However, the production of cell lysates is difficult to implement on a larger scale due to large time requirements, cultivation costs, and the supplementation of cell-free reactions with energy regeneration systems. Consequently, the methylotrophic yeast Pichia pastoris, which is widely used in recombinant protein production, was utilized in the present study to realize cell-free synthesis in a cost-effective manner. Sensitive disruption conditions were evaluated, and appropriate signal sequences for translocation into ER vesicles were identified. An alternative energy regeneration system based on fructose-1,6-bisphosphate was developed and a ~2-fold increase in protein production was observed. Using a statistical experiment design, the optimal composition of the cell-free reaction milieu was determined. Moreover, functional ion channels could be produced, and a G-protein-coupled receptor was site-specifically modified using the novel cell-free system. Finally, the established P. pastoris cell-free protein production system can economically produce complex proteins for biotechnological applications in a short time.

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2024 – Discovery and optimization of dihydropteridone derivatives as novel PLK1 and BRD4 dual inhibitor for the treatment of cancer
SyncroPatch 384 Publication in Bioorganic & Medicinal Chemistry (2024) Authors: Liu J., Huang J., Wang K., Li Y., Li C., Zhu Y., He X., Zhang Y., Zhao Y., Hu C., Xi Z., Tong M., Li Z., Gong P., Hou Y.

In this study, we have designed, synthesized and tested three series of novel dihydropteridone derivatives possessing isoindolin-1-one or isoindoline moieties as potent inhibitors of PLK1/BRD4. Remarkably, most of the compounds showed preferable inhibitory activity against PLK1 and BRD4. Compound SC10 exhibited excellent inhibitory activity with IC50 values of 0.3 nM and 60.8 nM against PLK1 and BRD4, respectively. Meanwhile, it demonstrated significant anti-proliferative activities against three tumor-derived cell lines (MDA-MB-231 IC50 = 17.3 nM, MDA-MB-361 IC50 = 8.4 nM, and MV4-11 IC50 = 5.4 nM). Moreover, SC10 exhibited moderate rat liver microsomal stability (CLint = 21.3 µL·min-1·mg-1), acceptable pharmacokinetic profile (AUC0-t = 657 ng·h·mL-1, oral bioavailability of 21.4%) in Sprague-Dawley rats, reduced hERG toxicity, acceptable PPB and CYP450 inhibition. Further research indicated that SC10 could induce MV4-11 cell arrest at the S phase and apoptosis in a dose-dependent manner. This investigation provided us with an initial point for developing novel anticancer agents as dual inhibitors of PLK1 and BRD4.

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2024 – Unraveling the kinetics and pharmacology of human PepT1 using solid supported membrane-based electrophysiology
SURFE2R N1 and SURFE2R 96SE Publication in Bioelectrochemistry (2024) Authors: Körner A., Bazzone A., Wichert M., Barthmes M., Dondapati S.K., Fertig N., Kubick S.

The human Peptide Transporter 1 (hPepT1) is known for its broad substrate specificity and its ability to transport (pro-)drugs. Here, we present an in-depth comprehensive study of hPepT1 and its interactions with various substrates via solid supported membrane-based electrophysiology (SSME). Using hPepT1-containing vesicles, we could not identify any peptide induced pre-steady-state currents, indicating that the recorded peak currents reflect steady-state transport. Electrogenic co-transport of H+/glycylglycine (GlyGly) was observed across a pH range of 5.0 to 9.0. The pH dependence is described by a bell-shaped activity curve and two pK values. KM and relative Vmax values of various canonical and non-canonical peptide substrates were contextualized with current mechanistic understandings of hPepT1. Finally, specific inhibition was observed for various inhibitors in a high throughput format, and IC50 values are reported. Taken together, these findings contribute to promoting the design and analysis of pharmacologically relevant substances.

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2024 – Cryo-electron tomography reveals how COPII assembles on cargo-containing membranes
Vesicle Prep Pro Pre-Print Publication in BioRxiv (2024) Authors: Pyle E., Zanetti G.

Proteins traverse the eukaryotic secretory pathway via membrane trafficking between organelles. The COPII coat mediates the anterograde transport of newly synthesised proteins from the endoplasmic reticulum, engaging cargoes with wide ranges of sizes and biophysical properties. The native architecture of the COPII coat and the cargo-dependent regulation of its assembly remain poorly understood. Here, we have reconstituted COPII-coated membrane carriers using purified S. cerevisiae proteins and cell-derived microsomes as a native membrane source. Using cryo-electron tomography with subtomogram averaging, we demonstrate that the COPII coat binds cargo and forms largely spherical vesicles from native membranes. We reveal the architecture of the inner and outer coat layers and shed light on how spherical carriers are formed. Our results provide novel insights into the architecture and regulation of the COPII coat and challenge our current understanding of how membrane curvature is generated.

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2024 – Propofol directly binds and inhibits skeletal muscle ryanodine receptor 1 (RyR1)
Orbit mini Pre-Print in bioRxiv. (2024) Authors: Joseph T., Bu W., Haji-Ghassemi O., Chen Y., Woll K., Allen P., Brannigan G., van Petegem F., Eckenhoff R.

As the primary Ca2+ release channel in skeletal muscle sarcoplasmic reticulum (SR), mutations in the type 1 ryanodine receptor (RyR1) or its binding partners underlie a constellation of muscle disorders, including malignant hyperthermia (MH). In patients with MH mutations, exposure to triggering drugs such as the
halogenated volatile anesthetics biases RyR1 to an open state, resulting in uncontrolled Ca2+ release, sarcomere tension and heat production. Restoration of Ca2+ into the SR also consumes ATP, generating a further untenable metabolic load. When anesthetizing patients with known MH mutations, the non-triggering intravenous general anesthetic propofol is commonly substituted for triggering anesthetics. Evidence of direct binding of anesthetic agents to RyR1 or its binding partners is scant, and the atomic-level interactions of propofol with RyR1 are entirely unknown. Here, we show that propofol decreases RyR1 opening in heavy SR vesicles and planar lipid bilayers, and that it inhibits activator-induced Ca2+ release from SR in human skeletal muscle. In addition to confirming direct binding, photoaffinity labeling using m-azipropofol (AziPm) revealed several putative propofol binding sites on RyR1. Prediction of binding affinity by molecular dynamics simulation suggests that propofol binds at least one of these sites at clinical concentrations. These findings invite the hypothesis that in addition to propofol not triggering MH, it may also be protective against MH by inhibiting induced Ca2+ flux through RyR1.

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2024 – No Influence of Asundexian on Cardiac Repolarization
Patchliner Publication in Clinical Pharmacology in Drug Development (2024) Authors: Brase C., Kanefendt F., Loewen S., Himmel H., Schmitz S.

Inhibition of activated factor XI reduces thrombogenesis while maintaining physiological hemostasis, with the expectation of reduced bleeding risk compared with standard of care in the clinical setting. Asundexian (BAY 2433334), an activated factor XI inhibitor, is in clinical development for the prevention of thromboembolic events. The effect of asundexian and its plasma metabolite M10 on cardiac repolarization and potential interactions with the hNav1.5 sodium, hCav1.2 calcium, and human ether-à-go-go-related gene (hERG) potassium channels was investigated in vitro. Additionally, asundexian effects on cardiac parameters and electrocardiogram were examined in telemetered beagle dogs. A randomized, placebo-controlled, 4-way crossover, thorough QT study in healthy adults evaluated the influence of 50 and 150 mg of asundexian on the corrected QT interval, including 400 mg of moxifloxacin as positive control. Across all studies, asundexian and M10 were not associated with any effects on cardiac repolarization. The largest in vitro effects of asundexian (approximately 20% inhibition) were seen for hCav1.2 and hERG. Throughout the thorough QT study, the upper limits of the one-sided 95% confidence interval of placebo-corrected mean changes from baseline in Fridericia corrected QT for 50 and 150 mg of asundexian were below Δ = 10 milliseconds. Asundexian demonstrated favorable safety and tolerability profiles.

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2024 – Functional and structural asymmetry suggest a unifying principle for catalysis in membrane-bound pyrophosphatases
SURFE2R N1 Publication in EMBO Rep. (2024) Authors: Strauss J., Wilkinson C., Vidilaseris K., de Castro Ribeiro O.M., Liu J., Hillier J., Wichert M., Malinen A.M., Gehl B., Jeuken L.J.C., Pearson A.R., Goldman A.

Membrane-bound pyrophosphatases (M-PPases) are homodimeric primary ion pumps that couple the transport of Na+- and/or H+ across membranes to the hydrolysis of pyrophosphate. Their role in the virulence of protist pathogens like Plasmodium falciparum makes them an intriguing target for structural and functional studies. Here, we show the first structure of a K+-independent M-PPase, asymmetric and time-dependent substrate binding in time-resolved structures of a K+-dependent M-PPase and demonstrate pumping-before-hydrolysis by electrometric studies. We suggest how key residues in helix 12, 13, and the exit channel loops affect ion selectivity and K+-activation due to a complex interplay of residues that are involved in subunit-subunit communication. Our findings not only explain ion selectivity in M-PPases but also why they display half-of-the-sites reactivity. Based on this, we propose, for the first time, a unified model for ion-pumping, hydrolysis, and energy coupling in all M-PPases, including those that pump both Na+ and H+.

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2023 – Structure and electromechanical coupling of a voltage-gated Na+/H+ exchanger
SURFE2R N1 Publication in Nature (2023) Authors: Yeo H., Mehta V., Gulati A., Drew D.

Voltage-sensing domains control the activation of voltage-gated ion channels, with a few exceptions. One such exception is the sperm-specific Na+/H+ exchanger SLC9C1, which is the only known transporter to be regulated by voltage-sensing domains. After hyperpolarization of sperm flagella, SLC9C1 becomes active, causing pH alkalinization and CatSper Ca2+ channel activation, which drives chemotaxis. SLC9C1 activation is further regulated by cAMP, which is produced by soluble adenyl cyclase (sAC). SLC9C1 is therefore an essential component of the pH–sAC–cAMP signalling pathway in metazoa, required for sperm motility and fertilization. Despite its importance, the molecular basis of SLC9C1 voltage activation is unclear. Here we report cryo-electron microscopy (cryo-EM) structures of sea urchin SLC9C1 in detergent and nanodiscs. We show that the voltage-sensing domains are positioned in an unusual configuration, sandwiching each side of the SLC9C1 homodimer. The S4 segment is very long, 90 Å in length, and connects the voltage-sensing domains to the cytoplasmic cyclic-nucleotide-binding domains. The S4 segment is in the up configuration—the inactive state of SLC9C1. Consistently, although a negatively charged cavity is accessible for Na+ to bind to the ion-transporting domains of SLC9C1, an intracellular helix connected to S4 restricts their movement. On the basis of the differences in the cryo-EM structure of SLC9C1 in the presence of cAMP, we propose that, upon hyperpolarization, the S4 segment moves down, removing this constriction and enabling Na+/H+ exchange.

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2024 – Plural molecular and cellular mechanisms of pore domain KCNQ2 encephalopathy
SyncroPatch 768 PE (a predecessor model of the SyncroPatch 384) Pre-Print Publication in BioRxiv (2024) Authors: Abreo T., Thompson E., Madabushi A., Soh H., Varghese N., Vanoye C., Springer K., Park K., Johnson J., Sims S., Ji Z., Chavez A., Jankovic M., Habte B., Zuberi A., Lutz C., Wang Z., Krishnan V., Dudler L., Einsele-Scholz S., Noebels J., George A., Maheshwari A., Tzingounis A, Cooper E.

KCNQ2 variants in children with neurodevelopmental impairment are difficult to assess due their heterogeneity and unclear pathogenic mechanisms. We describe a child with neonatal-onset epilepsy, developmental impairment of intermediate severity, and KCNQ2 G256W heterozygosity. Analyzing prior KCNQ2 channel cryoelectron microscopy models revealed G256 as keystone of an arch-shaped non-covalent bond network linking S5, the pore turret, and the ion path. Co-expression with G256W dominantly suppressed conduction by wild-type subunits in heterologous cells. Ezogabine partly reversed this suppression. G256W/+ mice have epilepsy leading to premature deaths. Hippocampal CA1 pyramidal cells from G256W/+ brain slices showed hyperexcitability. G256W/+ pyramidal cell KCNQ2 and KCNQ3 immunolabeling was significantly shifted from axon initial segments to neuronal somata. Despite normal mRNA levels, G256W/+ mouse KCNQ2 protein levels were reduced by about 50%. Our findings indicate that G256W pathogenicity results from multiplicative effects, including reductions in intrinsic conduction, subcellular targeting, and protein stability. These studies reveal pore “turret arch” bonding as a KCNQ structural novelty and introduce a valid animal model of KCNQ2 encephalopathy. Our results, spanning structure to behavior, may be broadly applicable because the majority of KCNQ2 encephalopathy patients share variants near the selectivity filter.

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2024 – Pharmacologic Characterization of LTGO-33, a Selective Small Molecule Inhibitor of the Voltage-Gated Sodium Channel NaV1.8 with a Unique Mechanism of Action
SyncroPatch 768 PE (a predecessor model of the SyncroPatch 384) Publication in Mol. Pharmacol. (2024) Authors: Gilchrist J.M., Yang N-D., Jiang V., Moyer B.D.

Discovery and development of new molecules directed against validated pain targets is required to advance the treatment of pain disorders. Voltage-gated sodium channels (NaVs) are responsible for action potential initiation and transmission of pain signals. NaV1.8 is specifically expressed in peripheral nociceptors and has been genetically and pharmacologically validated as a human pain target. Selective inhibition of NaV1.8 can ameliorate pain while minimizing effects on other NaV isoforms essential for cardiac, respiratory, and central nervous system physiology. Here we present the pharmacology, interaction site, and mechanism of action of LTGO-33, a novel NaV1.8 small molecule inhibitor. LTGO-33 inhibited NaV1.8 in the nM potency range and exhibited over 600-fold selectivity against human NaV1.1-NaV1.7 and NaV1.9. Unlike prior reported NaV1.8 inhibitors that preferentially interacted with an inactivated state via the pore region, LTGO-33 was state-independent with similar potencies against closed and inactivated channels. LTGO-33 displayed species specificity for primate NaV1.8 over dog and rodent NaV1.8 and inhibited action potential firing in human dorsal root ganglia neurons. Using chimeras combined with mutagenesis, the extracellular cleft of the second voltage-sensing domain was identified as the key site required for channel inhibition. Biophysical mechanism of action studies demonstrated that LTGO-33 inhibition was relieved by membrane depolarization, suggesting the molecule stabilized the deactivated state to prevent channel opening. LTGO-33 equally inhibited wild-type and multiple NaV1.8 variants associated with human pain disorders. These collective results illustrate LTGO-33 inhibition via both a novel interaction site and mechanism of action previously undescribed in NaV1.8 small molecule pharmacologic space.

Significance Statement NaV1.8 sodium channels primarily expressed in peripheral pain-sensing neurons represent a validated target for the development of novel analgesics. Here we present the selective small molecule NaV1.8 inhibitor LTGO-33 that interdicts a distinct site in a voltage-sensor domain to inhibit channel opening. These results inform the development of new analgesics for pain disorders.

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2023 – Electric Potential at the Interface of Membraneless Organelles Gauged by Graphene
Vesicle Prep Pro Publication in Nano Lett. (2023) Authors: Hoffmann C., Murastov G., Tromm J., Moog J., Aslam M., Matkovic A., Milovanovic D.

Eukaryotic cells contain membrane-bound and membrane-less organelles that are often in contact with each other. How the interface properties of membrane-less organelles regulate their interactions with membranes remains challenging to assess. Here, we employ graphene-based sensors to investigate the electrostatic properties of synapsin 1, a major synaptic phosphoprotein, either in a single phase or after undergoing phase separation to form synapsin condensates. Using these graphene-based sensors, we discover that synapsin condensates generate strong electrical responses that are otherwise absent when synapsin is present as a single phase. By introducing atomically thin dielectric barriers, we show that the electrical response originates in an electric double layer whose formation governs the interaction between synapsin condensates and graphene. Our data indicate that the interface properties of the same protein are substantially different when the protein is in a single phase versus within a biomolecular condensate, unraveling that condensates can harbor ion potential differences at their interface.

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2023 – Nanopore Signatures of Nucleoside Drugs
Orbit mini Publication in Nano Lett. (2023) Authors: Jia W., Ouyang Y., Zhang S., Du X., Zhang P., Huang S.

Nucleoside drugs, which are analogues of natural nucleosides, have been widely applied in the clinical treatment of viral infections and cancers. The development of nucleoside drugs, repurposing of existing drugs, and combined use of multiple drug types have made the rapid sensing of nucleoside drugs urgently needed. Nanopores are emerging single-molecule sensors that have high resolution to resolve even minor structural differences between chemical compounds. Here, an engineered Mycobacterium smegmatis porin A hetero-octamer was used to perform general nucleoside drug analysis. Ten nucleoside drugs were simultaneously detected and fully discriminated. An accuracy of >99.9% was consequently reported. This sensing capacity was further demonstrated in direct nanopore analysis of ribavirin buccal tablets, confirming its sensing reliability against complex samples and environments. No sample separation is needed, however, significantly minimizing the complexity of the measurement. This technique may inspire nanopore applications in pharmaceutical production and pharmacokinetics measurements.

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2023 – A high-throughput electrophysiology assay to study the response of PIEZO1 to mechanical stimulation
SyncroPatch 384 Publication in J Gen Physiol (2023) Authors: Murciano N., Rotordam M., Becker N., Ludlow M. , Parsonage G. , Darras A., Kaestner L. , Beech D., George M., Fertig N., Rapedius M., Brüggemann A.

PIEZO1 channels are mechanically activated cation channels that play a pivotal role in sensing mechanical forces in various cell types. Their dysfunction has been associated with numerous pathophysiological states, including generalized lymphatic dysplasia, varicose vein disease, and hereditary xerocytosis. Given their physiological relevance, investigating PIEZO1 is crucial for the pharmaceutical industry, which requires scalable techniques to allow for drug discovery. In this regard, several studies have used high-throughput automated patch clamp (APC) combined with Yoda1, a specific gating modifier of PIEZO1 channels, to explore the function and properties of PIEZO1 in heterologous expression systems, as well as in primary cells. However, a combination of solely mechanical stimulation (M-Stim) and high-throughput APC has not yet been available for the study of PIEZO1 channels. Here, we show that optimization of pipetting parameters of the SyncroPatch 384 coupled with multihole NPC-384 chips enables M-Stim of PIEZO1 channels in high-throughput electrophysiology. We used this approach to explore differences between the response of mouse and human PIEZO1 channels to mechanical and/or chemical stimuli. Our results suggest that applying solutions on top of the cells at elevated pipetting flows is crucial for activating PIEZO1 channels by M-Stim on the SyncroPatch 384. The possibility of comparing and combining mechanical and chemical stimulation in a high-throughput patch clamp assay facilitates investigations on PIEZO1 channels and thereby provides an important experimental tool for drug development.

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2023 – A BODIPY-Based Molecular Rotor in Giant Unilamellar Vesicles: A Case Study by Polarization-Resolved TimeResolved Emission and Transient Absorption Spectroscopy
Vesicle Prep Pro Publication in ChemPhotoChem (2023) Authors: Jha K., Prabhakaran A., Spantzel L., Sia R., Pérez I., Arellano-Reyes R., Elmanova A., Dasgupta A., Eggeling C., Börsch M., Guthmuller J., Presselt M., Keyes T., Dietzek-Ivanšić B.

BODIPY and BODIPY-derived systems are widely applied as fluorophores and as probes for viscosity detection in solvents and biological media. Their orientational and rotational dynamics in biological media are thus of vital mechanistic importance and extensively investigated. In this contribution, polarization-resolved confocal microscopy is used to determine the orientation of an amphiphilic BODIPY-cholesterol derivative in homogeneous giant unilamellar vesicles (GUV) made from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). The BODIPY-moiety of the molecule is placed near the polar headgroups, and the cholesterol moiety is embedded in the membrane along the acyl chain of the lipids. The rotational relaxation of fluorophore is conventionally investigated by time-resolved emission anisotropy (TEA); and this method is also used here. However, TEA depends on the emission of the fluorophore and may not be useful to probe rotational dynamics of the non-emissive triplet states. Thus, we employ femtosecond transient absorption anisotropy (TAA), that relies on the absorption of the molecule to complement the studies of the amphiphilic BODIPY in DCM and GUV. The photoinduced anisotropy of the BODIPY molecule in DCM decays tri-exponentially, the decay components (sub-5 ps, 43 ps and 440 ps) of anisotropy are associated with the non-spherical shape of the BODIPY molecule. However, the anisotropy decay in homogenous GUVs follows a biexponential decay; which arises from the wobbling-in-a-cone motion of the non-spherical molecule in the high viscous lipid bilayer media. The observations for the BODIPY-chol molecule in the GUV environment by TAA will extend to the investigation of non-emissive molecules in cellular environment since GUV structure and size resembles the membrane of a biological cell.

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2023 – Mechanistic Insight into the Early Stages of Toroidal Pore Formation by the Antimicrobial Peptide Smp24
Port-a-Patch, Vesicle Prep Pro Publication in Pharmaceutics (2023) Authors: Bertelsen M., Lacey M., Nichol T., Miller, K.

The antimicrobial peptide Smp24, originally derived from the venom of Scorpio maurus palmatus, is a promising candidate for further drug development. However, before doing so, greater insight into the mechanism of action is needed to construct a reliable structure–activity relationship. The aim of this study was to specifically investigate the critical early stages of peptide-induced membrane disruption. Single-channel current traces were obtained via planar patch-clamp electrophysiology, with multiple types of pore-forming events observed, unlike those expected from the traditional, more rigid mechanistic models. To better understand the molecular-level structures of the peptide-pore assemblies underlying these observed conductance events, molecular dynamics simulations were used to investigate the peptide structure and orientation both before and during pore formation. The transition of the peptides to transmembrane-like states within disordered toroidal pores occurred due to a peptide-induced bilayer-leaflet asymmetry, explaining why pore stabilization does not always follow pore nucleation in the experimental observations. To fully grasp the structure–activity relationship of antimicrobial peptides, a more nuanced view of the complex and dynamic mechanistic behaviour must be adopted

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2023 – Design, synthesis, and biological evaluation of a novel series of 1,2,4-oxadiazole inhibitors of SLACK potassium channels: Identification of in vitro tool VU0935685
SyncroPatch 384 Publication in Bioorganic & Medicinal Chemistry (2023) Authors: Qunies A., Spitznagel B., Du Y., Weaver C., Emmitte K.

Malignant migrating partial seizure of infancy (MMPSI) is a devastating and pharmacoresistant form of infantile epilepsy. MMPSI has been linked to multiple gain-of-function (GOF) mutations in the KCNT1 gene, which encodes for a potassium channel often referred to as SLACK. SLACK channels are sodium-activated potassium channels distributed throughout the central nervous system (CNS) and the periphery. The investigation described here aims to discover SLACK channel inhibitor tool compounds and profile their pharmacokinetic and pharmacodynamic properties. A SLACK channel inhibitor VU0531245 (VU245) was identified via a high-throughput screen (HTS) campaign. Structure-activity relationship (SAR) studies were conducted in five distinct regions of the hit VU245. VU245 analogs were evaluated for their ability to affect SLACK channel activity using a thallium flux assay in HEK-293 cells stably expressing wild-type (WT) human SLACK. Selected analogs were tested for metabolic stability in mouse liver microsomes and plasma-protein binding in mouse plasma. The same set of analogs was tested via thallium flux for activity versus human A934T SLACK and other structurally related potassium channels, including SLICK and Maxi-K. In addition, potencies for selected VU245 analogs were obtained using whole-cell electrophysiology (EP) assays in CHO cells stably expressing WT human SLACK through an automated patch clamp system. Results revealed that this scaffold tolerates structural changes in some regions, with some analogs demonstrating improved SLACK inhibitory activity, good selectivity against the other channels tested, and modest improvements in metabolic clearance. Analog VU0935685 represents a new, structurally distinct small-molecule inhibitor of SLACK channels that can serve as an in vitro tool for studying this target.

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2024 – Acute antiarrhythmic effects of SGLT2 inhibitors–dapagliflozin lowers the excitability of atrial cardiomyocytes
SyncroPatch 384 Publication in Basic Research in Cardiology (2024) Authors:  Paasche A., Wiedmann F., Kraft M., Seibertz F., Herlt V., Blochberger PL., Jávorszky N., Beck M., Weirauch L., Seeger T., Blank A., Haefeli W.E., Arif R., Meyer A.L., Warnecke G., Karck M., Voigt N., Frey N., Schmidt C.
In recent years, SGLT2 inhibitors have become an integral part of heart failure therapy, and several mechanisms contributing to cardiorenal protection have been identified. In this study, we place special emphasis on the atria and investigate acute electrophysiological effects of dapagliflozin to assess the antiarrhythmic potential of SGLT2 inhibitors. Direct electrophysiological effects of dapagliflozin were investigated in patch clamp experiments on isolated atrial cardiomyocytes. Acute treatment with elevated-dose dapagliflozin caused a significant reduction of the action potential inducibility, the amplitude and maximum upstroke velocity. The inhibitory effects were reproduced in human induced pluripotent stem cell-derived cardiomyocytes, and were more pronounced in atrial compared to ventricular cells. Hypothesizing that dapagliflozin directly affects the depolarization phase of atrial action potentials, we examined fast inward sodium currents in human atrial cardiomyocytes and found a significant decrease of peak sodium current densities by dapagliflozin, accompanied by a moderate inhibition of the transient outward potassium current. Translating these findings into a porcine large animal model, acute elevated-dose dapagliflozin treatment caused an atrial-dominant reduction of myocardial conduction velocity in vivo. This could be utilized for both, acute cardioversion of paroxysmal atrial fibrillation episodes and rhythm control of persistent atrial fibrillation. In this study, we show that dapagliflozin alters the excitability of atrial cardiomyocytes by direct inhibition of peak sodium currents. In vivo, dapagliflozin exerts antiarrhythmic effects, revealing a potential new additional role of SGLT2 inhibitors in the treatment of atrial arrhythmias.
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2024 – Revisiting the urease production of MICP-relevant bacterium Sporosarcina pasteurii during cultivation
CardioExcyte 96 Publication in Biocatalysis and Agricultural Biotechnology (2024) Authors: Lapierre FM. Huber R.

The ureolytic bacterium Sporosarcina pasteurii is commonly used for Microbial Induced Calcium Carbonate Precipitation (MICP). This process has a variety of applications in the fields of construction, geotechnical engineering, and environmental remediation. However, the factors influencing urease activity of S. pasteurii during cultivation are not yet fully understood. Even though there is debate over whether higher urease activity is in fact beneficial for MICP applications, knowledge about urease production can help provide ureolytic biomass more cost- effectively, reducing overall production costs. This study revisits the effect of various cultivation conditions on growth and urease activity by using an automated high-throughput microbioreactor system combined with a novel system for high-throughput urease activity quantification. This experimental set-up allows for unprecedented data density and enables new insights into urease production of S. pasteurii. Several factors were tested, including oxygen limitation, the feeding of urea, choice of ammonium salt, the proportion of ammonium salt in the medium, and lowering the pH during cultivation; none of these had a notable impact on urease production. Only the addition of a broad spectrum of nutrients through regular feeding with highly concentrated yeast extract and glucose led to a 70 % increased specific urease activity compared to a batch culture. These results can improve the understanding of the regulatory mechanisms governing urease expression in S. pasteurii during cultivation, and allow to adapt the cultivation process accordingly. Additionally, the new system for automated high-throughput enzyme activity determination presented in this study could be applied to optimize bioprocesses involving other ion producing enzymes.

 

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2023 – The Potential Mechanisms behind Loperamide-Induced Cardiac Arrhythmias Associated with Human Abuse and Extreme Overdose
SyncroPatch 384 Publication in Biomolecules (2023) Authors: Lu H., Damiano B., Kreir M., Rohrbacher J., van der Linde H., Saidov T., Teisman A., Gallacher D.

Loperamide has been a safe and effective treatment for diarrhea for many years. However, many cases of cardiotoxicity with intentional abuse of loperamide ingestion have recently been reported. We evaluated loperamide in in vitro and in vivo cardiac safety models to understand the mechanisms for this cardiotoxicity. Loperamide slowed conduction (QRS-duration) starting at 0.3 µM [~1200-fold (×) its human Free Therapeutic Plasma Concentration; FTPC] and reduced the QT-interval and caused cardiac arrhythmias starting at 3 µM (~12,000× FTPC) in an isolated rabbit ventricular-wedge model. Loperamide also slowed conduction and elicited Type II/III A-V block in anesthetized guinea pigs at overdose exposures of 879× and 3802× FTPC. In ion-channel studies, loperamide inhibited hERG (IKr), INa, and ICa currents with IC50 values of 0.390 µM, 0.526 µM, and 4.091 µM, respectively (i.e., >1560× FTPC). Additionally, in silico trials in human ventricular action potential models based on these IC50s confirmed that loperamide has large safety margins at therapeutic exposures (≤600× FTPC) and confirmed repolarization abnormalities in the case of extreme doses of loperamide. The studies confirmed the large safety margin for the therapeutic use of loperamide but revealed that at the extreme exposure levels observed in human overdose, loperamide can cause a combination of conduction slowing and alterations in repolarization time, resulting in cardiac proarrhythmia. Loperamide’s inhibition of the INa channel and hERG-mediated IKr are the most likely basis for this cardiac electrophysiological toxicity at overdose exposures. The cardiac toxic effects of loperamide at the overdoses could be aggravated by co-medication with other drug(s) causing ion channel inhibition.

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2023 – A simple quantitative model of neuromodulation, Part I: Ion flow through neural ion channels
Buffer Solutions Publication in Journal of the Mechanics and Physics of Solids (2023) Authors: Werneck L., Han M., Yildiz E., Keip M., Sitti M., Ortiz M.

We develop a simple model of ionic current through neuronal membranes as a function of membrane potential and extracellular ion concentration. The model combines a simplified Poisson–Nernst–Planck (PNP) model of ion transport through individual ion channels with channel activation functions calibrated from ad hoc in-house experimental data. The simplified PNP model is validated against bacterial gramicidin A ion channel data. The calibrated model accounts for the transport of calcium, sodium, potassium, and chloride and exhibits remarkable agreement with the experimentally measured current–voltage curves for the differentiated human neural cells. All relevant data and code related to the ion flow models are available at Werneck et al. (2023)

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2023 – Membrane Tension Inhibits Lipid Mixing by Increasing the Hemifusion Stalk Energy
Vesicle Prep Pro Publication in ACS Nano (2023) Authors: Shendrick P., Golani G., Dharan R., Schwarz U., Sorkin R.

Fusion of biological membranes is fundamental in various physiological events. The fusion process involves several intermediate stages with energy barriers that are tightly dependent on the mechanical and physical properties of the system, one of which is membrane tension. As previously established, the late stages of fusion, including hemifusion diaphragm and pore expansions, are favored by membrane tension. However, a current understanding of how the energy barrier of earlier fusion stages is affected by membrane tension is lacking. Here, we apply a newly developed experimental approach combining micropipette-aspirated giant unilamellar vesicles and optically trapped membrane-coated beads, revealing that membrane tension inhibits lipid mixing. We show that lipid mixing is 6 times slower under a tension of 0.12 mN/m compared with tension-free membranes. Furthermore, using continuum elastic theory, we calculate the dependence of the hemifusion stalk formation energy on membrane tension and intermembrane distance and find the increase in the corresponding energy barrier to be 1.6 kBT in our setting, which can explain the increase in lipid mixing time delay. Finally, we show that tension can be a significant factor in the stalk energy if the pre-fusion intermembrane distance is on the order of several nanometers, while for membranes that are tightly docked, tension has a negligible effect.

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2023 – Determination of single-molecule transport activity of OATP2B1 by measuring the number of transporter molecules using electrophysiological approach
Port-a-Patch Publication in Journal of Pharmacological Sciences (2023) Authors: Yajima K., Akiyoshi T., Sakamoto K., Suzuki Y., Oka T., Imaoka A., Yamamura H., Kurokawa J., Ohtani H.

Transporter-mediated clearance is determined by two factors, its single-molecule clearance, and expression level. However, no reliable method has been developed to evaluate them separately. This
study aimed to develop a reliable method for evaluating the single-molecule activity of membrane transporters, such as organic anion transporting polypeptide (OATP) 2B1. HEK293 cells that co-expressed large conductance calcium-activated potassium (BK) channel and OATP2B1 were established and used for the following experiments. i) BK channel-mediated whole-cell conductance was measured using patch-clamp technique and divided by its unitary conductance to estimate the number of channels on plasma membrane (QI). ii) Using plasma membrane fraction, quantitative targeted absolute proteomics determined the stoichiometric ratio (r) of OATP2B1 to BK channel. iii) The uptake of estrone 3-sulfate was evaluated to calculate the Michaelis constant and uptake clearance (CL) per cell. Single-molecule clearance (CLint) was calculated by dividing CL by QIxp. QI and r values were estimated to be 916 and 2.16, respectively, yielding CLint of 5.23 fL/min/molecule. We successfully developed a novel method to reliably measure the single-molecule activity of a transporter, which could be used to evaluate the influences of factors such as genetic variations and post-translational modifications on the intrinsic activity of transporters

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2023 – Synergy between Winter Flounder antimicrobial peptides
Vesicle Prep Pro, Port-a-Patch Publication in Nature npj Antimicrobials and Resistance (2023) Authors: Clarke M., Hind C., Ferguson P., Manzo G., Mistry B., Yue B., Romanopulos J., Clifford M., Bui T., Drake A., Lorenz C., Sutton M., Mason J.

Some antimicrobial peptides (AMPs) have potent bactericidal activity and are being considered as potential alternatives to classical antibiotics. In response to an infection, such AMPs are often produced in animals alongside other peptides with low or no perceivable antimicrobial activity, whose role is unclear. Here we show that six AMPs from the Winter Flounder (WF) act in synergy against a range of bacterial pathogens and provide mechanistic insights into how this increases the cooperativity of the dose-dependent bactericidal activity and potency that enable therapy. Only two WF AMPs have potent antimicrobial activity when used alone but we find a series of two-way combinations, involving peptides which otherwise have low or no activity, yield potent antimicrobial activity. Weakly active WF AMPs modulate the membrane interactions of the more potent WF AMPs and enable therapy in a model of Acinetobacter baumannii burn wound infection. The observed synergy and emergent behaviour may explain the evolutionary benefits of producing a family of related peptides and are attractive properties to consider when developing AMPs towards clinical applications.

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2024 – Functional Analysis of Human GRIN2A Mutations Associated with Schizophrenia and Neurodevelopmental Disorders Reveals Distinct Pathological Mechanism
SyncroPatch 384 Publication in Scentific Reports (2024) Authors: Shepard N., Baez-Nieto D., Iqbal S., Kurgano E., Budnik N., Campbell A., Pan J., Sheng M., Farsi Z.

Human genetic studies have revealed rare missense and protein-truncating variants in GRIN2A, encoding for the GluN2A subunit of the NMDA receptors, that confer significant risk for schizophrenia (SCZ). Mutations in GRIN2A are also associated with epilepsy and developmental delay/intellectual disability (DD/ID). However, it remains enigmatic how alterations to the same protein can result in diverse clinical phenotypes. Here, we performed functional characterization of human GluN1/GluN2A heteromeric NMDA receptors that contain SCZ-linked GluN2A variants, and compared them to NMDA receptors with GluN2A variants associated with epilepsy or DD/ID. Our findings demonstrate that SCZ-associated GRIN2A variants were predominantly loss-of-function (LoF), whereas epilepsy and DD/ID-associated variants resulted in both gain- and loss-of-function phenotypes. We additionally show that M653I and S809R, LoF GRIN2A variants associated with DD/ID, exert a dominant-negative effect when co-expressed with a wild-type GluN2A, whereas E58Ter and Y698C, SCZ-linked LoF variants, and A727T, an epilepsy-linked LoF variant, do not. These data offer a potential mechanism by which SCZ/epilepsy and DD/ID-linked variants can cause different effects on receptor function and therefore result in divergent pathological outcomes.

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2023 – A Comparative Study on the Lysosomal Cation Channel TMEM175 Using Automated Whole-Cell Patch-Clamp, Lysosomal Patch-Clamp, and Solid Supported Membrane-Based Electrophysiology: Functional Characterization and High-Throughput Screening Assay Development
SURFE2R 96SE, SURFE2R N1, SyncroPatch 384 Publication in International Journal of Molecular Sciences (2023) Authors: Bazzone A., Barthmes M., George C., Brinkwirth N., Zerlotti R., Prinz V., Cole K., Friis S., Dickson A., Rice S., Lim J., Toh M., Mohammadi M. Pau D., Stone D., Renger J., Fertig N.
The lysosomal cation channel TMEM175 is a Parkinson’s disease-related protein and a promising drug target. Unlike whole-cell automated patch-clamp (APC), lysosomal patch-clamp (LPC) facilitates physiological conditions, but is not yet suitable for high-throughput screening (HTS) applications. Here, we apply solid supported membrane-based electrophysiology (SSME), which enables both direct access to lysosomes and high-throughput electrophysiological recordings. In SSME, ion translocation mediated by TMEM175 is stimulated using a concentration gradient at a resting potential of 0 mV. The concentration-dependent K+ response exhibited an I/c curve with two distinct slopes, indicating the existence of two conducting states. We measured H+ fluxes with a permeability ratio of PH/PK = 48,500, which matches literature findings from patch-clamp studies, validating the SSME approach. Additionally, TMEM175 displayed a high pH dependence. Decreasing cytosolic pH inhibited both K+ and H+ conductivity of TMEM175. Conversely, lysosomal pH and pH gradients did not have major effects on TMEM175. Finally, we developed HTS assays for drug screening and evaluated tool compounds (4-AP, Zn as inhibitors; DCPIB, arachidonic acid, SC-79 as enhancers) using SSME and APC. Additionally, we recorded EC50 data for eight blinded TMEM175 enhancers and compared the results across all three assay technologies, including LPC, discussing their advantages and disadvantages.
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2023 – Pathogenic SCN2A variants are associated with familial and sporadic hemiplegic migraine
SyncroPatch 384 Pre-print in Research Square (2023) Authors: Riant F., Thompson C., DeKeyser J., Abramova T., Gazal S., Moulin T.,Chaigne D., Kort L., Corpechot M., Tournier-Lasserve E., George A., Ducros A.

Background: Familial hemiplegic migraine is a severe autosomal dominant subtype of migraine with aura characterized by transient motor weakness during attacks. Previously identified genes CACNA1AATP1A2SCN1A and PRRT2 account for less than 20% of cases with hemiplegic migraine referred for genetic diagnosis.

Objectives and Methods: To identify a novel gene, we conducted a whole-genome linkage analysis combined with mini-exome sequencing in a four-generation pedigree with hemiplegic migraine. A candidate ion channel gene was analyzed for mutations in six other affected pedigrees comprising at least three available affected members, and in a large panel of unrelated probands with hemiplegic migraine referred for molecular diagnosis, all without mutations in the known genes. The functional consequences of the identified variants were determined.

Results: In the discovery pedigree, we identified a heterozygous missense mutation (c.4438A>G, p.Lys1480Glu) in the neuronal voltage-gated sodium channel gene SCN2A, which cosegregated with the hemiplegic migraine phenotype. We detected another mutation (c.769T>A, p.Phe257Ile) cosegregating with hemiplegic migraine in a second family, in which two members also had infantile seizures. A third variant (c.3955C>G, p.Arg1319Gly) was found in a sporadic hemiplegic migraine case. All three SCN2A variants were absent in the genome aggregation database gnomAD. Heterologous expression in HEK293T cells coupled with automated patch clamp recording demonstrated abnormal voltage-dependent and kinetic properties of all three SCN2A variants.

Conclusions:Dysfunction of the neuronal sodium channel SCN2A can be associated with familial and sporadic hemiplegic migraine. Our finding expands the genetic landscape for migraine and contributes to the diverse genotype-phenotype spectrum associated with SCN2A.

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2023 – A previously unrecognized superfamily of macro-conotoxins includes an inhibitor of the sensory neuron calcium channel Cav2.3
Patchliner Publication in PLoS Biol (2023) Authors: Hackney C., Flórez Salcedo P., Mueller E., Lund Koch T., Kjelgaard L., Watkins M., Zachariassen L., Sønderby Tuelung P., McArthur J., Adams D., Kristensen A., Olivera B., Finol-Urdaneta R., Safavi-Hemami H., Preben Morth J., Ellgaard L.

Animal venom peptides represent valuable compounds for biomedical exploration. The venoms of marine cone snails constitute a particularly rich source of peptide toxins, known as conotoxins. Here, we identify the sequence of an unusually large conotoxin, Mu8.1, which defines a new class of conotoxins evolutionarily related to the well-known con-ikot-ikots and 2 additional conotoxin classes not previously described. The crystal structure of recombinant Mu8.1 displays a saposin-like fold and shows structural similarity with con-ikot-ikot. Functional studies demonstrate that Mu8.1 curtails calcium influx in defined classes of murine somatosensory dorsal root ganglion (DRG) neurons. When tested on a variety of recombinantly expressed voltage-gated ion channels, Mu8.1 displayed the highest potency against the R-type (Cav2.3) calcium channel. Ca2+ signals from Mu8.1-sensitive DRG neurons were also inhibited by SNX-482, a known spider peptide modulator of Cav2.3 and voltage-gated K+ (Kv4) channels. Our findings highlight the potential of Mu8.1 as a molecular tool to identify and study neuronal subclasses expressing Cav2.3. Importantly, this multidisciplinary study showcases the potential of uncovering novel structures and bioactivities within the largely unexplored group of macro-conotoxins.

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2023 – Structure and function of a YeeE-YeeD complex for sophisticated thiosulfate uptake
SURFE2R N1 Publication in bioRxiv (2023) Authors: Ikei M., Miyazaki R., Monden K., Naito Y., Takeuchi A., Takahashi Y., Tanaka Y., Mori T., Ichikawa M., Tsukazaki T.

Uptake of thiosulfate ions as an inorganic sulfur source from the environment is important for bacterial sulfur assimilation. Recently, a selective thiosulfate uptake pathway involving membrane protein YeeE (TsuA) was characterized. However, the precise function of YeeE and a putative cofactor in the
thiosulfate ion uptake pathway remained unclear. Here, we assessed selective thiosulfate transport via YeeE in vitro and characterized YeeD (TsuB) as an adjacent and essential cofactor for YeeE-mediated thiosulfate uptake in vivo. We further showed that YeeD possesses thiosulfate decomposition activity
and that a conserved cysteine in YeeD was modified in several forms in the presence of thiosulfate. Finally, the crystal structure of a YeeE-YeeD fusion protein at 2.6-Å resolution revealed their interactions. The association was evaluated by a binding assay using purified proteins. Based on these results, a model of the sophisticated uptake of thiosulfate ions by YeeE and YeeD is proposed.

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2023 – De novo design of diverse small molecule binders and sensors using Shape Complementary Pseudocycles
Orbit 16 TC Pre-Print Publication in BioRxiv (2023) Authors: An L., Said M., Tran L., Majumder S., Goreshnik I., Lee G.R., Juergens D., Dauparas J., Anishchenko I., Coventry B., Bera A.K., Kang A., Levine P.M., Alvarez V., Pillai A., Norn C., Feldman D., Zorine D., Hicks D.R., Li X., Sanchez M.G., Vafeados D.K., Salveson P.J., Vorobieva A.A., Baker D.

A general method for designing proteins to bind and sense any small molecule of interest would be widely useful. Due to the small number of atoms to interact with, binding to small molecules with high affinity requires highly shape complementary pockets, and transducing binding events into signals is challenging. Here we describe an integrated deep learning and energy based approach for designing high shape complementarity binders to small molecules that are poised for downstream sensing applications. We employ deep learning generated psuedocycles with repeating structural units surrounding central pockets; depending on the geometry of the structural unit and repeat number, these pockets span wide ranges of sizes and shapes. For a small molecule target of interest, we extensively sample high shape complementarity pseudocycles to generate large numbers of customized potential binding pockets; the ligand binding poses and the interacting interfaces are then optimized for high affinity binding. We computationally design binders to four diverse molecules, including for the first time polar flexible molecules such as methotrexate and thyroxine, which are expressed at high levels and have nanomolar affinities straight out of the computer. Co-crystal structures are nearly identical to the design models. Taking advantage of the modular repeating structure of pseudocycles and central location of the binding pockets, we constructed low noise nanopore sensors and chemically induced dimerization systems by splitting the binders into domains which assemble into the original pseudocycle pocket upon target molecule addition.

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2023 – Multi-site validation of a functional assay to adjudicate SCN5A Brugada Syndrome-associated variants
SyncroPatch 384 Pre-Print Publication in MedRxiv (2023) Authors: Ma J.G., O’Neill M.J., Richardson E., Thomson K.L., Ingles J., Muhammad A., Solus Joseph F., Davogustto G., Anderson K.C., Shoemaker M.B., Stergachis A.B., Floyd B.J., Dunn K., Parikh V.N., Chubb H., Perrin M.J., Roden D.M., Vandenberg J.I., Ng C-A., Glazer A.M.

Brugada Syndrome (BrS) is an inheritable arrhythmia condition that is associated with rare, loss-of-function variants in the cardiac sodium channel gene, SCN5A. Interpreting the pathogenicity of SCN5A missense variants is challenging and ∼79% of SCN5A missense variants in ClinVar are currently classified as Variants of Uncertain Significance (VUS). An in vitro SCN5A-BrS automated patch clamp assay was generated for high-throughput functional studies of NaV1.5. The assay was independently studied at two separate research sites – Vanderbilt University Medical Center and Victor Chang Cardiac Research Institute – revealing strong correlations, including peak INa density (R2=0.86). The assay was calibrated according to ClinGen Sequence Variant Interpretation recommendations using high-confidence variant controls (n=49). Normal and abnormal ranges of function were established based on the distribution of benign variant assay results. The assay accurately distinguished benign controls (24/25) from pathogenic controls (23/24). Odds of Pathogenicity values derived from the experimental results yielded 0.042 for normal function (BS3 criterion) and 24.0 for abnormal function (PS3 criterion), resulting in up to strong evidence for both ACMG criteria. The calibrated assay was then used to study SCN5A VUS observed in four families with BrS and other arrhythmia phenotypes associated with SCN5A loss-of-function. The assay revealed loss-of-function for three of four variants, enabling reclassification to likely pathogenic. This validated APC assay provides clinical-grade functional evidence for the reclassification of current VUS and will aid future SCN5A-BrS variant classification.

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2023 – Sculpting conducting nanopore size and shape through de novo protein design
Orbit 16 TC Pre-Print Publication in BioRxiv (2023) Authors: Berhanu S., Majumder S., Müntener T., Whitehouse J., Berner C., Bera A.K., Kang A., Liang B., Khan G.N., Sankaran B., Tamm L.K., Brockwell D.J., Hiller S., Radford S.E., Baker D., Vorobieva A.A.

Transmembrane β-barrels (TMBs) are widely used for single molecule DNA and RNA sequencing and have considerable potential for a broad range of sensing and sequencing applications. Current engineering approaches for nanopore sensors are limited to naturally occurring channels such as CsgG, which have evolved to carry out functions very different from sensing, and hence provide sub-optimal starting points. In contrast, de novo protein design can in principle create an unlimited number of new nanopores with any desired properties. Here we describe a general approach to the design of transmembrane β-barrel pores with different diameter and pore geometry. NMR and crystallographic characterization shows that the designs are stably folded with structures close to the design models. We report the first examples of de novo designed TMBs with 10, 12 and 14 stranded β-barrels. The designs have distinct conductances that correlate with their pore diameter, ranging from 110 pS (∼0.5 nm pore diameter) to 430 pS (∼1.1 nm pore diameter), and can be converted into sensitive small-molecule sensors with high signal to noise ratio. The capability to generate on demand β-barrel pores of defined geometry opens up fundamentally new opportunities for custom engineering of sequencing and sensing technologies.

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2023 – Deep Learning-Assisted Single-Molecule Detection of Protein Post-translational Modifications with a Biological Nanopore
Orbit mini Publication in ACS Nano (2023) Authors: Cao C.,MagalhaesP., Krapp L.F.,Bada J.F.J, Mayer S.F., Rukes V., Chiki A., Lashuel H.A., Dal Peraro M.

Protein post-translational modifications (PTMs) play a crucial role in countless biological processes, profoundly modulating protein properties on both spatial and temporal scales. Protein PTMs have also emerged as reliable biomarkers for several diseases. However, only a handful of techniques are available to accurately measure their levels, capture their complexity at a single molecule level, and characterize their multifaceted roles in health and disease. Nanopore sensing provides high sensitivity for the detection of low-abundance proteins, holding the potential to impact single-molecule proteomics and PTM detection, in particular. Here, we demonstrate the ability of a biological nanopore, the pore-forming toxin aerolysin, to detect and distinguish α-synuclein-derived peptides bearing single or multiple PTMs, namely, phosphorylation, nitration, and oxidation occurring at different positions and in various combinations. The characteristic current signatures of the α-synuclein peptide and its PTM variants could be confidently identified by using a deep learning model for signal processing. We further demonstrate that this framework can quantify α-synuclein peptides at picomolar concentrations and detect the C-terminal peptides generated by digestion of full-length α-synuclein. Collectively, our work highlights the advantage of using nanopores as a tool for simultaneous detection of multiple PTMs and facilitates their use in biomarker discovery and diagnostics.

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Cardiotox., cardio-oncology – “Chronic cardiotoxicity study on H9C2 cells using HESI stem cell working group reference compounds”
AtlaZ application note
Publication link
2023 – Functional Characterization of the Lysosomal Peptide/Histidine Transporter PHT1 (SLC15A4) by Solid Supported Membrane Electrophysiology (SSME)
SURFE2R N1 Publication in Preprints.org (2023) Authors: Pujol-Giménez J., Hediger M.A.

Peptide/Histidine Transporter PHT1 (SLC15A4) is expressed in lysosomal membranes of immune cells where it plays an important role in metabolic and inflammatory signaling. PHT1 is an H+-coupled/histidine symporter that can transport a broad range of oligopeptides, including a variety of bacterial-derived peptides. Moreover, it enables the scaffolding of various metabolic signaling molecules and interacts with key regulatory elements of the immune response. Therefore, it is not surprising that PHT1 is associated with the development of autoimmune diseases, such as systemic lupus erythematosus (SLE). Unfortunately, the pharmacological development of PHT1 has been hampered by the lack of appropriate transport assays. With the aim to address this shortcoming, a novel transport assay based on solid supported membrane-based electrophysiology (SSME) is presented. Key findings of the present SSME studies include the first recordings of electrophysiological properties, a pH dependence analysis, an assessment of PHT1 substrate selectivity, as well as the transport kinetics of the identified substrates. In contrast to previous works, PHT1 is studied its native lysosomal environment. Moreover, observed substrate selectivity is validated by molecular docking. Overall, this new SSME-based assay is expected to contribute to unlock the pharmacological potential of PHT1 and to deepen the understanding of its functional properties.

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2023 – Peptide toxins that target vertebrate voltage-gated sodium channels underly the painful stings of harvester ants
SyncroPatch 384 publication in JBC (2023) Authors: Robinson SD., Deuis JR., Niu P., Touchard A., Mueller A., Schendel V., Brinkwirth N., King GF., Vetter I., Schmidt JO.

Harvester ants (genus Pogonomyrmex) are renowned for their stings which cause intense, long-lasting pain and other neurotoxic symptoms in vertebrates. Here we show that harvester ant venoms are relatively simple and composed largely of peptide toxins. One class of peptides is primarily responsible for the long-lasting local pain of envenomation via activation of peripheral sensory neurons. These hydrophobic, cysteine-free peptides potently modulate mammalian voltage-gated sodium (NaV) channels, reducing the voltage threshold for activation and inhibiting channel inactivation. These toxins appear to have evolved specifically to deter vertebrates.

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2023 – Epileptic Encephalopathy GABRB Structural Variants Share Common Gating and Trafficking Defects
SyncroPatch 384 Publication in Biomolecules (2023) Authors: Hernandez C.C., Hu N., Shen W., Macdonald R.L.

Variants in the GABRB gene, which encodes the β subunit of the GABAA receptor, have been implicated in various epileptic encephalopathies and related neurodevelopmental disorders such as Dravet syndrome and Angelman syndrome. These conditions are often associated with early-onset seizures, developmental regression, and cognitive impairments. The severity and specific features of these encephalopathies can differ based on the nature of the genetic variant and its impact on GABAA receptor function. These variants can lead to dysfunction in GABAA receptor-mediated inhibition, resulting in an imbalance between neuronal excitation and inhibition that contributes to the development of seizures. Here, 13 de novo EE-associated GABRB variants, occurring as missense mutations, were analyzed to determine their impact on protein stability and flexibility, channel function, and receptor biogenesis. Our results showed that all mutations studied significantly impact the protein structure, altering protein stability, flexibility, and function to varying degrees. Variants mapped to the GABA-binding domain, coupling zone, and pore domain significantly impact the protein structure, modifying the β+/α− interface of the receptor and altering channel activation and receptor trafficking. Our study proposes that the extent of loss or gain of GABAA receptor function can be elucidated by identifying the specific structural domain impacted by mutation and assessing the variability in receptor structural dynamics. This paves the way for future studies to explore and uncover links between the incidence of a variant in the receptor topology and the severity of the related disease.

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SB Drug Discovery and Nanion: Accelerating Growth for Success
Application protocol
Contractility Analysis of iCell Cardiomyocytes2 : Evaluating iCell Cardiomyocytes2 in co-culture with iCell Cardiac Fibroblasts on the FLEXcyte 96 System
CardioExcyte 96, FLEXcyte 96, collaboration with FUJIFILM Cellular Dynamics, Inc. and InnoVitro
Publication link
2023 – Exome sequencing of ATP1A3-negative cases of alternating hemiplegia of childhood reveals SCN2A as a novel causative gene
SyncroPatch 384 Publication in Eur. J. Hum. Gen Basic Research in Cardiology (2023) Authors: Panagiotakaki E., Tiziano F.D., Mikati M.A., Vijfhuizen L.S., Nicole S., Lesca G., Abiusi E., Novelli A., Di Pietro L., I.B.AHC Consortium, IAHCRC Consortium, Harder A.V.E., Walley N.M., De Grandis E., Poulat A-L., Des Portes V., Lépine A., Nassogne M-C., Arzimanoglou A., Vavassori R., Koenderink J., Thompson C.H., George Jr. A.L., Gurrieri F., van den Maagdenberg A.M.J.M., Heinzen E.L.

Alternating hemiplegia of childhood (AHC) is a rare neurodevelopment disorder that is typically characterized by debilitating episodic attacks of hemiplegia, seizures, and intellectual disability. Over 85% of individuals with AHC have a de novo missense variant in ATP1A3 encoding the catalytic α3 subunit of neuronal Na+/K+ ATPases. The remainder of the patients are genetically unexplained. Here, we used next-generation sequencing to search for the genetic cause of 26 ATP1A3-negative index patients with a clinical presentation of AHC or an AHC-like phenotype. Three patients had affected siblings. Using targeted sequencing of exonic, intronic, and flanking regions of ATP1A3 in 22 of the 26 index patients, we found no ultra-rare variants. Using exome sequencing, we identified the likely genetic diagnosis in 9 probands (35%) in five genes, including RHOBTB2 (n = 3), ATP1A2 (n = 3), ANK3 (n = 1), SCN2A (n = 1), and CHD2 (n = 1). In follow-up investigations, two additional ATP1A3-negative individuals were found to have rare missense SCN2A variants, including one de novo likely pathogenic variant and one likely pathogenic variant for which inheritance could not be determined. Functional evaluation of the variants identified in SCN2A and ATP1A2 supports the pathogenicity of the identified variants. Our data show that genetic variants in various neurodevelopmental genes, including SCN2A, lead to AHC or AHC-like presentation. Still, the majority of ATP1A3-negative AHC or AHC-like patients remain unexplained, suggesting that other mutational mechanisms may account for the phenotype or that cases may be explained by oligo- or polygenic risk factors.

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2023 – Therapeutic efficacy of AAV-mediated restoration of PKP2 in arrhythmogenic cardiomyopathy
SyncroPatch 384 Publication in Nature Cardiovascular Research (2023) Authors: Kyriakopoulou E., Versteeg D., de Ruiter H., Perini I., Seibertz F., Döring Y., Zentilin L., Tsui H., van Kampen S.J., Tiburcy M.,, Meyer T., Voigt N, Tintelen v.J.P., Zimmermann W.H., Giacca M., van Rooij E.

Arrhythmogenic cardiomyopathy is a severe cardiac disorder characterized by lethal arrhythmias and sudden cardiac death, with currently no effective treatment. Plakophilin 2 (PKP2) is the most frequently affected gene. Here we show that adeno-associated virus (AAV)-mediated delivery of PKP2 in PKP2c.2013delC/WT induced pluripotent stem cell-derived cardiomyocytes restored not only cardiac PKP2 levels but also the levels of other junctional proteins, found to be decreased in response to the mutation. PKP2 restoration improved sodium conduction, indicating rescue of the arrhythmic substrate in PKP2 mutant induced pluripotent stem cell-derived cardiomyocytes. Additionally, it enhanced contractile function and normalized contraction kinetics in PKP2 mutant engineered human myocardium. Recovery of desmosomal integrity and cardiac function was corroborated in vivo, by treating heterozygous Pkp2c.1755delA knock-in mice. Long-term treatment with AAV9–PKP2 prevented cardiac dysfunction in 12-month-old Pkp2c.1755delA/WT mice, without affecting wild-type mice. These findings encourage clinical exploration of PKP2 gene therapy for patients with PKP2 haploinsufficiency.

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NaV, KV, GABAA – “Stem cell derived neurons recorded on Nanion’s Patchliner”
Patchliner application note: hiPS cell-derived neurons (iCell® Neurons) kindly provided by Cellular Dynamics International

Stem cell–derived neurons provide a novel and unique model for studying human drug targets in their physiologically relevant environment of terminally differentiated, postmitotic cells. It has been increasingly recognized that associated proteins modulate the physiology and pharmacology of neuronal proteins. Therefore, assays that investigate neuronal toxicity, drug effects, or basic cellular functions of neurons can mostly benefit from the development of human neurons derived from induced pluripotent stem cells (hiPSC-neurons). In 2011, Fujifilm Cellular Dynamics International (CDI) announced the commercial launch of human iCell® Neurons for use in neuroscience drug discovery, neurotoxicity screens, and other health research. This was the first commercially available iPSC-derived neuronal type available3. It was a mixture of GABAergic and glutamatergic post-mitotic neurons which rapidly regenerate post-thaw. Over the years, Fujifilm Cellular Dynamics International has developed a range of hiPSC-neurons with either healthy or diseased models. These can be used to study a number of different neurodegenerative diseases including Alzheimer’s Disease, one of the leading causes on death in the United States5.

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Kv7.2-7.3- “Cross-site study of neuronal KV7.2/7.3 heteromers on the Patchliner”
Patchliner application note: in collaboration with ApconiX

The KV7 channels are a family of voltage-gated potassium ion channels with five members (KV7.1 - 7.5) encoded by the KCNQ1-5 genes1. The channels exist as tetramers, with each subunit containing six transmembrane domains with cytoplasmic N- and C-termini. The long intracellular terminus is essential for tetramerization as well as interaction with critical regulators such as PIP2, calmodulin, protein kinase C and ankyrin G2. KV7-mediated currents are voltage activated, slowly activating and non-inactivating and are involved in repolarization of the cell membrane potential, thereby controlling cell excitability. KV7.1 channels are primarily expressed in cardiac cells whereas KV7.2, KV7.3 and KV7.5 are widely distributed in neuronal and primary sensory cells2. KV7.2/7.3 heteromeric channels primarily underlie the neuronal M-current (IKM)which plays a crucial role in repolarizing neuronal membrane potential after a depolarizing input which limits repetitive firing and is, therefore, a key mechanism in spike frequency adaptation.

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2023 – GABA(A) receptor activation drives GABARAP-Nix mediated autophagy to radiation-sensitize primary and brain-metastatic lung adenocarcinoma tumors
Port-a-Patch Pre-Print Publication in BioRxiv (2023) Authors: Bhattacharya D., Barille R., Toukam D.K., Gawali V.S., Kallay L., Ahmed T., Brown H., Rezvanian S., Karve A., Desai P.B., Medvedovic M., Wang K., Ionascu D., Harun N., Wang C., Baschnagel A.M., Kritzer J.A., Cook J.M., Krummel D.A.P., Sengupta S.

In non-small cell lung cancer (NSCLC) treatment, targeted therapies benefit only a subset of NSCLC, while radiotherapy responses are not durable and toxicity limits therapy. We find that a GABA(A) receptor activator, AM-101, impairs viability and clonogenicity of NSCLC primary and brain metastatic cells. Employing an ex vivo ‘chip’, AM-101 is as efficacious as the chemotherapeutic docetaxel, which is used with radiotherapy for advanced-stage NSCLC. In vivo, AM-101 potentiates radiation, including conferring a survival benefit to mice bearing NSCLC intracranial tumors. GABA(A) receptor activation stimulates a selective-autophagic response via multimerization of GABA(A) Receptor-Associated Protein (GABARAP), stabilization of mitochondrial receptor Nix, and utilization of ubiquitin-binding protein p62. A targeted-peptide disrupting Nix binding to GABARAP inhibits AM-101 cytotoxicity. This supports a model of GABA(A) receptor activation driving a GABARAP-Nix multimerization axis triggering autophagy. In patients receiving radiotherapy, GABA(A) receptor activation may improve tumor control while allowing radiation dose de-intensification to reduce toxicity.

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2023 – CRAFTing Delivery of Membrane Proteins into Protocells using Nanodiscs
Vesicle Prep Pro, Port-a-Patch Publication in ACS Appl. Mater. Interfaces (2023) Authors: Stępień P., Świątek S., Robles M.Y.Y., Markiewicz-Mizera J., Balakrishnan D., Inaba-Inoue S., De Vries A.H., Beis K., Marrink S.J., HeddleJ.G.

For the successful generative engineering of functional artificial cells, a convenient and controllable means of delivering membrane proteins into membrane lipid bilayers is necessary. Here we report a delivery system that achieves this by employing membrane protein-carrying nanodiscs and the calcium-dependent fusion of phosphatidylserine lipid membranes. We show that lipid nanodiscs can fuse a transported lipid bilayer with the lipid bilayers of small unilamellar vesicles (SUVs) or giant unilamellar vesicles (GUVs) while avoiding recipient vesicles aggregation. This is triggered by a simple, transient increase in calcium concentration, which results in efficient and rapid fusion in a one-pot reaction. Furthermore, nanodiscs can be loaded with membrane proteins that can be delivered into target SUV or GUV membranes in a detergent-independent fashion while retaining their functionality. Nanodiscs have a proven ability to carry a wide range of membrane proteins, control their oligomeric state, and are highly adaptable. Given this, our approach may be the basis for the development of useful tools that will allow bespoke delivery of membrane proteins to protocells, equipping them with the cell-like ability to exchange material across outer/subcellular membranes.

Application Note PDF
iPSC-CM – “Functional and pharmacological differences between the contractility of axoCellsTM iPSC-derived atrial and ventricular cardiomyocytes assessed on the FLEXcyte 96”
FLEXcyte 96 application note

In this application note, InnoVitro GmbH and Axol Biosciences Limited show that distinct pharmacological effects can be observed between axoCellsTM atrial and ventricular hiPSC-derived cardiomyocytes when using the FLEXcyte 96 platform to assess contractility, providing a powerful tool to perform in vitro drug screening and disease modelling on subtype-specific hiPSC-derived cardiomyocytes.

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2023 – Development of automated patch clamp assays to overcome the burden of variants of uncertain significance in inheritable arrhythmia syndromes
Patchliner and SyncroPatch 384 Publication (Review) in Front. Physiol. (2023) Authors: Ma J.G., Vandenberg J.I., Ng C-A.

Advances in next-generation sequencing have been exceptionally valuable for identifying variants in medically actionable genes. However, for most missense variants there is insufficient evidence to permit definitive classification of variants as benign or pathogenic. To overcome the deluge of Variants of Uncertain Significance, there is an urgent need for high throughput functional assays to assist with the classification of variants. Advances in parallel planar patch clamp technologies has enabled the development of automated high throughput platforms capable of increasing throughput 10- to 100-fold compared to manual patch clamp methods. Automated patch clamp electrophysiology is poised to revolutionize the field of functional genomics for inheritable cardiac ion channelopathies. In this review, we outline i) the evolution of patch clamping, ii) the development of high-throughput automated patch clamp assays to assess cardiac ion channel variants, iii) clinical application of these assays and iv) where the field is heading.

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2023 – Recombinant cellular model system for human muscle‑type nicotinic acetylcholine receptor α1(2)β1δε
Patchliner and Buffer Solution Publication in Cell Stress and Chaperones (2023) Authors: Brockmöller S., Seeger T., Worek F., Rothmiller S.

The human muscle-type nicotinic acetylcholine receptor α12β1δε (nAChR) is a complex transmembrane receptor needed for drug screening for disorders like congenital myasthenic syndromes and multiple pterygium syndrome. Until today, most models are still using the nAChR from Torpedo californica electric ray. A simple reproducible cellular system expressing functional human muscle-type nAChR is still missing. This study addressed this issue and further tested the hypothesis that different chaperones, both biological and chemical, and posttranslational modification supporting substances as well as hypothermic incubation are able to increase the nAChR yield. Therefore, Gibson cloning was used to generate transfer plasmids carrying the sequence of nAChR or chosen biological chaperones to support the nAChR folding in the cellular host. Viral transduction was used for stable integration of these transgenes in Chinese hamster ovary cells (CHO). Proteins were detected with Western blot, in-cell and on-cell Western, and the function of the receptor with voltage clamp analysis. We show that the internalization of nAChR into plasma membranes was sufficient for detection and function. Additional transgenic overexpression of biological chaperones did result in a reduced nAChR expression. Chemical chaperones, posttranslational modification supporting substances, and hypothermic conditions are well-suited supporting applications to increase the protein levels of different subunits. This study presents a stable and functional cell line that expresses human muscle-type nAChR and yields can be further increased using the chemical chaperone nicotine without affecting cell viability. The simplified access to this model system should enable numerous applications beyond drug development.

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2023 – Baicalein attenuates rotenone-induced SH-SY5Y cell apoptosis through binding to SUR1 and activating ATP-sensitive potassium channels
Patchliner Publication in Acta Pharmacologica Sinica (2023) Authors: Kong D., Du L., Liu R., Yuan T., Wang S., Wang Y., Lu Y., Fang L., Du G.

Dopaminergic neurons in the substantia nigra (SN) expressing SUR1/Kir6.2 type ATP-sensitive potassium channels (K-ATP) are more vulnerable to rotenone or metabolic stress, which may be an important reason for the selective degeneration of neurons in Parkinson’s disease (PD). Baicalein has shown neuroprotective effects in PD animal models. In this study, we investigated the effect of baicalein on K-ATP channels and the underlying mechanisms in rotenone-induced apoptosis of SH-SY5Y cells. K-ATP currents were recorded from SH-SY5Y cells using whole-cell voltage-clamp recording. Drugs dissolved in the external solution at the final concentration were directly pipetted onto the cells. We showed that rotenone and baicalein opened K-ATP channels and increased the current amplitudes with EC50 values of 0.438 μM and 6.159 μM, respectively. K-ATP channel blockers glibenclamide (50 μM) or 5-hydroxydecanoate (5-HD, 250 μM) attenuated the protective effects of baicalein in reducing reactive oxygen species (ROS) content and increasing mitochondrial membrane potential and ATP levels in rotenone-injured SH-SY5Y cells, suggesting that baicalein protected against the apoptosis of SH-SY5Y cells by regulating the effect of rotenone on opening K-ATP channels. Administration of baicalein (150, 300 mg·kg−1·d−1, i.g.) significantly inhibited rotenone-induced overexpression of SUR1 in SN and striatum of rats. We conducted surface plasmon resonance assay and molecular docking, and found that baicalein had a higher affinity with SUR1 protein (KD = 10.39 μM) than glibenclamide (KD = 24.32 μM), thus reducing the sensitivity of K-ATP channels to rotenone. Knockdown of SUR1 subunit reduced rotenone-induced apoptosis and damage of SH-SY5Y cells, confirming that SUR1 was an important target for slowing dopaminergic neuronal degeneration in PD. Taken together, we demonstrate for the first time that baicalein attenuates rotenone-induced SH-SY5Y cell apoptosis through binding to SUR1 and activating K-ATP channels.

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2023 – Confirming Silent Translocation through Nanopores with Simultaneous Single-Molecule Fluorescence and Single-Channel Electrical Recordings
Orbit mini Publication in Anal. Chem. (2023) Authors: Burden D.L., Meyer J.J., Michael R.D., Anderson S.C., Burden H.M., Pena S.M., Leong-Fern K.J., Ye L.A.V., Meyer E.C, Keranen-Burden L.M.

Most of what is known concerning the luminal passage of materials through nanopores arises from electrical measurements. Whether nanopores are biological, solid-state, synthetic, hybrid, glass-capillary-based, or protein ion channels in cells and tissues, characteristic signatures embedded in the flow of ionic current are foundational to understanding functional behavior. In contrast, this work describes passage through a nanopore that occurs without producing an electrical signature. We refer to the phenomenon as “silent translocation.” By definition, silent translocations are invisible to the standard tools of electrophysiology and fundamentally require a simultaneous ancillary measurement technique for positive identification. As a result, this phenomenon has been largely unexplored in the literature. Here, we report on a derivative of Cyanine 5 (sCy5a) that passes through the α-hemolysin (αHL) nanopore silently. Simultaneously acquired single-molecule fluorescence and single-channel electrical recordings from bilayers formed over a closed microcavity demonstrate that translocation does indeed take place, albeit infrequently. We report observations of silent translocation as a function of time, dye concentration, and nanopore population in the bilayer. Lastly, measurement of the translocation rate as a function of applied potential permits estimation of an effective energy barrier for transport through the pore as well as the effective charge on the dye, all in the absence of an information-containing electrical signature.

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2023 – Molecular and Cellular Context Influences SCN8A Variant Function
SyncroPatch 768 (a predecessor of the SyncroPatch 384) Pre-Publication in BioRxiv (2023) Authors: Vanoye C.G., Abramova T.V., DeKeyser J-M., Ghabra N.F., Oudin M.J., Burge C.B., Helbig I., Thompson C.H., George Jr. A.L.

Pathogenic variants in SCN8A, which encodes the voltage-gated sodium (NaV) channel NaV1.6, are associated with neurodevelopmental disorders including epileptic encephalopathy. Previous approaches to determine SCN8A variant function may be confounded by the use of a neonatal-expressed alternatively spliced isoform of NaV1.6 (NaV1.6N), and engineered mutations to render the channel tetrodotoxin (TTX) resistant. In this study, we investigated the impact of SCN8A alternative splicing on variant function by comparing the functional attributes of 15 variants expressed in two developmentally regulated splice isoforms (NaV1.6N, NaV1.6A). We employed automated patch clamp recording to enhance throughput, and developed a novel neuronal cell line (ND7/LoNav) with low levels of endogenous NaV current to obviate the need for TTX-resistance mutations. Expression of NaV1.6N or NaV1.6A in ND7/LoNav cells generated NaV currents that differed significantly in voltage-dependence of activation and inactivation. TTX-resistant versions of both isoforms exhibited significant functional differences compared to the corresponding wild-type (WT) channels. We demonstrated that many of the 15 disease-associated variants studied exhibited isoform-dependent functional effects, and that many of the studied SCN8A variants exhibited functional properties that were not easily classified as either gain- or loss-of-function. Our work illustrates the value of considering molecular and cellular context when investigating SCN8A variants.

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2023 – Efficient and sustained optogenetic control of nervous and cardiac systems
SyncroPatch 384 and Optogenetic Stimulation Tool Pre-Publication in BioRxiv (2023) Authors: Zerche M., Hunniford V., Alekseev A., El May F., Vavakou A., Siegenthaler D., Hüser M.A., Kiehn S.M., Garrido-Charles A., Alvanos T., Witzke I., Trenholm S., Macé E., Kusch K., Bruegmann T., Wolf B.J., Mager T., Moser T.

Optogenetic control of cells is a key life sciences method and promises novel therapies. Here we report on ChReef, an improved variant of the channelrhodopsin ChRmine, enabling efficient (nano-Joule) and sustained optogenetic stimulation of excitable cells. ChReef offers minimal photocurrent desensitization, a unitary conductance of 80 fS and closing kinetics of 30 ms, which together enable reliable optogenetic control of cardiac and nervous systems at low light levels with good temporal fidelity. We demonstrate efficient and reliable red-light pacing and depolarization block of ChReef-expressing cardiomyocyte clusters. ChReef-expression in the optic nerve restores visual function in blind mice with light sources as weak as an iPad screen. ChReef enables stimulation of the auditory nerve at up to 50-100 Hz with good temporal precision and low pulse energy threshold (170 nJ) close that of electrical stimulation (50 nJ). Thus, ChReef outperforms ChRmine and bears great potential for life sciences and clinical application.

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2023 – Discovery of Novel 8-Hydroxyquinoline Derivatives with Potent In Vitro and In Vivo Antifungal Activity
SyncroPatch 384 Publication in J. Med.Chem (2023) Authors: Li L., Wu H., Wang J., Ji Z., Fang T., Lu H., Yan L., Shen F., Zhang D., Jiang Y., Ni T.

Fungal pathogens can cause life-threatening infections, yet current antifungals are inadequate at treating many of these, highlighting the importance of novel drug discovery. Here, we report hit compound L14, a novel 8-hydroxyquinoline derivative with potent and broad-spectrum antifungal activity. In vitro experiments exhibited that L14 had better activity and lower cytotoxicity than that of clioquinol and showed synergy in combination with fluconazole (FLC). In a Candida albicans-infected murine model, L14 at 2 mg/kg showed better in vivo efficacy than clioquinol at reducing fungal burden and extending the survival of C. albicans-infected mice. In addition, L14 alone or in combination with FLC had significant inhibitory activity against hypha and biofilm formation. Overall, our data indicated that 8-hydroxyquinoline derivative L14 has favorable pharmacokinetics and acceptable safety profiles and could be further investigated as a promising antifungal hit compound.

Application Note PDF
iPSC-CM – “FLEXcyte 96 recordings in comparison to in vivo and in vitro cardiac muscle contraction via MUSCLEMOTION data”
FLEXcyte 96 application note

In order to reduce cardiovascular safety liabilities of new therapeutic agents, there is an urgent need to integrate human-relevant platforms/approaches into drug development1. Optimizing baseline function of human induced pluripotent stem cell-derived cardiomyocytes (hiPSCCMs) is essential for their effective application in models of cardiac toxicity and disease2. Here, hiPSC-CMs were cultured on flexible substrates using the FLEXcyte 96 system. The promaturation environment enables observation of inotropic and chronotropic compound effects, which are typically hard to detect with 2D monolayers on overly stiff substrates3. For example, the beta-adrenergic agonist isoprenaline, or isoproterenol, is well known for its positive inotropic effects on the human heart, although common hiPSC-CM in vitro assays fail to display this physiological response by this compound.

Poster PDF
2023 – AtlaZ as a High-Throughput System for Advanced Functional Cell Analysis to Develop Immunotherapies
AtlaZ Poster
Poster PDF
2023 – Pharmacology of P2X3and P2X2/3receptors in cell lines and hiPSC-derived neurons: An automated patch clamp study
SyncroPatch 384, Patchliner, Port-a-Patch Poster
Poster PDF
2023 – Functional characterization of human GAT-1 using solid supported membrane
SURFE2R N1 and Patchliner Poster
Publication link
2023 – An integrated approach for early in vitro seizure prediction utilizing hiPSC neurons and human ion channel assays
Patchliner Publication in Toxicological Sciences (2023) Authors: Rockley K., Roberts R., Jennings H., Jones K., Davis M., Levesque P., Morton M.

Seizure liability remains a significant cause of attrition throughout drug development. Advances in stem cell biology coupled with an increased understanding of the role of ion channels in seizure offer an opportunity for a new paradigm in screening. We assessed the activity of 15 pro-seizurogenic compounds (7 CNS active therapies, 4 GABA receptor antagonists, and 4 other reported seizurogenic compounds) using automated electrophysiology against a panel of 14 ion channels (Nav1.1, Nav1.2, Nav1.6, Kv7.2/7.3, Kv7.3/7.5, Kv1.1, Kv4.2, KCa4.1, Kv2.1, Kv3.1, KCa1.1, GABA α1β2γ2, nicotinic α4β2, NMDA 1/2A). These were selected based on linkage to seizure in genetic/pharmacological studies. Fourteen compounds demonstrated at least one “hit” against the seizure panel and 11 compounds inhibited 2 or more ion channels. Next, we assessed the impact of the 15 compounds on electrical signaling using human-induced pluripotent stem cell neurons in microelectrode array (MEA). The CNS active therapies (amoxapine, bupropion, chlorpromazine, clozapine, diphenhydramine, paroxetine, quetiapine) all caused characteristic changes to electrical activity in key parameters indicative of seizure such as network burst frequency and duration. The GABA antagonist picrotoxin increased all parameters, but the antibiotics amoxicillin and enoxacin only showed minimal changes. Acetaminophen, included as a negative control, caused no changes in any of the parameters assessed. Overall, pro-seizurogenic compounds showed a distinct fingerprint in the ion channel/MEA panel. These studies highlight the potential utility of an integrated in vitro approach for early seizure prediction to provide mechanistic information and to support optimal drug design in early development, saving time and resources.

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2023 – Structural flexibility of apolipoprotein E-derived arginine-rich peptides improves their cell penetration capability
Orbit 16 TC Publication in Scientific Reports (2023) Authors: Takechi-Haraya Y., Ohgita T., Usui A., Nishitsuji K., Uchimura K., Abe Y., Kawano R., Konaklieva M.I., Reimund M., Remaley A.T., Sato Y., Izutsu K., Saito H.

Amphipathic arginine-rich peptide, A2-17, exhibits moderate perturbation of lipid membranes and the highest cell penetration among its structural isomers. We investigated the direct cell-membrane penetration mechanism of the A2-17 peptide while focusing on structural flexibility. We designed conformationally constrained versions of A2-17, stapled (StpA2-17) and stitched (StchA2-17), whose α-helical conformations were stabilized by chemical crosslinking. Circular dichroism confirmed that StpA2-17 and StchA2-17 had higher α-helix content than A2-17 in aqueous solution. Upon liposome binding, only A2-17 exhibited a coil-to-helix transition. Confocal microscopy revealed that A2-17 had higher cell penetration efficiency than StpA2-17, whereas StchA2-17 remained on the cell membrane without cell penetration. Although the tryptophan fluorescence analysis suggested that A2-17 and its analogs had similar membrane-insertion positions between the interface and hydrophobic core, StchA2-17 exhibited a higher membrane affinity than A2-17 or StpA2-17. Atomic force microscopy demonstrated that A2-17 reduced the mechanical rigidity of liposomes to a greater extent than StpA2-17 and StchA2-17. Finally, electrophysiological analysis showed that A2-17 induced a higher charge influx through transient pores in a planer lipid bilayer than StpA2-17 and StchA2-17. These findings indicate that structural flexibility, which enables diverse conformations of A2-17, leads to a membrane perturbation mode that contributes to cell membrane penetration.

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2023 – Atrial fibrillation-associated electrical remodelling in human induced pluripotent stem cell-derived atrial cardiomyocytes: a novel pathway for antiarrhythmic therapy development
SyncroPatch Publication in Cardiovascular Research (2023) Authors: Seibertz F., Rubio T., Springer R., Popp F., Ritter M., Liutkute A., Bartelt L., Stelzer L., Haghighi F., Pietras J., Windel H., Díaz i Pedrosa N., Rapedius M., Doering Y:, Solano R., Hindmarsh R., Shi R., Tiburcy M., Bruegmann T., Kutschka I., Streckfuss-Bömeke K., Kensah G., Cyganek L., Zimmermann W., Voigt N.
Aims
Atrial fibrillation (AF) is associated with tachycardia-induced cellular electrophysiology alterations which promote AF chronification and treatment resistance. Development of novel antiarrhythmic therapies is hampered by the absence of scalable experimental human models that reflect AF-associated electrical remodelling. Therefore, we aimed to assess if AF-associated remodelling of cellular electrophysiology can be simulated in human atrial-like cardiomyocytes derived from induced pluripotent stem cells in the presence of retinoic acid (iPSC-aCM), and atrial-engineered human myocardium (aEHM) under short term (24 h) and chronic (7 days) tachypacing (TP).
Methods and results
First, 24-h electrical pacing at 3 Hz was used to investigate whether AF-associated remodelling in iPSC-aCM and aEHM would ensue. Compared to controls (24 h, 1 Hz pacing) TP-stimulated iPSC-aCM presented classical hallmarks of AF-associated remodelling: (i) decreased L-type Ca2+ current (ICa,L) and (ii) impaired activation of acetylcholine-activated inward-rectifier K+ current (IK,ACh). This resulted in action potential shortening and an absent response to the M-receptor agonist carbachol in both iPSC-aCM and aEHM subjected to TP. Accordingly, mRNA expression of the channel-subunit Kir3.4 was reduced. Selective IK,ACh blockade with tertiapin reduced basal inward-rectifier K+ current only in iPSC-aCM subjected to TP, thereby unmasking an agonist-independent constitutively active IK,ACh. To allow for long-term TP, we developed iPSC-aCM and aEHM expressing the light-gated ion-channel f-Chrimson. The same hallmarks of AF-associated remodelling were observed after optical-TP. In addition, continuous TP (7 days) led to (i) increased amplitude of inward-rectifier K+ current (IK1), (ii) hyperpolarization of the resting membrane potential, (iii) increased action potential-amplitude and upstroke velocity as well as (iv) reversibly impaired contractile function in aEHM.
Conclusions
Classical hallmarks of AF-associated remodelling were mimicked through TP of iPSC-aCM and aEHM. The use of the ultrafast f-Chrimson depolarizing ion channel allowed us to model the time-dependence of AF-associated remodelling in vitro for the first time. The observation of electrical remodelling with associated reversible contractile dysfunction offers a novel platform for human-centric discovery of antiarrhythmic therapies.
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2023 – A new look at Hsp70 activity in phosphatidylserine-enriched membranes: chaperone-induced quasi-interdigitated lipid phase.
Vesicle Prep Pro Publication in Scientific Reports (2023) Authors: Tagaeva R., Efmova S., Ischenko A., Zhakhov A., Shevtsov M., Ostroumova O.

70 kDa heat shock protein Hsp70 (also termed HSP70A1A) is the major stress-inducible member of the HSP70 chaperone family, which is present on the plasma membranes of various tumor cells, but not on the membranes of the corresponding normal cells. The exact mechanisms of Hsp70 anchoring in the membrane and its membrane-related functions are still under debate, since the protein does not contain consensus signal sequence responsible for translocation from the cytosol to the lipid bilayer. The present study was focused on the analysis of the interaction of recombinant human Hsp70 with the model phospholipid membranes. We have confirmed that Hsp70 has strong specificity toward membranes composed of negatively charged phosphatidylserine (PS), compared to neutral phosphatidylcholine membranes. Using differential scanning calorimetry, we have shown for the first time that Hsp70 affects the thermotropic behavior of saturated PS and leads to the interdigitation that controls membrane thickness and rigidity. Hsp70-PS interaction depended on the lipid phase state; the protein stabilized ordered domains enriched with high-melting PS, increasing their area, probably due to formation of quasi-interdigitated phase. Moreover, the ability of Hsp70 to form ion-permeable pores in PS membranes may also be determined by the bilayer thickness. These observations contribute to a better understanding of Hsp70-PS interaction and biological functions of membrane-bound Hsp70 in cancer cells.

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2023 – Biophysical characterization of chloride intracellular channel 6 (CLIC6)
SyncroPatch 384 Publication in JBC (2023) Authors: Loyo-Celis V., Patel D., Sanghvi S., Kaur K., Ponnalagu D., Zheng Y., Bindra S., Bhachu HR., Deschenes I., Gururaja Rao S., Singh H.

Chloride intracellular channels (CLICs) are a family of proteins that exist in soluble and transmembrane forms. The newest discovered member of the family CLIC6 is implicated in breast, ovarian, lung gastric, and pancreatic cancers and is also known to interact with dopamine-(D(2)-like) receptors. The soluble structure of the channel has been resolved, but the exact physiological role of CLIC6, biophysical characterization, and the membrane structure remain unknown. Here, we aimed to characterize the biophysical properties of this channel using a patch-clamp approach. To determine the biophysical properties of CLIC6, we expressed CLIC6 in HEK-293 cells. On ectopic expression, CLIC6 localizes to the plasma membrane of HEK-293 cells. We established the biophysical properties of CLIC6 by using electrophysiological approaches. Using various anions and potassium (K+) solutions, we determined that CLIC6 is more permeable to chloride-(Cl) as compared to bromide-(Br), fluoride-(F), and K+ ions. In the whole-cell configuration, the CLIC6 currents were inhibited after the addition of 10 μM of IAA-94 (CLIC-specific blocker). CLIC6 was also found to be regulated by pH and redox potential. We demonstrate that the histidine residue at 648 (H648) in the C terminus and cysteine residue in the N terminus (C487) are directly involved in the pH-induced conformational change and redox regulation of CLIC6, respectively. Using qRT-PCR, we identified that CLIC6 is most abundant in the lung and brain, and we recorded the CLIC6 current in mouse lung epithelial cells. Overall, we have determined the biophysical properties of CLIC6 and established it as a Cl channel.

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2023 – Gating Of the Channel Pore of Ionotropic Glutamate Receptors with Bacterial Substrate Binding Proteins: Functional Coupling of the Ectoine Binding Protein Ehub to Glur0
Port-a-Patch Publication in Journal of Biotechnology and Biomedicine. (2023) Authors: Bernhard M., Laube B

Mammalian neuronal tetrameric ionotropic glutamate receptors (iGluRs) are thought to have originally arisen from the fusion of a bacterial substrate binding protein (SBP) with an inverted potassium channel. This hypothesis is based on structural and sequential similarities between the ligand binding and channel domains of iGluR subunits with SBPs and potassium channels. Ligand binding occurs at the interface between two lobed domains in both ligand binding domains (LBDs) and leads to closure of the shell-like structure, which is considered to be a key element in the transition from ligand recognition to ion channel gating in iGluRs. Here we report the functional coupling of the ectoine-binding protein EhuB to the channel pore of the GluR0 receptor. Fusion of an unmodified EhuB-binding protein to the transmembrane domain of GluR0 did not result in activation of the channel pore. Only by stabilizing the inserted EhuB-binding domain with a dimerization interface the resulting chimera was activated by ectoine, resembling the activation properties of other iGluRs. These results demonstrate the functional compatibility of SBPs to the gate of the channel pore of an iGluR and highlight the role of LBD dimerization in the functional evolution of iGluRs. Based on the high specificity and affinity of SBPs for an incredible variety of substrates, our results demonstrate the competence of SBP/ion channel chimeras for the development of new Biosensors for specific recognition of analytes by functionally linking a bacterial binding protein to the channel pore of an iGluR.

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2023 – SARS-CoV-2 spike protein S1 activates Cx43 hemichannels and disturbs intracellular Ca2+ dynamics
Patchliner Publication in Biological Research (2023) Authors: Prieto-Villalobos J., Lucero C.M., Rovegno M., Gómez G.I., Retamal M.A. Orellana J.A.

Background

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the ongoing coronavirus disease 2019 (COVID-19). An aspect of high uncertainty is whether the SARS-CoV-2 per se or the systemic inflammation induced by viral infection directly affects cellular function and survival in different tissues. It has been postulated that tissue dysfunction and damage observed in COVID-19 patients may rely on the direct effects of SARS-CoV-2 viral proteins. Previous evidence indicates that the human immunodeficiency virus and its envelope protein gp120 increase the activity of connexin 43 (Cx43) hemichannels with negative repercussions for cellular function and survival. Here, we evaluated whether the spike protein S1 of SARS-CoV-2 could impact the activity of Cx43 hemichannels.

Results

We found that spike S1 time and dose-dependently increased the activity of Cx43 hemichannels in HeLa-Cx43 cells, as measured by dye uptake experiments. These responses were potentiated when the angiotensin-converting enzyme 2 (ACE2) was expressed in HeLa-Cx43 cells. Patch clamp experiments revealed that spike S1 increased unitary current events with conductances compatible with Cx43 hemichannels. In addition, Cx43 hemichannel opening evoked by spike S1 triggered the release of ATP and increased the [Ca2+]i dynamics elicited by ATP.

Conclusions

We hypothesize that Cx43 hemichannels could represent potential pharmacological targets for developing therapies to counteract SARS-CoV-2 infection and their long-term consequences.

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2023 – Analysis of the effect of the scorpion toxin AaH-II on action potential generation in the axon initial segment
SyncroPatch 384 Pre-Publication in BioRxiv (2023) Authors: Abbas F., Blömer L.A., Millet H., Montnach J., De Waard M., Canepari M.

The toxin AaH-II, from the scorpion Androctonus australis Hector venom, is a 64 amino acid peptide that targets voltage-gated Na+ channels (VGNCs) and slows their inactivation. While at macroscopic cellular level AaH-II prolongs the action potential (AP), a functional analysis of the effect of the toxin in the axon initial segment (AIS), where VGNCs are highly expressed, was never performed so far. Here, we report an original analysis of the effect of AaH-II on the AP generation in the AIS of neocortical layer-5 pyramidal neurons from mouse brain slices. After determining that AaH-II does not discriminate between Nav1.2 and Nav1.6, i.e. between the two VGNC isoforms expressed in this neuron, we established that 7 nM was the smallest toxin concentration producing a minimal detectable deformation of the somatic AP after local delivery of the toxin. Using membrane potential imaging, we found that, at this minimal concentration, AaH-II substantially widened the AP in the AIS. Using ultrafast Na+ imaging, we found that local application of 7 nM AaH-II caused a large increase in the slower component of the Na+ influx in the AIS. Finally, using ultrafast Ca2+ imaging, we observed that 7 nM AaH-II produces a spurious slow Ca2+ influx via Ca2+-permeable VGNCs. Molecules targeting VGNCs, including peptides, are proposed as potential therapeutic tools. Thus, the present analysis in the AIS can be considered a general proof-of-principle on how high-resolution imaging techniques can disclose drug effects that cannot be observed when tested at the macroscopic level.

Case study PDF
32-well mode – “Advancing cardiac research: 32-wells at a time”
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2023 – The Gárdos Channel and Piezo1 Revisited: Comparison between Reticulocytes and Mature Red Blood Cells
Patchliner and SyncroPatch 384 Pre-Print Publication in Preprints (2023) Authors: Petkova-Kirova P., Murciano N., Jansen J., Simionato G., Iacono G., Rotordam M.G., John T., Qiao M., Hertz L., Hoogendijk A.J., Becker N., Wagner C., von Lindern M., Egee S., Van den Akker E., Kaestner L.

(1) Background: The Gárdos channel (KCNN4) and Piezo1 are the best-known ion channels in the red blood cell (RBC) membrane. Nevertheless, the quantitative electrophysiological behavior of RBCs and its heterogeneity are still not completely understood.

(2) Methods: Here we use state-of-the-art biochemical methods to probe for the abundance of the channels in RBCs. Furthermore, we utilize automated patch-clamp, based on planar chips, to compare the activity of the two channels in reticulocytes and mature RBCs. Besides this characterization, we performed membrane potential measurements to demonstrate the effect of channel activity and interplay on the RBC properties.

(3) Results: Both, Gárdos channel and Piezo1, albeit their average copy number of activatable channels per cell is in the single digit range, can be detected by transcriptome analysis of reticulocytes. Proteomics analysis of RBCs could only detect Piezo1 but not the Gárdos channel. Furthermore, they can be reliably measured in the whole-cell configuration of the patch-clamp method. While for the Gárdos channel the activity is higher in reticulocytes compared to mature RBCs, for Piezo1 the tendency is the opposite. While the interplay between Piezo1 and Gárdos channel cannot be followed using the patch-clamp measurements, it could be proved based on membrane potential measurements in populations of intact RBCs.

(4) Conclusions: We discuss the Gárdos channel and Piezo1 abundance, interdependencies and interactions in the context of their proposed physiological and pathophysiological functions, which are the passing of small constrictions, e.g., in the spleen, and their active participation in blood clot formation and thrombosis.

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2023 – Structure-based development and preclinical evaluation of the SARS-CoV-2 3C-like protease inhibitor simnotrelvir
Patchliner Publication in Nature Communications (2023) Authors: Jiang X., Su H., Shang W., Zhou F., Zhang Y., Zhao W, Zhang Q., Xie H., Jiang L., Nie T., Yang F., Xiong M., Huang X., Li M., Chen P., Peng S., Xiao G., Jiang H., Tang R., Zhang L., Shen J., Xu Y.

The persistent pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants accentuates the great demand for developing effective therapeutic agents. Here, we report the development of an orally bioavailable SARS-CoV-2 3C-like protease (3CLpro) inhibitor, namely simnotrelvir, and its preclinical evaluation, which lay the foundation for clinical trials studies as well as the conditional approval of simnotrelvir in combination with ritonavir for the treatment of COVID-19. The structure-based optimization of boceprevir, an approved HCV protease inhibitor, leads to identification of simnotrelvir that covalently inhibits SARS-CoV-2 3CLpro with an enthalpy-driven thermodynamic binding signature. Multiple enzymatic assays reveal that simnotrelvir is a potent pan-CoV 3CLpro inhibitor but has high selectivity. It effectively blocks replications of SARS-CoV-2 variants in cell-based assays and exhibits good pharmacokinetic and safety profiles in male and female rats and monkeys, leading to robust oral efficacy in a male mouse model of SARS-CoV-2 Delta infection in which it not only significantly reduces lung viral loads but also eliminates the virus from brains. The discovery of simnotrelvir thereby highlights the utility of structure-based development of marked protease inhibitors for providing a small molecule therapeutic effectively combatting human coronaviruses.

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2023 – Unravelling Novel SCN5A Mutations Linked to Brugada Syndrome: Functional, Structural, and Genetic Insights
Patchliner Publication in Int. J. Mol. Sci. (2023) Authors: Frosio A., Micaglio E., Polsinelli I., Calamaio S., Melgari D., Prevostini R., Ghiroldi A., Binda A., Carrera P., Villa M., Mastrocinque F., Presi S., Salerno R., Boccellino A., Anastasia L., Ciconte G.,Ricagno S., Pappone C., Rivolta I.

Brugada Syndrome (BrS) is a rare inherited cardiac arrhythmia causing potentially fatal ventricular tachycardia or fibrillation, mainly occurring during rest or sleep in young individuals without heart structural issues. It increases the risk of sudden cardiac death, and its characteristic feature is an abnormal ST segment elevation on the ECG. While BrS has diverse genetic origins, a subset of cases can be conducted to mutations in the SCN5A gene, which encodes for the Nav1.5 sodium channel. Our study focused on three novel SCN5A mutations (p.A344S, p.N347K, and p.D349N) found in unrelated BrS families. Using patch clamp experiments, we found that these mutations disrupted sodium currents: p.A344S reduced current density, while p.N347K and p.D349N completely abolished it, leading to altered voltage dependence and inactivation kinetics when co-expressed with normal channels. We also explored the effects of mexiletine treatment, which can modulate ion channel function. Interestingly, the p.N347K and p.D349N mutations responded well to the treatment, rescuing the current density, while p.A344S showed a limited response. Structural analysis revealed these mutations were positioned in key regions of the channel, impacting its stability and function. This research deepens our understanding of BrS by uncovering the complex relationship between genetic mutations, ion channel behavior, and potential therapeutic interventions.

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2023 – Evidence of Cytolysin A nanopore incorporation in mammalian cells assessed by a graphical user interface
Patchliner Publication in Nanoscale (2023) Authors: Lucas F.L.R., Finol-Urdaneta R.K., Van Thillo T., McArthur J.R., van der Heide N.J., Maglia G., Dedecker P., Strauss O., Wloka C.

Technologies capable of assessing cellular metabolites with high precision and temporal resolution are currently limited. Recent developments in the field of nanopore sensor sallow the non-stochastic quantification of metabolites, where a nanopore is acting as an electrical transducer for selective substrate binding proteins (SBPs). Here we show that incorporation of the pore-forming toxin Cytolysin A (ClyA) into the plasma membrane of Chinese hamster ovary cells (CHO-K1) results in the appearance of single-channel conductance amenable to multiplexed automated patch-clamp (APC) electrophysiology. In CHO-K1 cells, SBPs modify the ionic current flowing though ClyA nanopores, thus demonstrating its potential for metabolite sensing of living cells. Moreover, we developed a graphical user interface for the analysis of the complex signals resulting from multiplexed APC recordings. This system lays the foundation to bridge the gap between recent advances in the nanopore field (e.g., proteomic and transcriptomic) and potential cellular applications.

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2023 – Formation of Giant Unilamellar Vesicles Assisted byFluorinated Nanoparticles
Vesicle Prep Pro Publication in Advanced Science (2023) Authors: Waeterschoot J., Gosselé W., Zeinabad H.A., Lammertyn J., Koos E., Casadevall i Solvas X.

In the quest to produce artificial cells, one key challenge that remains to besolved is the recreation of a complex cellular membrane. Among the existingmodels, giant unilamellar vesicles (GUVs) are particularly interesting due totheir intrinsic compartmentalisation ability and their resemblance in size andshape to eukaryotic cells. Many techniques have been developed to produceGUVs all having inherent advantages and disadvantages. Here, the authorsshow that fluorinated silica nanoparticles (FNPs) used to form Pickeringemulsions in a fluorinated oil can destabilise lipid nanosystems to templatethe formation of GUVs. This technique enables GUV production across abroad spectrum of buffer conditions, while preventing the leakage of theencapsulated components into the oil phase. Furthermore, a simplecentrifugation process is sufficient for the release of the emulsion-trappedGUVs, bypassing the need to use emulsion-destabilising chemicals. Withfluorescent FNPs and transmission electron microscopy, the authors confirmthat FNPs are efficiently removed, producing contaminant-free GUVs. Furtherexperiments assessing the lateral diffusion of lipids and unilamellarity of theGUVs demonstrate that they are comparable to GUVs produced viaelectroformation. Finally, the ability of incorporating transmembrane proteinsis demonstrated, highlighting the potential of this method for the productionof GUVs for artificial cell applications.

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2023 – Encoding extracellular modification of artificial cell membranes using engineered self-translocating proteins
Orbit mini Pre-Print Publication in BioRxiv (2023) Authors: Harjung A., Fracassi A., Devaraj N.

A common method of generating artificial cells is to encapsulate protein expression systems within lipid vesicles. However, to communicate with the external environment, protein translocation across lipid membranes must take place. In living cells, protein transport across membranes is achieved with the aid of complex translocase systems which are difficult to reconstitute into artificial cells. Thus, there is need for simple mechanisms by which proteins can be encoded and expressed inside synthetic compartments yet still be externally displayed. Here we present a genetically encodable membrane functionalization system based on mutants of pore-forming proteins. We show that the membrane translocating loop of α-hemolysin can be engineered to translocate functional peptides up to 52 amino acids across lipid membranes. Engineered hemolysins can be used for genetically programming artificial cells to display interacting peptide pairs, enabling their assembly into artificial tissue-like structures capable of signal transduction.

Application Note PDF
CAR T, Cancer – “CAR T cell-mediated killing of cancer cells”
AtlaZ application note

The US Food and Drug Administration has approved a number of chimeric antigen receptor (CAR) T-cell therapies. Due to the nature of CAR T cells as “living drugs”, they display a unique toxicity profile. As CAR T-cell therapy is extending towards multiple diseases and being broadly employed in hematology and oncology, being able to reliably predict treatment efficacy and a quantification of responses are of high relevance. Furthermore, for continued breakthroughs, novel CAR designs are needed. This includes different antigenbinding domains such as antigen-ligand binding partners and variable lymphocyte receptors (1). Now, after amazing advances for treating blook cancers, CAR T cell therapy is showing promise for solid tumors.

In general, identifying T cells that kill cancer cells in vivo is critical to the development of successful cell therapies. The label-free AtlaZ immune cell killing assay can be used to measure rate of killing at Effector:Target (E:T) ratios to predict in vivo activity. In order to gain a deeper understanding of cancer cells, real-time and continuous monitoring is necessary to access kinetic and phenotypic information. Such monitoring captures also unique toxicity profiles of CAR T cells.

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2023 – A Small Multidrug Resistance Transporter in Pseudomonas aeruginosa Confers Substrate-Specific Resistance or Susceptibility
SURFE2R N1 Pre-Print Publication in BioRxiv (2023) Authors: Wegrzynowicz A.K., Heelan W.J., Demas S.P., McLean M.S., Peters J.M., Henzler-Wildman K.A.

Voltage-sensing domains control the activation of voltage-gated ion channels, with a few exceptions. One such exception is the sperm-specific Na+/H+ exchanger SLC9C1, which is the only known transporter to be regulated by voltage-sensing domains. After hyperpolarization of sperm flagella, SLC9C1 becomes active, causing pH alkalinization and CatSper Ca2+ channel activation, which drives chemotaxis. SLC9C1 activation is further regulated by cAMP, which is produced by soluble adenyl cyclase (sAC). SLC9C1 is therefore an essential component of the pH–sAC–cAMP signalling pathway in metazoa, required for sperm motility and fertilization. Despite its importance, the molecular basis of SLC9C1 voltage activation is unclear. Here we report cryo-electron microscopy (cryo-EM) structures of sea urchin SLC9C1 in detergent and nanodiscs. We show that the voltage-sensing domains are positioned in an unusual configuration, sandwiching each side of the SLC9C1 homodimer. The S4 segment is very long, 90 Å in length, and connects the voltage-sensing domains to the cytoplasmic cyclic-nucleotide-binding domains. The S4 segment is in the up configuration—the inactive state of SLC9C1. Consistently, although a negatively charged cavity is accessible for Na+ to bind to the ion-transporting domains of SLC9C1, an intracellular helix connected to S4 restricts their movement. On the basis of the differences in the cryo-EM structure of SLC9C1 in the presence of cAMP, we propose that, upon hyperpolarization, the S4 segment moves down, removing this constriction and enabling Na+/H+ exchange.

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2023 – Target-based discovery of a broad spectrum flukicide
Patchliner Publication Pre-print in bioRxiv (2023) Authors: Sprague D.J., Park S-K., Gramberg S., Bauer L., Rohr C.M., Chulkov E.G., Smith E., Scampavia L., Spicer T.P., Haeberlein S., Marchant J.S.

Diseases caused by parasitic flatworms impart a considerable healthcare burden worldwide. Many of these diseases – for example, the parasitic blood fluke infection, schistosomiasis – are treated with the drug praziquantel (PZQ). However, PZQ is ineffective against disease caused by liver flukes from the genus Fasciola. This is due to a single amino acid change within the target of PZQ, a transient receptor potential ion channel (TRPMPZQ), in Fasciola species. Here we identify benzamidoquinazolinone analogs that are active against Fasciola TRPMPZQ. Structure-activity studies define an optimized ligand (BZQ) that caused protracted paralysis and damage to the protective tegument of these liver flukes. BZQ also retained activity against Schistosoma mansoni comparable to PZQ and was active against TRPMPZQ orthologs in all profiled species of parasitic fluke. This broad spectrum activity was manifest as BZQ adopts a pose within the binding pocket of TRPMPZQ dependent on a ubiquitously conserved residue. BZQ therefore acts as a universal activator of trematode TRPMPZQ and a first-in-class, broad spectrum flukicide.

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2023 – Enhancing the performance of a mutant pyrrolysyl-tRNA synthetase to create a highly versatile eukaryotic cell-free protein synthesis tool
Orbit 16 (a predecessor model of the Orbit 16 TC) Publication in Scientific Reports (2023) Authors: Schloßhauer J., Zemella A., Dondapati S., Thoring L., Meyer M., Stefan Kubick S.

Modification of proteins with a broad range of chemical functionalities enables the investigation of protein structure and activity by manipulating polypeptides at single amino acid resolution. Indeed, various functional groups including bulky non-canonical amino acids like strained cyclooctenes could be introduced by the unique features of the binding pocket of the double mutant pyrrolysyl-tRNA synthetase (Y306A, Y384F), but the instable nature of the enzyme limits its application in vivo. Here, we constructed a cell-free protein production system, which increased the overall enzyme stability by combining different reaction compartments. Moreover, a co-expression approach in a one-pot reaction allowed straightforward site-specific fluorescent labeling of the functional complex membrane protein cystic fibrosis transmembrane conductance regulator. Our work provides a versatile platform for introducing various non-canonical amino acids into difficult-to-express proteins for structural and fluorescence based investigation of proteins activity.

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2023 – Pharmacological characterization of SAGE-718, a novel positive allosteric modulator of N-methyl-d-aspartate receptors
SyncroPatch 384 Publication in British Journal of Pharmacology (2023) Authors: Beckley J., Aman T., Ackley M., Kazdoba T., Lewis M., Smith A., Farley B., Dai J., Deats W., Hoffmann E., Robichaud A., Doherty J., Quirk M.

Background and Purpose

Select neuroactive steroids tune neural activity by modulating excitatory and inhibitory neurotransmission, including the endogenous cholesterol metabolite 24(S)-hydroxycholesterol (24(S)-HC), which is an N-methyl-d-aspartate (NMDA) receptor positive allosteric modulator (PAM). NMDA receptor PAMs are potentially an effective pharmacotherapeutic strategy to treat conditions associated with NMDA receptor hypofunction.

Experimental Approach

Using in vitro and in vivo electrophysiological recording experiments and behavioural approaches, we evaluated the effect of SAGE-718, a novel neuroactive steroid NMDA receptor PAM currently in clinical development for the treatment of cognitive impairment, on NMDA receptor function and endpoints that are altered by NMDA receptor hypoactivity and assessed its safety profile.

Key Results

SAGE-718 potentiated GluN1/GluN2A-D NMDA receptors with equipotency and increased NMDA receptor excitatory postsynaptic potential (EPSP) amplitude without affecting decay kinetics in striatal medium spiny neurons. SAGE-718 increased the rate of unblock of the NMDA receptor open channel blocker ketamine on GluN1/GluN2A in vitro and accelerated the rate of return on the ketamine-evoked increase in gamma frequency band power, as measured with electroencephalogram (EEG), suggesting that PAM activity is driven by increased channel open probability. SAGE-718 ameliorated deficits due to NMDA receptor hypofunction, including social deficits induced by subchronic administration of phencyclidine, and behavioural and electrophysiological deficits from cholesterol and 24(S)-HC depletion caused by 7-dehydrocholesterol reductase inhibition. Finally, SAGE-718 did not produce epileptiform activity in a seizure model or neurodegeneration following chronic dosing.

Conclusions and Implications

These findings provide strong evidence that SAGE-718 is a neuroactive steroid NMDA receptor PAM with a mechanism that is well suited as a treatment for conditions associated with NMDA receptor hypofunction.

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2023 – PI-(3,5)P2-mediated oligomerization of the endosomal sodium/proton exchanger NHE9
SURFE2R N1 Pre-Print Publication in BioRxiv (2023) Authors: Kokane S., Meier P.F., Gulati A., Matsuoka R., Pipatpolkai T., Delemotte L., Drew D.

Na+/H+ exchangers (NHE) are found in all cells to regulate intracellular pH, sodium levels and cell volume. The NHE isoform 9 (SLC9A9) fine-tunes endosomal pH, and its activity is linked to glioblastoma, epilepsy, autism spectrum and attention-deficit-hyperactivity disorders. Here, we report cryo-EM structures of horse NHE9 and a cysteine-variant at 3.6 and 3.1 Å resolution, respectively. We show how lysine residues, from a previously unresolved TM2-TM3 β-hairpin loop domain, are positioned above the dimerization interface and interact with the endosomal-specific PI-(3,5)P2 lipid, together with residues located on dimer domain helices. Thermal-shift assays, solid-state membrane (SSM) electrophysiology and MD simulations, corroborates that NHE9 can specifically bind PI-(3,5)P2, and that its addition stabilizes the homodimer and enhances NHE9 activity. We have further determined the cryo-EM structure of E. coli NhaA, confirming the expected coordination of cardiolipin at the dimerization interface, solidifying the concept that Na+/H+ exchanger dimerization and transporter activity can be regulated by specific lipids. Taken together, we propose that the activity of NHE9 is regulated by the PI-(3,5)P2 lipid upon reaching endosomes, which we refer to as an lipid-activation-upon-arrival model.

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2023 – ParSE-seq: A Calibrated Multiplexed Assay to Facilitate the Clinical Classification of Putative Splice-altering Variants
SyncroPatch 384 Pre-Publication in MedRxiv (2023) Authors: O’Neill M.J., Yang T., Laudeman J., Calandranis M., Solus J., Roden D.M., Glazer A.M.

Background:

Interpreting the clinical significance of putative splice-altering variants outside 2-base pair canonical splice sites remains difficult without functional studies.

Methods:

We developed Parallel Splice Effect Sequencing (ParSE-seq), a multiplexed minigene-based assay, to test variant effects on RNA splicing quantified by high-throughput sequencing. We studied variants in SCN5A, an arrhythmia-associated gene which encodes the major cardiac voltage-gated sodium channel. We used the computational tool SpliceAI to prioritize exonic and intronic candidate splice variants, and ClinVar to select benign and pathogenic control variants. We generated a pool of 284 barcoded minigene plasmids, transfected them into Human Embryonic Kidney (HEK293) cells and induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), sequenced the resulting pools of splicing products, and calibrated the assay to the American College of Medical Genetics and Genomics scheme. Variants were interpreted using the calibrated functional data, and experimental data were compared to SpliceAI predictions. We further studied some splice-altering missense variants by cDNA-based automated patch clamping (APC) in HEK cells and assessed splicing and sodium channel function in CRISPR-edited iPSC-CMs.

Results:

ParSE-seq revealed the splicing effect of 224 SCN5A variants in iPSC-CMs and 244 variants in HEK293 cells. The scores between the cell types were highly correlated (R2=0.84). In iPSCs, the assay had concordant scores for 21/22 benign/likely benign and 24/25 pathogenic/likely pathogenic control variants from ClinVar. 43/112 exonic variants and 35/70 intronic variants with determinate scores disrupted splicing. 11 of 42 variants of uncertain significance were reclassified, and 29 of 34 variants with conflicting interpretations were reclassified using the functional data. SpliceAI computational predictions correlated well with experimental data (AUC = 0.96). We identified 20 unique SCN5A missense variants that disrupted splicing, and 2 clinically observed splice-altering missense variants of uncertain significance had normal function when tested with the cDNA-based APC assay. A splice-altering intronic variant detected by ParSE-seq, c.1891–5C>G, also disrupted splicing and sodium current when introduced into iPSC-CMs at the endogenous locus by CRISPR editing.

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2023 – Cell-Free Synthesis and Electrophysiological Analysis of Multipass Voltage-Gated Ion Channels Tethered in Microsomal Membranes
Orbit 16TC Publication in Advances in Biochemical Engineering/Biotechnology (2023) Authors: Pandey Y., Dondapati S., Wüstenhagen D., Kubick S.

Cell-free protein synthesis (CFPS) has emerged as a powerful tool for the rapid synthesis and analysis of various structurally and functionally distinct proteins. These include ‘difficult-to-express’ membrane proteins such as large multipass ion channel receptors. Owing to their membrane localization, eukaryotic CFPS supplemented with endoplasmic reticulum (ER)-derived microsomal vesicles has proven to be an efficient system for the synthesis of functional membrane proteins. Here we demonstrate the applicability of the eukaryotic cell-free systems based on lysates from the mammalian Chinese Hamster Ovary (CHO) and insect Spodoptera frugiperda (Sf21) cells. We demonstrate the efficiency of the systems in the de novo cell-free synthesis of the human cardiac ion channels: ether-a-go-go potassium channel (hERG) KV11.1 and the voltage-gated sodium channel hNaV1.5.

Read more in the publication here.

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2023 – Nicotine-mediated effects in neuronal and mouse models of synucleinopathy
Patchliner Publication in Front. Neurosci. (2023) Authors: Fares M.B., Alijevic O., Johne S., Overk C., Hashimoto M., Kondylis A., Adame A., Dulize R., Peric D., Nury C., Battey J., Guedj E., Sierro N., Mc Hugh D., Rockenstein E., Kim C. Rissman R.A., Hoeng J., Peitsch M.C., Masliah E., Mathis C.

Introduction: Alpha-synuclein (α-Syn) aggregation, transmission, and contribution to neurotoxicity represent central mechanisms underlying Parkinson’s disease. The plant alkaloid “nicotine” was reported to attenuate α-Syn aggregation in different models, but its precise mode of action remains unclear.

Methods: In this study, we investigated the effect of 2-week chronic nicotine treatment on α-Syn aggregation, neuroinflammation, neurodegeneration, and motor deficits in D-line α-Syn transgenic mice. We also established a novel humanized neuronal model of α-Syn aggregation and toxicity based on treatment of dopaminergic neurons derived from human induced pluripotent stem cells (iPSC) with α-Syn preformed fibrils (PFF) and applied this model to investigate the effects of nicotine and other compounds and their modes of action.

Results and discussion: Overall, our results showed that nicotine attenuated α-Syn-provoked neuropathology in both models. Moreover, when investigating the role of nicotinic acetylcholine receptor (nAChR) signaling in nicotine’s neuroprotective effects in iPSC-derived dopaminergic neurons, we observed that while α4-specific antagonists reduced the nicotine-induced calcium response, α4 agonists (e.g., AZD1446 and anatabine) mediated similar neuroprotective responses against α-Syn PFF-provoked neurodegeneration. Our results show that nicotine attenuates α-Syn-provoked neuropathology in vivo and in a humanized neuronal model of synucleinopathy and that activation of α4β2 nicotinic receptors might mediate these neuroprotective effects.

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2023 – Photophysics of Conjugated Oligoelectrolytes Relevant to Two-Photon Fluorescence-Lifetime Imaging Microscopy
Vesicle Prep Pro Publication in Advanced Functional Materials (2023) Authors: Zhu J-Y., Mikhailovsky A., Wei S.C.J., Moreland A., Limwongyut J., Guarrotxena N., Bazan G.C.

Conjugated oligoelectrolytes (COEs) comprise a class of cell-membrane intercalating molecules that serve as effective optical reporters. However, little is known about the photophysical properties of COEs in biological environments such as buffers, cell membranes, and intracellular organelles, which is critical to optimize performance. Herein, how COE self-assembly depends on the dielectric environment (polarity and ion content) is explored based on the representative molecule 6-ring phenylenevinylene (PV) conjugated oligoelectrolyte (COE-S6), and its optical properties within mammalian cells are subsequently studied. Two-photon fluorescence lifetime imaging microscopy (FLIM), confocal laser scanning microscopy, and optical properties in solutions are brought together to obtain information about the location, accumulation, and characteristics of the local surroundings. FLIM imaging lifetime phasor plots, decays, and fluorescence spectra on stained mammalian cells provide evidence of successful COE-S6 internalization via endocytosis. The fluorescence lifetime of COE-S6 is identical when in A549 mammalian cells and in giant unilamellar vesicle model membranes, thereby providing a correlation between living system and artificial constructs.

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2023 – Plasticity of the binding pocket in peptide transporters underpins promiscuous substrate recognition
SURFE2R N1 Publication in Cell Reports (2023) Authors: Kotov V. Killer M. Jungnickel K.E.J., Lei J., Finocchio G., Steinke J., Bartels K., Strauss J., Dupeux F., Humm A-S., Cornaciu I., Márquez J.A., Pardon E., Steyaert J., Löw C.

Proton-dependent oligopeptide transporters (POTs) are promiscuous transporters of the major facilitator superfamily that constitute the main route of entry for a wide range of dietary peptides and orally administrated peptidomimetic drugs. Given their clinical and pathophysiological relevance, several POT homologs have been studied extensively at the structural and molecular level. However, the molecular basis of recognition and transport of diverse peptide substrates has remained elusive. We present 14 X-ray structures of the bacterial POT DtpB in complex with chemically diverse di- and tripeptides, providing novel insights into the plasticity of the conserved central binding cavity. We analyzed binding affinities for more than 80 peptides and monitored uptake by a fluorescence-based transport assay. To probe whether all 8400 natural di- and tripeptides can bind to DtpB, we employed state-of-the-art molecular docking and machine learning and conclude that peptides with compact hydrophobic residues are the best DtpB binders.

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2023 – Epilepsy-associated SCN2A (NaV1.2) Variants Exhibit Diverse and Complex Functional Properties
SyncroPatch 768PE (a predecessor model of the SyncroPatch 384) Publication in J. Gen. Physiol. (2023) Authors: Thompson C.H., Potet F., Abramova T.V., DeKeyser J.M., Ghabra N.F., Vanoye C.G., Millichap J.,George, Jr. A.L.

Pathogenic variants in voltage-gated sodium (NaV) channel genes including SCN2A, encoding NaV1.2, are discovered frequently in neurodevelopmental disorders with or without epilepsy. SCN2A is also a high-confidence risk gene for autism spectrum disorder (ASD) and nonsyndromic intellectual disability (ID). Previous work to determine the functional consequences of SCN2A variants yielded a paradigm in which predominantly gain-of-function variants cause neonatal-onset epilepsy, whereas loss-of-function variants are associated with ASD and ID. However, this framework was derived from a limited number of studies conducted under heterogeneous experimental conditions, whereas most disease-associated SCN2A variants have not been functionally annotated. We determined the functional properties of SCN2A variants using automated patch-clamp recording to demonstrate the validity of this method and to examine whether a binary classification of variant dysfunction is evident in a larger cohort studied under uniform conditions. We studied 28 disease-associated variants and 4 common variants using two alternatively spliced isoforms of NaV1.2 expressed in HEK293T cells. Automated patch-clamp recording provided a valid high throughput method to ascertain detailed functional properties of NaV1.2 variants with concordant findings for variants that were previously studied using manual patch clamp. Many epilepsy-associated variants in our study exhibited complex patterns of gain- and loss-of-functions that are difficult to classify by a simple binary scheme. The higher throughput achievable with automated patch clamp enables study of variants with greater standardization of recording conditions, freedom from operator bias, and enhanced experimental rigor. This approach offers an enhanced ability to discern relationships between channel dysfunction and neurodevelopmental disorders.

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2023 – Transport of metformin metabolites by guanidinium exporters of the Small Multidrug Resistance family
SURFE2R N1 Pre-Print Publication in BioRxiv (2023) Authors: Lucero R.M., Demirer K., Yeh T.J., Stockbridge R.B.

Proteins from the Small Multidrug Resistance (SMR) family are frequently associated with horizontally transferred multidrug resistance gene arrays found in bacteria from wastewater and the human-adjacent biosphere. Recent studies suggest that a subset of SMR transporters might participate in metabolism of the common pharmaceutical metformin by bacterial consortia. Here, we show that both genomic and plasmid-associated transporters of the SMRGdx functional subtype export byproducts of microbial metformin metabolism, with particularly high export efficiency for guanylurea. We use solid supported membrane electrophysiology to evaluate the transport kinetics for guanylurea and native substrate guanidinium by four representative SMRGdx homologues. Using an internal reference to normalize independent electrophysiology experiments, we show that transport rates are comparable for genomic and plasmid-associated SMRGdx homologues, and using a proteoliposome-based transport assay, we show that 2 proton:1 substrate transport stoichiometry is maintained. Additional characterization of guanidinium and guanylurea export properties focuses on the structurally characterized homologue, Gdx-Clo, for which we examined the pH dependence and thermodynamics of substrate binding and solved an x-ray crystal structure with guanylurea bound. Together, these experiments contribute in two main ways. By providing the first detailed kinetic examination of the structurally characterized SMRGdx homologue Gdx-Clo, they provide a functional framework that will inform future mechanistic studies of this model transport protein. Second, this study casts light on a potential role for SMRGdx transporters in microbial handling of metformin and its microbial metabolic byproducts, providing insight into how native transport physiologies are co-opted to contend with new selective pressures.

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2023 – Discovery of novel triazoles containing benzyloxy phenyl isoxazole side chain with potent and broad-spectrum antifungal activity
SyncroPatch 384 Publication in Biomolecules (2023) Authors: Chi X., Xie F., Li L., Hao Y., Wu H., Li X., Xia G., Yan L., Zhang D., Jiang Y., Ni T.

As a continuation study, 29 novel triazoles containing benzyloxy phenyl isoxazole side chain were designed and synthesized based on our previous work. The majority of the compounds exhibited high potency in vitro antifungal activities against eight pathogenic fungi. The most active compounds 13, 20 and 27 displayed outstanding antifungal activity with MIC values ranging from <0.008 μg/mL to 1 μg/mL, and showed potent activity against six drug-resistant Candida auris isolates. Growth curve assays further confirmed the high potency of these compounds. Moreover, compounds 13, 20 and 27 showed a potent inhibitory activity on biofilm formation of C. albicans SC5314 and C. neoformans H99. Notably, compound 13 showed no inhibition of human CYP1A2 and low inhibitory activity against CYP2D6 and CYP3A4, suggesting a low risk of drug-drug interactions. With high potency in vitro and in vivo and good safety profiles, compound 13 will be further investigated as a promising candidate.

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2023 – Insight into the mechanism of H+-coupled nucleobase transport
SURFE2R N1 Publication in PNAS (2023) Authors: Weng J., Zhou X., Wiriyasermkul P., Ren Z., Chen K., Gil-Iturbe E., Zhou M., Quick M.

Members of the nucleobase/ascorbic acid transporter (NAT) gene family are found in all kingdoms of life. In mammals, the concentrative uptake of ascorbic acid (vitamin C) by members of the NAT family is driven by the Na+ gradient, while the uptake of nucleobases in bacteria is powered by the H+ gradient. Here, we report the structure and function of PurTCp, a NAT family member from Colwellia psychrerythraea. The structure of PurTCp was determined to 2.80 Å resolution by X-ray crystallography. PurTCp forms a homodimer, and each protomer has 14 transmembrane segments folded into a transport domain (core domain) and a scaffold domain (gate domain). A purine base is present in the structure and defines the location of the substrate binding site. Functional studies reveal that PurTCp transports purines but not pyrimidines and that purine binding and transport is dependent on the pH. Mutation of a conserved aspartate residue close to the substrate binding site reveals the critical role of this residue in H+-dependent transport of purines. Comparison of the PurTCp structure with transporters of the same structural fold suggests that rigid-body motions of the substrate-binding domain are central for substrate translocation across the membrane.

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2023 – Cold and warmth intensify pain-linked sodium channel gating effects and persistent currents
SyncroPatch 384 Publication in J Gen Physiol (2023) Authors: Kriegeskorte S., Bott R., Hampl M., Korngreen A., Hausmann R., Lampert A.

Voltage-gated sodium channels (Nav) are key players in excitable tissues with the capability to generate and propagate action potentials. Mutations in the genes encoding Navs can lead to severe inherited diseases, and some of these so-called channelopathies show temperature-sensitive phenotypes, for example, paramyotonia congenita, Brugada syndrome, febrile seizure syndromes, and inherited pain syndromes like erythromelalgia (IEM) and paroxysmal extreme pain disorder (PEPD). Nevertheless, most investigations of mutation-induced gating effects have been conducted at room temperature, and thus the role of cooling or warming in channelopathies remains poorly understood. Here, we investigated the temperature sensitivity of four Nav subtypes: Nav1.3, Nav1.5, Nav1.6, and Nav1.7, and two mutations in Nav1.7 causing IEM (Nav1.7/L823R) and PEPD (Nav1.7/I1461T) expressed in cells of the human embryonic kidney cell line using an automated patch clamp system. Our experiments at 15°C, 25°C, and 35°C revealed a shift of the voltage dependence of activation to more hyperpolarized potentials with increasing temperature for all investigated subtypes. Nav1.3 exhibited strongly slowed inactivation kinetics compared with the other subtypes that resulted in enhanced persistent current, especially at 15°C, indicating a possible role in cold-induced hyperexcitability. Impaired fast inactivation of Nav1.7/I1461T was significantly enhanced by a cooling temperature of 15°C. The subtype-specific modulation as well as the intensified mutation-induced gating changes stress the importance to consider temperature as a regulator for channel gating and its impact on cellular excitability as well as disease phenotypes.

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2023 – Berberine attenuates sunitinib-induced cardiac dysfunction by normalizing calcium regulation disorder via SGK1 activation
CardioExcyte 96 Publication in Food and Chemical Toxicology (2023) Authors: Li C., Wu W., Xing J., Yan W., Zhang J., Sun J., Zhang Z., Qiu Z., Xu Y., Wang X.

Sunitinib (SNT)-induced cardiotoxicity is associated with abnormal calcium regulation caused by phosphoinositide 3 kinase inhibition in the heart. Berberine (BBR) is a natural compound that exhibits cardioprotective effects and regulates calcium homeostasis. We hypothesized that BBR ameliorates SNT-induced cardiotoxicity by normalizing the calcium regulation disorder via serum and glucocorticoid-regulated kinase 1 (SGK1) activation. Mice, neonatal rat cardiomyocytes (NRVMs), and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were used to study the effects of BBR-mediated SGK1 activity on the calcium regulation disorder caused by SNT as well as the underlying mechanism. BBR offered prevention against SNT-induced cardiac systolic dysfunction, QT interval prolongation, and histopathological changes in mice. After the oral administration of SNT, the Ca2+ transient and contraction of cardiomyocytes was significantly inhibited, whereas BBR exhibited an antagonistic effect. In NRVMs, BBR was significantly preventive against the SNT-induced reduction of calcium transient amplitude, prolongation of calcium transient recovery, and decrease in SERCA2a protein expression; however, SGK1 inhibitors resisted the preventive effects of BBR. In hiPSC-CMs, BBR pretreatment significantly prevented SNT from inhibiting the contraction, whereas coincubation with SGK1 inhibitors antagonized the effects of BBR. These findings indicate that BBR attenuates SNT-induced cardiac dysfunction by normalizing the calcium regulation disorder via SGK1 activation.

 

Read more in the publication here.

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2023 – MFN2 deficiency promotes cardiac response to hypobaric hypoxia by reprogramming cardiomyocyte metabolism
CardioExcyte 96 Publication in Acta Physiologica (2023) Authors: Zhang R., Yang A., Zhang L., He L., Gu X., Yu C., Lu Z., Wang C., Zhou F., Li F., Ji L., Xing J., Guo H.

Aim

Under hypobaric hypoxia (HH), the heart triggers various defense mechanisms including metabolic remodeling against lack of oxygen. Mitofusin 2 (MFN2), located at the mitochondrial outer membrane, is closely involved in the regulation of mitochondrial fusion and cell metabolism. To date, however, the role of MFN2 in cardiac response to HH has not been explored.

Methods

Loss- and gain-of-function approaches were used to investigate the role of MFN2 in cardiac response to HH. In vitro, the function of MFN2 in the contraction of primary neonatal rat cardiomyocytes under hypoxia was examined. Non-targeted metabolomics and mitochondrial respiration analyses, as well as functional experiments were performed to explore underlying molecular mechanisms.

Results

Our data demonstrated that, following 4 weeks of HH, cardiac-specific MFN2 knockout (MFN2 cKO) mice exhibited significantly better cardiac function than control mice. Moreover, restoring the expression of MFN2 clearly inhibited the cardiac response to HH in MFN2 cKO mice. Importantly, MFN2 knockout significantly improved cardiac metabolic reprogramming during HH, resulting in reduced capacity for fatty acid oxidation (FAO) and oxidative phosphorylation, and increased glycolysis and ATP production. In vitro data showed that down-regulation of MFN2 promoted cardiomyocyte contractility under hypoxia. Interestingly, increased FAO through palmitate treatment decreased contractility of cardiomyocyte with MFN2 knockdown under hypoxia. Furthermore, treatment with mdivi-1, an inhibitor of mitochondrial fission, disrupted HH-induced metabolic reprogramming and subsequently promoted cardiac dysfunction in MFN2-knockout hearts.

Conclusion

Our findings provide the first evidence that down-regulation of MFN2 preserves cardiac function in chronic HH by promoting cardiac metabolic reprogramming.

 

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2023 – Triggering the Amphotericin B Pore-Forming Activity by Phytochemicals
VPP Publication in Membranes (2023) Authors: Efimova S., Malykhina A., Ostroumova O.

The macrolide polyene antibiotic amphotericin B (AmB), remains a valuable drug to treat systemic mycoses due to its wide antifungal activity and low probability of developing resistance. The high toxicity of AmB, expressed in nephropathy and hemolysis, could be partially resolved by lowering therapeutic AmB concentration while maintaining efficacy. This work discusses the possibility of using plant polyphenols and alkaloids to enhance the pore-forming and consequently antifungal activity of AmB. We demonstrated that phloretin, phlorizin, naringenin, taxifolin, quercetin, biochanin A, genistein, resveratrol, and quinine led to an increase in the integral AmB-induced transmembrane current in the bilayers composed of palmitoyloleoylphosphocholine and ergosterol, while catechin, colchicine, and dihydrocapsaicin did not practically change the AmB activity. Cardamonin, 4′-hydroxychalcone, licochalcone A, butein, curcumin, and piperine inhibited AmB-induced transmembrane current. Absorbance spectroscopy revealed no changes in AmB membrane concentration with phloretin addition. A possible explanation of the potentiation is related to the phytochemical-produced changes in the elastic membrane properties and the decrease in the energy of formation of the lipid mouth of AmB pores, which is partially confirmed by differential scanning microcalorimetry. The possibility of AmB interaction with cholesterol in the mammalian cell membranes instead of ergosterol in fungal membranes, determines its high toxicity. The replacement of ergosterol with cholesterol in the membrane lipid composition led to a complete loss or a significant decrease in the potentiating effects of tested phytochemicals, indicating low potential toxicity of these compounds and high therapeutic potential of their combinations with the antibiotic. The discovered combinations of AmB with plant molecules that enhance its pore-forming ability in ergosterol-enriched membranes, seem to be promising for further drug development in terms of the toxicity decrease and efficacy improvement.

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2023 – Neuroprotection in an Experimental Model of Multiple Sclerosis via Opening of Big Conductance, Calcium-Activated Potassium Channels
SyncroPatch 384 Publication in Pharmaceuticals (2023) Authors: Pryce G., Sisay S., Giovannoni G., Selwood D., Baker D.

Big conductance calcium-activated (BK) channel openers can inhibit pathologically driven neural hyperactivity to control symptoms via hyperpolarizing signals to limit neural excitability. We hypothesized that BK channel openers would be neuroprotective during neuroinflammatory, autoimmune disease. The neurodegenerative disease was induced in a mouse experimental autoimmune encephalomyelitis model with translational value to detect neuroprotection in multiple sclerosis. Following the treatment with the BK channel openers, BMS-204253 and VSN16R, neuroprotection was assessed using subjective and objective clinical outcomes and by quantitating spinal nerve content. Treatment with BMS-204253 and VSN16R did not inhibit the development of relapsing autoimmunity, consistent with minimal channel expression via immune cells, nor did it change leukocyte levels in rodents or humans. However, it inhibited the accumulation of nerve loss and disability as a consequence of autoimmunity. Therefore, in addition to symptom control, BK channel openers have the potential to save nerves from excitotoxic damage and could be useful as either stand-alone neuroprotective agents or as add-ons to current disease-modifying treatments that block relapsing MS but do not have any direct neuroprotective activity.

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2023 – Structure-function relationship of new peptides activating human Nav1.1
SyncroPatch 384 Publication in Biomedicine & Pharmacotherapy (2023) Authors: Lopez L., De Waard S., Meudal H., Caumes C., Khakh K., Peigneur S., Oliveira-Mendes B., Lin S., De Waele J., Montnach J., Cestèle S., Tessier A., Johnson J., Mantegazza M., Tytgat J., Cohen C., Béroud C., Bosmans F., Landon C., De Waard M.

Nav1.1 is an important pharmacological target as this voltage-gated sodium channel is involved in neurological and cardiac syndromes. Channel activators are actively sought to try to compensate for haploinsufficiency in several of these pathologies. Herein we used a natural source of new peptide compounds active on ion channels and screened for drugs capable to inhibit channel inactivation as a way to compensate for decreased channel function. We discovered that JzTx-34 is highly active on Nav1.1 and subsequently performed a full structure-activity relationship investigation to identify its pharmacophore. These experiments will help interpret the mechanism of action of this and formerly identified peptides as well as the future identification of new peptides. We also reveal structural determinants that make natural ICK peptides active against Nav1.1 challenging to synthesize. Altogether, the knowledge gained by this study will help facilitate the discovery and development of new compounds active on this critical ion channel target.

 

Read more in the publication here.

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2023 – Brevetoxin versus Brevenal Modulation of Human Nav1 Channels
Patchliner Publication in Mar. Drugs (2023) Authors: Finol-Urdaneta R., Zhorov B., Baden D., Adams D.

Brevetoxins (PbTx) and brevenal are marine ladder-frame polyethers. PbTx binds to and activates voltage-gated sodium (Nav) channels in native tissues, whereas brevenal antagonizes these actions. However, the effects of PbTx and brevenal on recombinant Nav channel function have not been systematically analyzed. In this study, the PbTx-3 and brevenal modulation of tissue-representative Nav channel subtypes Nav1.2, Nav1.4, Nav1.5, and Nav1.7 were examined using automated patch-clamp. While PbTx-3 and brevenal elicit concentration-dependent and subtype-specific modulatory effects, PbTx-3 is >1000-fold more potent than brevenal. Consistent with effects observed in native tissues, Nav1.2 and Nav1.4 channels were PbTx-3- and brevenal-sensitive, whereas Nav1.5 and Nav1.7 appeared resistant. Interestingly, the incorporation of brevenal in the intracellular solution caused Nav channels to become less sensitive to PbTx-3 actions. Furthermore, we generated a computational model of PbTx-2 bound to the lipid-exposed side of the interface between domains I and IV of Nav1.2. Our results are consistent with competitive antagonism between brevetoxins and brevenal, setting a basis for future mutational analyses of Nav channels’ interaction with brevetoxins and brevenal. Our findings provide valuable insights into the functional modulation of Nav channels by brevetoxins and brevenal, which may have implications for the development of new Nav channel modulators with potential therapeutic applications.

Read more about the publication here.

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2022 – Full opening of helix bundle crossing does not lead to NaK channel activation
SURFE2R N1 Publication in J. Gen. Physiol. (2022) Authors: Kurauskas V., Tonelli M., Henzler-Wildman K.

A critical part of ion channel function is the ability to open and close in response to stimuli and thus conduct ions in a regulated fashion. While x-ray diffraction studies of ion channels suggested a general steric gating mechanism located at the helix bundle crossing (HBC), recent functional studies on several channels indicate that the helix bundle crossing is wide-open even in functionally nonconductive channels. Two NaK channel variants were crystallized in very different open and closed conformations, which served as important models of the HBC gating hypothesis. However, neither of these NaK variants is conductive in liposomes unless phenylalanine 92 is mutated to alanine (F92A). Here, we use NMR to probe distances at near-atomic resolution of the two NaK variants in lipid bicelles. We demonstrate that in contrast to the crystal structures, both NaK variants are in a fully open conformation, akin to Ca2+-bound MthK channel structure where the HBC is widely open. While we were not able to determine what a conductive NaK structure is like, our further inquiry into the gating mechanism suggests that the selectivity filter and pore helix are coupled to the M2 helix below and undergo changes in the structure when F92 is mutated. Overall, our data show that NaK exhibits coupling between the selectivity filter and HBC, similar to K+ channels, and has a more complex gating mechanism than previously thought, where the full opening of HBC does not lead to channel activation.

Read more in the publication here.

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2023 – PO-01-173 Ibrutinib treatment results in increased phosphorylation of the ERK1/2 signaling pathway in atrial-specific human iPSC-derived cardiomyocytes
CardioExcyte 96 Publication in Heart Rhythm (2023) Authors: Fleming M., O'Neill M., Knollmann B., Moslehi J., Roden D.

Background

Cardiovascular sequelae of targeted cancer therapies may provide novel insights into cardiovascular biology and disease pathogenesis, including atrial fibrillation (AF). The Bruton tyrosine kinase (BTK) inhibitor ibrutinib has revolutionized treatment for B-cell malignancies but increases the incidence of AF compared with conventional chemotherapy. Recent data in mouse models have shown that ibrutinib mediated-AF likely results not from inhibition of BTK (“on target” effect), but from off-target inhibition of a different kinase, C-terminal Src kinase (CSK). The signaling pathway by which CSK inhibition results in AF remains unknown and thus may represent a novel mechanism for AF molecular pathogenesis.

Objective

To test the hypothesis that ibrutinib generates atrial fibrillation in susceptible patients by targeting signaling pathways downstream from CSK.

Methods

We studied cellular monolayers of hiPSC-aCMs using extracellular field potential (EFP) recordings obtained by the Nanion CardioExcyte 96 system after 72-hour exposure to ibrutinib, the second-generation BTK inhibitor acalabrutinib, or vehicle. As an initial proteomic approach to determining which kinase signaling pathways are altered upon inhibition of CSK and hence may predispose to AF arrhythmogenesis, we used a human phospho-antibody array to study the relative phosphorylation state of 37 kinases in hiPSC-aCMs treated with ibrutinib or vehicle.

Results

EFP recordings from cellular monolayers of hiPSC-aCMs exposed to ibrutinib for 24 hours develop a striking increase in spontaneous beat-to-beat variability, an in vitro correlate of arrhythmogenic behavior. This effect was not seen with vehicle control or the second-generation BTK inhibitor acalabrutinib, which is less arrhythmogenic in clinical trials. Treatment with ibrutinib for 72 hours resulted in increased phosphorylation of multiple kinases in the Erk1/2 pathway, including Erk1 T202/Y204 and Erk2 T185/Y187, in human phospho-kinase array studies. Traditional Western Blotting confirmed these results and showed that ibrutinib, but not acalabrutinib, resulted in increased Erk1/2 phosphorylation.

Conclusion

Off-target inhibition of CSK by ibrutinib may result in increased AF through the ERK1/2 pathway, consistent with previous reports implicating increased phosphorylation in the ERK1/2 pathway in the pathogenesis of AF. The downstream mechanisms by which this occurs remain unknown and thus represents a novel potential target for AF therapeutics.
Read more in the publication here.
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2023 – Structure-activity relationship of dihydropyridines for rhabdomyosarcoma
Patchliner Publication in Biochem. & Biophys. Res. Comms. (2023) Authors: Chauhan S., Woods A.D., Bharathy N., Lian X., Ricker C.A., Mantz A., Zuercher W.J., Price L.H., Morton M.J., Durrant E., Corbel S.Y., Sampath S.C., Sampath S.C., Joslin J., Keller C.

Childhood muscle-related cancer rhabdomyosarcoma is a rare disease with a 50-year unmet clinical need for the patients presented with advanced disease. The rarity of ∼350 cases per year in North America generally diminishes the viability of large-scale, pharmaceutical industry driven drug development efforts for rhabdomyosarcoma. In this study, we performed a large-scale screen of 640,000 compounds to identify the dihydropyridine (DHP) class of anti-hypertensives as a priority compound hit. A structure-activity relationship was uncovered with increasing cell growth inhibition as side chain length increases at the ortho and para positions of the parent DHP molecule. Growth inhibition was consistent across n = 21 rhabdomyosarcoma cell line models. Anti-tumor activity in vitro was paralleled by studies in vivo. The unexpected finding was that the action of DHPs appears to be other than on the DHP receptor (i.e., L-type voltage-gated calcium channel). These findings provide the basis of a medicinal chemistry program to develop dihydropyridine derivatives that retain anti-rhabdomyosarcoma activity without anti-hypertensive effects.

Product video
2023 – Port-a-Patch: The world´s smallest patch clamp rig
The Port-a-Patch is a turn-key miniaturized patch clamp system enabling the user to rapidly generate high quality data, regardless of experience.

The Port-a-Patch is a small and easy-to-use complete patch clamp setup with multiple add-ons available to make sure you get the right configuration for your application.

Publication link
2023 – A humoral stress response protects Drosophila tissues from antimicrobial peptides
Orbit 16 TC Pre-Print Publication in BioRxiv (2023) Authors: Rommelaere S., Carboni A., Juarez J.F.B., Boquete J-P., Abriata L.A., Meireles F.T.P., Rukes V., Vincent C., Kondo S., Dionne M.S., Peraro M.D., Cao C., Lemaitre B.

The immune response against an invading pathogen is generally associated with collateral tissue damage caused by the immune system itself. Consequently, several resilience mechanisms have evolved to attenuate the negative impacts of immune effectors. Antimicrobial peptides (AMPs) are small, cationic peptides that contribute to innate defenses by targeting negatively charged membranes of microbes. While being protective against pathogens, AMPs can be cytotoxic to host cells. Little is known of mechanisms that protect host tissues from AMP-induced immunopathology. Here, we reveal that a family of stress-induced proteins, the Turandots, protect Drosophila host tissues from AMPs, increasing resilience to stress. Deletion of several Turandot genes increases fly susceptibility to environmental stresses due to trachea apoptosis and poor oxygen supply. Tracheal cell membranes expose high levels of phosphatidylserine, a negatively charged phospholipid, sensitizing them to the action of AMPs. Turandots are secreted from the fat body upon stress and bind to tracheal cells to protect them against AMPs. In vitro, Turandot A binds to phosphatidylserine on membranes and inhibits the pore-forming activity of Drosophila and human AMPs on eukaryotic cells without affecting their microbicidal activity. Collectively, these data demonstrate that Turandot stress proteins mitigate AMP cytotoxicity to host tissues and therefore improve their efficacy. This provides a first example of a humoral mechanism used by animals limiting host-encoded AMP collateral damages.

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2023 – L-Carnitine Suppresses Transient Receptor Potential Vanilloid Type1 Activation in Human Corneal Epithelial Cells
Port-a-Patch Publication in Int. J. Mol. Sci. (2023) Authors: Lucius A., Chhatwal S., Valtink M., Reinach P.S. Li A., Pleyer U. Mergler S.

Tear film hyperosmolarity induces dry eye syndrome (DES) through transient receptor potential vanilloid type 1 (TRPV1) activation. L-carnitine is a viable therapeutic agent since it protects against this hypertonicity-induced response. Here, we investigated whether L-carnitine inhibits TRPV1 activation by blocking heat- or capsaicin-induced increases in Ca2+ influx or hyperosmotic stress-induced cell volume shrinkage in a human corneal epithelial cell line (HCE-T). Single-cell fluorescence imaging of calcein/AM-loaded cells or fura-2/AM-labeled cells was used to evaluate cell volume changes and intracellular calcium levels, respectively. Planar patch-clamp technique was used to measure whole-cell currents. TRPV1 activation via either capsaicin (20 µmol/L), hyperosmolarity (≈450 mosmol/L) or an increase in ambient bath temperature to 43 °C induced intracellular calcium transients and augmented whole-cell currents, whereas hypertonicity induced cell volume shrinkage. In contrast, either capsazepine (10 µmol/L) or L-carnitine (1–3 mmol/L) reduced all these responses. Taken together, L-carnitine and capsazepine suppress hypertonicity-induced TRPV1 activation by blocking cell volume shrinkage.

Product video
2023 – AtlaZ accelerates cellular research
AtlaZ accelerates cellular research by enabling the investigation of a large variety of effects in cells over time.

AtlaZ accelerates cellular research by enabling the investigation of a large variety of effects in cells over time. It offers label-free and real-time monitoring capabilities. It can simultaneously or independently record data from up to six 96-well plates.

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2023 – Optimization of a series of novel, potent and selective Macrocyclic SYK inhibitors
SyncroPatch 384 Publication in Bioorg. & Med. Chem. Lett (2023) Authors: Grimster N.P., Gingipalli L., Balazs A., Barlaam B., Boiko S., Boyd S., Dry H., Goldberg F.W., Ikeda T., Johnson T., Kawatkar S., Kemmitt P., Lamont S., Lorthioir O., Mfuh A., Patel J., Pike A., Read J., Romero R., Sarkar U., Sha L., Simpson I. Song K., Su Q., Wang H., Watson D., Wu A., Zehnder T.E., Zheng X.,Li S., Dong Z., Yang D., Song Y., Wang P., Liu X., Dowling .E., Edmondson S.D.

Spleen tyrosine kinase (SYK) is a non-receptor cytoplasmic kinase. Due to its pivotal role in B cell receptor and Fc-receptor signalling, inhibition of SYK has been a target of interest in a variety of diseases. Herein, we report the use of structure-based drug design to discover a series of potent macrocyclic inhibitors of SYK, with excellent kinome selectivity and in vitro metabolic stability. We were able to remove hERG inhibition through the optimization of physical properties, and utilized a pro-drug strategy to address permeability challenges.

Conference poster
2023 – Investigating lysosomal membrane proteins using SSM based electrophysiology: Improving amplification and accessibility
Poster presented at Physiology 2023 in Harrogate, UK
Conference poster
2023 – Mechanical stimulation of PIEZO 1 channels using high throughput automated patch clamp
Poster presented at Physiology 2023 in Harrogate, UK
Application Note PDF
ChR2 – “SyncroPatch 384 optogenetic stimulation and light-gated ion channels”
SyncroPatch 384 application note: Optogenetic stimulation tool
Case study PDF
Channelopathies – “A New Era is Emerging for Ion Channel and Channelopathy Research”
Case study PDF
Immune cells- “Immune cell – mediated killing of tumor cells with the AtlaZ platform”
Application Note PDF
TEER – “Monitoring cell – attachment and tight junctions in the same assay”
AtlaZ application note

The numerous different cell types in the human body are greatly specialized and often require a conjunctive action of a population of cells, for example in tissues. Cells are interconnected via cell junctions, multiprotein complexes found in the cell membrane of animal cells, and such cell junctions allow for a mechanical, chemical or electrical transmission of signals. These junctions can be subdivided into (I) tight junctions, (II) anchoring junctions or (III) gap junctions. Defects in cell–cell junctions give rise to a wide range of tissue abnormalities that disrupt homeostasis and are common in genetic abnormalities and cancers (1). The so-called tight junctions form the barrier in endothelial and epithelial cells. Classical transepithelial electrical resistance measurements are performed using microelectrodes, where trans- and para-cellular conductivities can be calculated (2).
Here the cells are grown on a porous filter membrane which is placed between two fluid compartments. Flux of solutes from one compartment to the other must pass the interfacial cell
layer then, and this is determined by the functional properties of the tight junctions.

Application Note PDF
Cancer, Immuno-oncology – “Immune cell-mediated killing of A549 cancer target cells in real-time”
AtlaZ application note

Cancer remains one of the leading causes of death, with, according to the World Health Organisation (WHO), around 10 million people dying due to the disease in 2020 (1). Chemo and
radio-therapy are still the dominant treatment types, but advancing therapies such as immuno-therapy have emerged as tools to fight against the disease. In general, identifying T cells that kill cancer cells in vivo is critical to the development of successful cell therapies. The label-free AtlaZ immune cell killing assay can be used to measure rate of killing at Effector : Target (E:T) ratios to predict in vivo activity. In order to gain a deeper understanding of cancer cells, real-time and continuous monitoring is necessary to access kinetic and phenotypic information.

The platform used here, AtlaZ, is a quantitative live-cell analysis system and allows for cellular research on cell adhesion and proliferation, cytotoxicity, GPCR, morphology and
barrier function, label-free and in real-time. Recordings can be performed in up to six 96-well plates simultaneously or independently. Electrical impedance spectroscopy (2,3) as
the methodology behind the AtlaZ system, in combination with the throughput of 6 x 96-wells allows for a so far unmet quantity and richness of information which can be gained from cells.

Product sheet PDF
AtlaZ – Product Sheet
Publication link
2023 – The Putative Role of the Transient Receptor Potential Ion Channel of Vanilloid Type 2 in Red Blood Cell Storage Lesions
Patchliner and SyncroPatch 384 Publication in Transfus. Med. Hemother. (2023) Authors: Murciano N., Kaestner L.

Recently, a review about big data and artificial intelligence was published in “Transfusion Medicine and Hemotherapy,” highlighting the importance and chances of quality control of stored red blood cells (RBCs). RBC quality is decreased over storage time in a donor-dependent manner. Here, we want to emphasize that besides quality control, one has to further think about improving the RBC quality during storage, i.e., addressing storage lesions. A component of the storage lesion is the dissipation of the cation gradients across the RBC membrane, i.e., K+ will leak out of the RBC and Na+ enters the cell. So far, the molecular cause of the cation gradient dissipation remains elusive. To this end, we like to present a hypothesis for the involvement of the transient receptor potential channel of vanilloid type 2 (TRPV2).

Publication link
2023 – Pain-causing stinging nettle toxins target TMEM233 to modulate NaV1.7 function
Patchliner Publication in Nature Communications (2023) Authors: Jami S., Deuis J., Klasfauseweh T., Cheng X., Kurdyukov S., Chung F., Okorokov A., Li S., Zhang J., Cristofori-Armstrong B., Israel M., Ju R., Robinson S., Zhao P., Ragnarsson L., Andersson Å., Tran P., Schendel V., McMahon K., Tran H., Chin Y., Zhu Y., Liu J., Crawford T., Purushothamvasan S., Habib A., Andersson D., Rash L., Wood J., Zhao J., Stehbens S., Mobli M., Leffler A., Jiang D., Cox J., Waxman S., Dib-Hajj S., Neely G., Durek T., Vetter I.

Voltage-gated sodium (NaV) channels are critical regulators of neuronal excitability and are targeted by many toxins that directly interact with the pore-forming α subunit, typically via extracellular loops of the voltage-sensing domains, or residues forming part of the pore domain. Excelsatoxin A (ExTxA), a pain-causing knottin peptide from the Australian stinging tree Dendrocnide excelsa, is the first reported plant-derived NaV channel modulating peptide toxin. Here we show that TMEM233, a member of the dispanin family of transmembrane proteins expressed in sensory neurons, is essential for pharmacological activity of ExTxA at NaV channels, and that co-expression of TMEM233 modulates the gating properties of NaV1.7. These findings identify TMEM233 as a previously unknown NaV1.7-interacting protein, position TMEM233 and the dispanins as accessory proteins that are indispensable for toxin-mediated effects on NaV channel gating, and provide important insights into the function of NaV channels in sensory neurons.

Read more in the publication here.

Publication link
2023 – Scanning mutagenesis of the voltage-gated sodium channel NaV1.2 using base editing
SyncroPatch 384 Publication in Cell Reports (2023) Authors: Pablo J., Cornett S., Wang L., Jo S., Brünger T., Budnik N., Hegde M., DeKeyser J., Thompson C., Doench J., Lal D., George Jr. A., Pan J.

It is challenging to apply traditional mutational scanning to voltage-gated sodium channels (NaVs) and functionally annotate the large number of coding variants in these genes. Using a cytosine base editor and a pooled viability assay, we screen a library of 368 guide RNAs (gRNAs) tiling NaV1.2 to identify more than 100 gRNAs that change NaV1.2 function. We sequence base edits made by a subset of these gRNAs to confirm specific variants that drive changes in channel function. Electrophysiological characterization of these channel variants validates the screen results and provides functional mechanisms of channel perturbation. Most of the changes caused by these gRNAs are classifiable as loss of function along with two missense mutations that lead to gain of function in NaV1.2 channels. This two-tiered strategy to functionally characterize ion channel protein variants at scale identifies a large set of loss-of-function mutations in NaV1.2.

Publication link
2023 – Ant venoms contain vertebrate-selective pain-causing sodium channel toxins
SyncroPatch 384 Publication in Nature Communications (2023) Authors: Robinson S., Deuis J., Touchard A., Keramidas A., Mueller A., Schroeder C., Barassé V., Walker A., Brinkwirth N., Jami S., Bonnafé E., Treilhou M., Undheim E., Schmidt J., King G., Vetter I.

Stings of certain ant species (Hymenoptera: Formicidae) can cause intense, long-lasting nociception. Here we show that the major contributors to these symptoms are venom peptides that modulate the activity of voltage-gated sodium (NaV) channels, reducing their voltage threshold for activation and inhibiting channel inactivation. These peptide toxins are likely vertebrate-selective, consistent with a primarily defensive function. They emerged early in the Formicidae lineage and may have been a pivotal factor in the expansion of ants.

Read more in the publication here.

Publication link
2023 – Assay for evaluation of proarrhythmic effects of herbal products: Case study with 12 Evodia preparations
Patchliner and SyncroPatch 384 Publication in Toxicology Reports (2023) Authors: Baltov B., Beyl S., Baburin I., Reinhardt J., Szkokan P., Garifulina A., Timin E., Kraushaar U., Potterat O., Hamburger M., Kügler P., Hering S.

Guidelines for preclinical drug development reduce the occurrence of arrhythmia-related side effects. Besides ample evidence for the presence of arrhythmogenic substances in plants, there is no consensus on a research strategy for the evaluation of proarrhythmic effects of herbal products. Here, we propose a cardiac safety assay for the detection of proarrhythmic effects of plant extracts based on the experimental approaches described in the Comprehensive In vitro Proarrhythmia Assay (CiPA). Microelectrode array studies (MEAs) and voltage sensing optical technique on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were combined with ionic current measurements in mammalian cell lines, In-silico simulations of cardiac action potentials (APs) and statistic regression analysis. Proarrhythmic effects of 12 Evodia preparations, containing different amounts of the hERG inhibitors dehydroevodiamine (DHE) and hortiamine were analysed. Extracts produced different prolongation of the AP, occurrence of early after depolarisations and triangulation of the AP in hiPSC-CMs depending on the contents of the hERG inhibitors. DHE and hortiamine dose-dependently prolonged the field potential duration in hiPSC-CMs studied with MEAs. In-silico simulations of ventricular AP support a scenario where proarrhythmic effects of Evodia extracts are predominantly caused by the content of the selective hERG inhibitors. Statistic regression analysis revealed a high torsadogenic risk for both compounds that was comparable to drugs assigned to the high-risk category in a CiPA study.

Read more in the publication here.

Publication link
2023 – Electrophysiological and calcium-handling development during long-term culture of human-induced pluripotent stem cell-derived cardiomyocytes
SyncroPatch 384 Publication in Basic Research in Cardiology (2023) Authors: Seibertz F., Sutanto H., Dülk R., Pronto J., Springer R., Rapedius M., Liutkute A., Ritter M., Jung P., Stelzer L., Hüsgen L., Klopp M., Rubio T., Fakuade F., Mason F., Hartmann N., Pabel S., Streckfuss-Bömeke K., Cyganek L., Sossalla S., Heijman J., Voigt N.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are increasingly used for personalised medicine and preclinical cardiotoxicity testing. Reports on hiPSC-CM commonly describe heterogenous functional readouts and underdeveloped or immature phenotypical properties. Cost-effective, fully defined monolayer culture is approaching mainstream adoption; however, the optimal age at which to utilise hiPSC-CM is unknown. In this study, we identify, track and model the dynamic developmental behaviour of key ionic currents and Ca2+-handling properties in hiPSC-CM over long-term culture (30–80 days). hiPSC-CMs > 50 days post differentiation show significantly larger ICa,L density along with an increased ICa,L-triggered Ca2+-transient. INa and IK1 densities significantly increase in late-stage cells, contributing to increased upstroke velocity and reduced action potential duration, respectively. Importantly, our in silico model of hiPSC-CM electrophysiological age dependence confirmed IK1 as the key ionic determinant of action potential shortening in older cells. We have made this model available through an open source software interface that easily allows users to simulate hiPSC-CM electrophysiology and Ca2+-handling and select the appropriate age range for their parameter of interest. This tool, together with the insights from our comprehensive experimental characterisation, could be useful in future optimisation of the culture-to-characterisation pipeline in the field of hiPSC-CM research.

Publication link
2023 – Nonclinical Cardiovascular Assessment of the Soluble Guanylate Cyclase Stimulator Vericiguat
Patchliner Publication in JPET (2023) Authors: Himmel H., Lagrutta A., Vömel M., Amin R.P., Imredy J.P., Johnson T., Vinzing M., Prescott J., Blaustein R.O.

Vericiguat and its metabolite M-1 were assessed for proarrhythmic risk in nonclinical in vitro and in vivo studies. In vitro manual voltage-clamp recordings at room temperature determined the effect of vericiguat on human Ether-a-go-go Related Gene (hERG) K+ channels. Effects of vericiguat and M-1 on hERG K+, Nav1.5, hCav1.2, hKvLQT1/1minK, and hKv4.3 channels were investigated via automated voltage-clamp recordings at ambient temperature. Effects of vericiguat and M-1 on hERG K+ and Nav1.5 channels at pathophysiological conditions were explored via manual voltage-clamp recordings at physiologic temperature. Single oral doses of vericiguat (0.6, 2.0, and 6.0 mg/kg) were assessed for in vivo proarrhythmic risk via administration to conscious telemetered dogs; electrocardiogram (ECG) and hemodynamic parameters were monitored. ECG recordings were included in 4- and 39-week dog toxicity studies. In manual voltage-clamp recordings, vericiguat inhibited hERG K+-mediated tail currents in a concentration-dependent manner (20% threshold inhibitory concentration ∼1.9 µM). In automated voltage-clamp recordings, neither vericiguat nor M-1 were associated with biologically relevant inhibition (>20%) of hNav1.5, hCav1.2, hKvLQT1, and hKv4.3. No clinically relevant observations were made for hNav1.5 and hKvLQT1 under simulated pathophysiological conditions. Vericiguat was associated with expected mode-of-action–related dose-dependent changes in systolic arterial blood pressure (up to −20%) and heart rate (up to +53%). At maximum vericiguat dose, corrected QT (QTc) interval changes from baseline varied slightly (−6 to +1%) depending on correction formula. Toxicity studies confirmed absence of significant QTc interval changes. There was no evidence of an increased proarrhythmic risk from nonclinical studies with vericiguat or M-1.

SIGNIFICANCE STATEMENT There was no evidence of an increased proarrhythmic risk from in vitro and in vivo nonclinical studies with vericiguat or M-1. The integrated risk assessment of these nonclinical data combined with existing clinical data demonstrate administration of vericiguat 10 mg once daily in patients with heart failure with reduced ejection fraction is not associated with a proarrhythmic risk.

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2023 – Allyl Isothiocianate Induces Ca2+ Signals and Nitric Oxide Release by Inducing Reactive Oxygen Species Production in the Human Cerebrovascular Endothelial Cell Line hCMEC/D3
Port-a-Patch Publication in Cells (2023) Authors: Berra-Romani R., Brunetti V., Pellavio G., Soda T., Laforenza U., Scarpellino G., Moccia F.

Nitric oxide (NO) represents a crucial mediator to regulate cerebral blood flow (CBF) in the human brain both under basal conditions and in response to somatosensory stimulation. An increase in intracellular Ca2+ concentrations ([Ca2+]i) stimulates the endothelial NO synthase to produce NO in human cerebrovascular endothelial cells. Therefore, targeting the endothelial ion channel machinery could represent a promising strategy to rescue endothelial NO signalling in traumatic brain injury and neurodegenerative disorders. Allyl isothiocyanate (AITC), a major active constituent of cruciferous vegetables, was found to increase CBF in non-human preclinical models, but it is still unknown whether it stimulates NO release in human brain capillary endothelial cells. In the present investigation, we showed that AITC evoked a Ca2+-dependent NO release in the human cerebrovascular endothelial cell line, hCMEC/D3. The Ca2+ response to AITC was shaped by both intra- and extracellular Ca2+ sources, although it was insensitive to the pharmacological blockade of transient receptor potential ankyrin 1, which is regarded to be among the main molecular targets of AITC. In accord, AITC failed to induce transmembrane currents or to elicit membrane hyperpolarization, although NS309, a selective opener of the small- and intermediate-conductance Ca2+-activated K+ channels, induced a significant membrane hyperpolarization. The AITC-evoked Ca2+ signal was triggered by the production of cytosolic, but not mitochondrial, reactive oxygen species (ROS), and was supported by store-operated Ca2+ entry (SOCE). Conversely, the Ca2+ response to AITC did not require Ca2+ mobilization from the endoplasmic reticulum, lysosomes or mitochondria. However, pharmacological manipulation revealed that AITC-dependent ROS generation inhibited plasma membrane Ca2+-ATPase (PMCA) activity, thereby attenuating Ca2+ removal across the plasma membrane and resulting in a sustained increase in [Ca2+]i. In accord, the AITC-evoked NO release was driven by ROS generation and required ROS-dependent inhibition of PMCA activity. These data suggest that AITC could be exploited to restore NO signalling and restore CBF in brain disorders that feature neurovascular dysfunction.

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2023 – Structure and sucrose binding mechanism of the plant SUC1 sucrose transporter
SURFE2R N1 Publication in Nature Plants (2023) Authors: Bavnhøj L., Driller J., Zuzic L., Dyrholm Stange A., Schiøtt B., Panyella Pedersen B.

Sucrose import from photosynthetic tissues into the phloem is mediated by transporters from the low-affinity sucrose transporter family (SUC/SUT family). Furthermore, sucrose redistribution to other tissues is driven by phloem sap movement, the product of high turgor pressure created by this import activity. Additionally, sink organs such as fruits, cereals and seeds that accumulate high concentrations of sugar also depend on this active transport of sucrose. Here we present the structure of the sucrose–proton symporter, Arabidopsis thaliana SUC1, in an outward open conformation at 2.7 Å resolution, together with molecular dynamics simulations and biochemical characterization. We identify the key acidic residue required for proton-driven sucrose uptake and describe how protonation and sucrose binding are strongly coupled. Sucrose binding is a two-step process, with initial recognition mediated by the glucosyl moiety binding directly to the key acidic residue in a stringent pH-dependent manner. Our results explain how low-affinity sucrose transport is achieved in plants, and pinpoint a range of SUC binders that help define selectivity. Our data demonstrate a new mode for proton-driven symport with links to cation-driven symport and provide a broad model for general low-affinity transport in highly enriched substrate environments.

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2023 – Three tyrosine kinase inhibitors cause cardiotoxicity by inducing endoplasmic reticulum stress and inflammation in cardiomyocytes
CardioExcyte 96 Publication in BMC Medicine (2023) Authors: Wang H., Wang Y., Li J., He Z., Boswell S., Chung M., You F., Han S.

Background

Tyrosine kinase inhibitors (TKIs) are anti-cancer therapeutics often prescribed for long-term treatment. Many of these treatments cause cardiotoxicity with limited cure. We aim to clarify molecular mechanisms of TKI-induced cardiotoxicity so as to find potential targets for treating the adverse cardiac complications.

Methods

Eight TKIs with different levels of cardiotoxicity reported are selected. Phenotypic and transcriptomic responses of human cardiomyocytes to TKIs at varying doses and times are profiled and analyzed. Stress responses and signaling pathways that modulate cardiotoxicity induced by three TKIs are validated in cardiomyocytes and rat hearts.

Results

Toxicity rank of the eight TKIs determined by measuring their effects on cell viability, contractility, and respiration is largely consistent with that derived from database or literature, indicating that human cardiomyocytes are a good cellular model for studying cardiotoxicity. When transcriptomes are measured for selected TKI treatments with different levels of toxicity in human cardiomyocytes, the data are classified into 7 clusters with mainly single-drug clusters. Drug-specific effects on the transcriptome dominate over dose-, time- or toxicity-dependent effects. Two clusters with three TKIs (afatinib, ponatinib, and sorafenib) have the top enriched pathway as the endoplasmic reticulum stress (ERS). All three TKIs induce ERS in rat primary cardiomyocytes and ponatinib activates the IRE1α-XBP1s axis downstream of ERS in the hearts of rats underwent a 7-day course of drug treatment. To look for potential triggers of ERS, we find that the three TKIs induce transient reactive oxygen species followed by lipid peroxidation. Inhibiting either PERK or IRE1α downstream of ERS blocks TKI-induced cardiac damages, represented by the induction of cardiac fetal and pro-inflammatory genes without causing more cell death.

Conclusions

Our data contain rich information about phenotypic and transcriptional responses of human cardiomyocytes to eight TKIs, uncovering potential molecular mechanisms in modulating cardiotoxicity. ER stress is activated by multiple TKIs and leads to cardiotoxicity through promoting expression of pro-inflammatory factors and cardiac fetal genes. ER stress-induced inflammation is a promising therapeutic target to mitigate ponatinib- and sorafenib-induced cardiotoxicity.

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2023 – Evaluation of Nanopore Sensor Design Using Electrical and Optical Analyses
Orbit mini Publication in ACS Nano (2023) Authors: Mayse L., Imran A., Wang Y., Ahmad M., Oot R., Wilkens S., Movileanu L.

Nanopores are currently utilized as powerful tools for single-molecule protein sensing. The reporting signal typically requires protein analytes to enter the nanopore interior, yet a class of these sensors has emerged that allows targeted detection free in solution. This tactic eliminates the spatial limitation of nanopore confinement. However, probing proteins outside the nanopore implies numerous challenges associated with transducing the physical interactions in the aqueous phase into a reliable electrical signature. Hence, it necessitates extensive engineering and tedious optimization routes. These obstacles have prevented the widespread adoption of these sensors. Here, we provide an experimental strategy by developing and validating single-polypeptide-chain nanopores amenable to single-molecule and bulk-phase protein detection approaches. We utilize protein engineering, as well as nanopore and nanodisc technologies, to create nanopore sensors that can be integrated with an optical platform in addition to traditional electrical recordings. Using the optical modality over an ensemble of detectors accelerates these sensors’ optimization process for a specific task. It also provides insights into how the construction of these single-molecule nanopore sensors influences their performance. These outcomes form a basis for evaluating engineered nanopores beyond the fundamental limits of the resistive-pulse technique.

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2023 – Cryo-EM analysis of scorpion toxin binding to Ryanodine Receptors reveals subconductance that is abolished by PKA phosphorylation
Orbit mini Publication in Science Advances (2023) Authors: Haji-Ghassemi O., Seby Chen Y., Woll K., Gurrola G., Valdivia C., Cai W., Li S., Valdivia H., Van Petegem F.

Calcins are peptides from scorpion venom with the unique ability to cross cell membranes, gaining access to intracellular targets. Ryanodine Receptors (RyR) are intracellular ion channels that control release of Ca2+ from the endoplasmic and sarcoplasmic reticulum. Calcins target RyRs and induce long-lived subconductance states, whereby single-channel currents are decreased. We used cryo–electron microscopy to reveal the binding and structural effects of imperacalcin, showing that it opens the channel pore and causes large asymmetry throughout the cytosolic assembly of the tetrameric RyR. This also creates multiple extended ion conduction pathways beyond the transmembrane region, resulting in subconductance. Phosphorylation of imperacalcin by protein kinase A prevents its binding to RyR through direct steric hindrance, showing how posttranslational modifications made by the host organism can determine the fate of a natural toxin. The structure provides a direct template for developing calcin analogs that result in full channel block, with potential to treat RyR-related disorder

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2023 – Structural mechanisms of TRPM7 activation and inhibition
Orbit mini Publication in Nature Communications (2023) Authors: Nadezhdin K., Correia L., Narangoda C., Patel D., Neuberger A., Gudermann T., Kurnikova M., Chubanov V., Sobolevsky A.

The transient receptor potential channel TRPM7 is a master regulator of the organismal balance of divalent cations that plays an essential role in embryonic development, immune responses, cell mobility, proliferation, and differentiation. TRPM7 is implicated in neuronal and cardiovascular disorders, tumor progression and has emerged as a new drug target. Here we use cryo-EM, functional analysis, and molecular dynamics simulations to uncover two distinct structural mechanisms of TRPM7 activation by a gain-of-function mutation and by the agonist naltriben, which show different conformational dynamics and domain involvement. We identify a binding site for highly potent and selective inhibitors and show that they act by stabilizing the TRPM7 closed state. The discovered structural mechanisms provide foundations for understanding the molecular basis of TRPM7 channelopathies and drug development.

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2023 – Activation energy for pore opening in lipid membranes under an electric field
Orbit mini Publication in PNAS (2023) Authors: Lafarge E., Muller P., Schroder A., Zaitseva E., Behrends J., Marques C.

The standard model of pore formation was introduced more than fifty years ago, and it has been since, despite some refinements, the cornerstone for interpreting experiments related to pores in membranes. A central prediction of the model concerning pore opening under an electric field is that the activation barrier for pore formation is lowered proportionally to the square of the electric potential. However, this has only been scarcely and inconclusively confronted to experiments. In this paper, we study the electropermeability of model lipid membranes composed of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) containing different fractions of POPC-OOH, the hydroperoxidized form of POPC, in the range 0 to 100 mol %. By measuring ion currents across a 50-μm-diameter black lipid membrane (BLM) with picoampere and millisecond resolution, we detect hydroperoxidation-induced changes to the intrinsic bilayer electropermeability and to the probability of opening angstrom-size or larger pores. Our results over the full range of lipid compositions show that the energy barrier to pore formation is lowered linearly by the absolute value of the electric field, in contradiction with the predictions of the standard model.

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SyncroPatch 384 – Interactive Brochure
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2023 – Omentin-1 drives cardiomyocyte cell cycle arrest and metabolic maturation by interacting with BMP7
CardioExcyte 96 Publication in Cell & Mol. Life Sci. (2023) Authors: Yang H., Song S., Li J., Li Y., Feng J., Sun Q., Qiu X., Chen Z., Bai X., Liu X., Lian H., Liu L., Bai Y., Zhang G., Nie Y.

Mammalian cardiomyocytes (CMs) undergo maturation during postnatal heart development to meet the increased demands of growth. Here, we found that omentin-1, an adipokine, facilitates CM cell cycle arrest and metabolic maturation. Deletion of omentin-1 causes mouse heart enlargement and dysfunction in adulthood and CM maturation retardation in juveniles, including delayed cell cycle arrest and reduced fatty acid oxidation. Through RNA sequencing, molecular docking analysis, and proximity ligation assays, we found that omentin-1 regulates CM maturation by interacting directly with bone morphogenetic protein 7 (BMP7). Omentin-1 prevents BMP7 from binding to activin type II receptor B (ActRIIB), subsequently decreasing the downstream pathways mothers against DPP homolog 1 (SMAD1)/Yes-associated protein (YAP) and p38 mitogen-activated protein kinase (p38 MAPK). In addition, omentin-1 is required and sufficient for the maturation of human embryonic stem cell-derived CMs. Together, our findings reveal that omentin-1 is a pro-maturation factor for CMs that is essential for postnatal heart development and cardiac function maintenance.

 

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Investigating PIEZO1 using the SyncroPatch 384
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2023 – Biological fractionation of lithium isotopes by cellular Na+/H+ exchangers unravels fundamental transport mechanisms
Orbit mini Publication in iScience (2023) Authors: Poet M., Vigier N., Bouret Y., Jarretou G., Gautier R., Bendahhou S., Balter V., Montanes M., Thibon F., Counillon L.

Lithium (Li) has a wide range of uses in science, medicine, and industry, but its isotopy is underexplored, except in nuclear science and in geoscience. 6Li and 7Li isotopic ratio exhibits the second largest variation on earth’s surface and constitutes a widely used tool for reconstructing past oceans and climates. As large variations have been measured in mammalian organs, plants or marine species, and as 6Li elicits stronger effects than natural Li (∼95% 7Li), a central issue is the identification and quantification of biological influence of Li isotopes distribution. We show that membrane ion channels and Na+-Li+/H+ exchangers (NHEs) fractionate Li isotopes. This systematic 6Li enrichment is driven by membrane potential for channels, and by intracellular pH for NHEs, where it displays cooperativity, a hallmark of dimeric transport. Evidencing that transport proteins discriminate between isotopes differing by one neutron opens new avenues for transport mechanisms, Li physiology, and paleoenvironments.

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2023 – Metamorphic proteins at the basis of human autophagy initiation and lipid transfer
Vesicle Prep Pro Publication in Mol. Cell (2023) Authors: Nguyen A., Lugarini F., David C., Hosnani P., Alagöz Ç., Friedrich A., Schlütermann D., Knotkova B., Patel A., Parfentev I., Urlaub H., Meinecke M., Stork B., Faesen A.C.

Autophagy is a conserved intracellular degradation pathway that generates de novo double-membrane autophagosomes to target a wide range of material for lysosomal degradation. In multicellular organisms, autophagy initiation requires the timely assembly of a contact site between the ER and the nascent autophagosome. Here, we report the in vitro reconstitution of a full-length seven-subunit human autophagy initiation supercomplex built on a core complex of ATG13-101 and ATG9. Assembly of this core complex requires the rare ability of ATG13 and ATG101 to switch between distinct folds. The slow spontaneous metamorphic conversion is rate limiting for the self-assembly of the supercomplex. The interaction of the core complex with ATG2-WIPI4 enhances tethering of membrane vesicles and accelerates lipid transfer of ATG2 by both ATG9 and ATG13-101. Our work uncovers the molecular basis of the contact site and its assembly mechanisms imposed by the metamorphosis of ATG13-101 to regulate autophagosome biogenesis in space and time.

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2023 – DNA Origami Signaling Units Transduce Chemical and Mechanical Signals in Synthetic Cells
Vesicle Prep Pro Publication in Advanced Functional Materials (2023) Authors: Jahnke K., Illig M., Scheffold M., Tran M., Mersdorf U., Göpfrich K.

Transmembrane proteins transmit chemical signals as well as mechanical cues. The latter is often achieved by coupling to the cytoskeleton. The incorporation of fully engineerable membrane-spanning structures for the transduction of chemical and, in particular, mechanical signals is therefore a critical aim for bottom-up synthetic biology. Here, a membrane-spanning DNA origami signaling units (DOSUs) is designed and mechanically coupled to DNA cytoskeletons encapsulated within giant unilamellar vesicles (GUVs). The incorporation of the DOSUs into the GUV membranes is verified and clustering upon external stimulation is achieved. Dye-influx assays reveal that clustering increases the insertion efficiency. The transmembrane-spanning DOSUs act as pores to allow for the transport of single-stranded DNA into the GUVs. This is employed to trigger the reconfiguration of DNA cytoskeletons within GUVs. In addition to chemical signaling, mechanical coupling of the DOSUs to the internal DNA cytoskeletons is induced. With chemical cues from the environment, clustering of the DOSUs is induced, which triggers a symmetry break in the organization of the DNA cytoskeleton inside of the GUV. DNA-based transmembrane structures are engineered that transduce signals without transporting the signaling molecule itself—providing a route toward signal processing and adaptive synthetic cells.

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2023 – Molecular orientation and optimization of membrane dyes based on conjugated oligoelectrolytes
Vesicle Prep Pro Publication in Cell Reports Physical Science (2023) Authors: Zhu J., Bazan G.

Conjugated oligoelectrolytes (COEs) are amphiphilic, fluorogenic molecules that spontaneously associate with lipid bilayer membranes and are gaining attention as molecular reporters, particularly for exosome detection by flow cytometry. Questions nonetheless remain on how to best design COEs for optimal performance and on the geometry of lipid bilayer intercalation. In response, we designed a series of oligo-phenylenevinylene COEs with varying lengths and numbers of charged groups to address these uncertainties. Examination of the organization within lipid bilayers through polarized fluorescence microscopy shows that the optical transition moments are perpendicular to the bilayer plane, with the conjugated segment flanked by hydrophobic phospholipid tails. COEs initially form a disorganized layer on the vesicle periphery, reflecting electrostatic association before intercalation. Uptake experiments show that longer dimensions and increased numbers of charges allow for a higher degree of cellular association. Both shorter core length and increased number of charges accelerate the rate needed to achieve emission saturation.

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2023 – Calcium-induced compaction and clustering of vesicles tracked with molecular resolution
Vesicle Prep Pro Publication in Biophys. J (2023) Authors: Saldanha O., Schiller L., Hauser K.

Theory and simulations predict the complex nature of calcium interaction with the lipid membrane. By maintaining the calcium concentrations at physiological conditions, herein we demonstrate experimentally the effect of Ca2+ in a minimalistic cell-like model. For this purpose, giant unilamellar vesicles (GUVs) with a neutral lipid DOPC are generated, and the ion-lipid interaction is observed with attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy providing molecular resolution. Firstly, Ca2+ encapsulated within the vesicle binds to the phosphate head groups of the inner leaflets and triggers vesicle compaction. This is tracked by changes in vibrational modes of the lipid groups. As the calcium  concentration within the GUV increases, IR intensities change indicating vesicle dehydration and lateral compression of the membrane. Secondly, by inducing a calcium gradient across the membrane up to a ratio of 1:20, interaction between several vesicles occurs as Ca2+ can bind to the outer leaflets leading to vesicle clustering. It is observed that larger calcium gradients induce stronger interactions. These findings with an exemplary biomimetic model reveal that divalent calcium ions not only cause local changes to the lipid packing but also have macroscopic implications to initiate vesicle-vesicle interaction.

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2023 – Modulators of cellular cholesterol homeostasis as antiproliferative and model membranes perturbing agents
Vesicle Prep Pro Publication in BBA - Biomembranes (2023) Authors: Błaszczyk M., Kozioł A., Palko-Łabuz A., Środa-Pomianek K., Wesołowska O.

Cholesterol is an important component of mammalian cell membranes affecting their fluidity and permeability. Together with sphingomyelin, cholesterol forms microdomains, called lipid rafts. They play important role in signal transduction forming platforms for interaction of signal proteins. Altered levels of cholesterol are known to be strongly associated with the development of various pathologies (e.g., cancer, atherosclerosis and cardiovascular diseases). In the present work, the group of compounds that share the property of affecting cellular homeostasis of cholesterol was studied. It contained antipsychotic and antidepressant drugs, as well as the inhibitors of cholesterol biosynthesis, simvastatin, betulin, and its derivatives. All compounds were demonstrated to be cytotoxic to colon cancer cells but not to non-cancerous cells. Moreover, the most active compounds decreased the level of free cellular cholesterol. The interaction of drugs with raft-mimicking model membranes was visualized. All compounds reduced the size of lipid domains, however, only some affected their number and shape. Membrane interactions of betulin and its novel derivatives were characterized in detail. Molecular modeling indicated that high dipole moment and significant lipophilicity were characteristic for the most potent antiproliferative agents. The importance of membrane interactions of cholesterol homeostasis-affecting compounds, especially betulin derivatives, for their anticancer potency was suggested.

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2023 – Systematic comparison of unilamellar vesicles reveals that archaeal core lipid membranes are more permeable than bacterial membranes
Vesicle Prep Pro Publication in PLoS Biol (2023) Authors: Łapińska U., Glover G., Kahveci Z., Irwin N., Milner D., Tourte M., Albers S., Santoro A., Richards T., Pagliara S.

One of the deepest branches in the tree of life separates the Archaea from the Bacteria. These prokaryotic groups have distinct cellular systems including fundamentally different phospholipid membrane bilayers. This dichotomy has been termed the lipid divide and possibly bestows different biophysical and biochemical characteristics on each cell type. Classic experiments suggest that bacterial membranes (formed from lipids extracted from Escherichia coli, for example) show permeability to key metabolites comparable to archaeal membranes (formed from lipids extracted from Halobacterium salinarum), yet systematic analyses based on direct measurements of membrane permeability are absent. Here, we develop a new approach for assessing the membrane permeability of approximately 10 μm unilamellar vesicles, consisting of an aqueous medium enclosed by a single lipid bilayer. Comparing the permeability of 18 metabolites demonstrates that diether glycerol-1-phosphate lipids with methyl branches, often the most abundant membrane lipids of sampled archaea, are permeable to a wide range of compounds useful for core metabolic networks, including amino acids, sugars, and nucleobases. Permeability is significantly lower in diester glycerol-3-phosphate lipids without methyl branches, the common building block of bacterial membranes. To identify the membrane characteristics that determine permeability, we use this experimental platform to test a variety of lipid forms bearing a diversity of intermediate characteristics. We found that increased membrane permeability is dependent on both the methyl branches on the lipid tails and the ether bond between the tails and the head group, both of which are present on the archaeal phospholipids. These permeability differences must have had profound effects on the cell physiology and proteome evolution of early prokaryotic forms. To explore this further, we compare the abundance and distribution of transmembrane transporter-encoding protein families present on genomes sampled from across the prokaryotic tree of life. These data demonstrate that archaea tend to have a reduced repertoire of transporter gene families, consistent with increased membrane permeation. These results demonstrate that the lipid divide demarcates a clear difference in permeability function with implications for understanding some of the earliest transitions in cell origins and evolution.

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2023 – The Specific Effect of Grapefruit Seed, Sea-Buckthorn Leaves, and Chaga Extracts on the Properties of Model Lipid Membranes
Vesicle Prep Pro Publication in Cell Tiss. Biol. (2023). Authors: Efimova S, Zakharova A., Chernyshova D., Ostroumova O.

The effect of extracts of grapefruit seeds (EGSs), sea-buckthorn leaves (EBLs), and chaga (ECs) on model lipid membranes has been investigated. It has been shown that the threshold concentrations of EGSs and ECs that induced destabilization of phosphatidylglycerol-enriched bilayers are 1.3–1.4 times lower than for phosphatidylcholine-containing membranes. It has been established that EGSs and EBLs reduce the boundary potential of membranes formed from a mixture of phosphatadylcholine and cholesterol (the changes reach 45 and 40 mV at concentrations of 60 and 800 μg/mL, respectively). ECs did not produce pronounced potential-modifying effect. It was shown that changes in the boundary potential in the presence of EBLs were due to the presence of flavonols, quercetin, and myricetin in its composition. Using the method of differential scanning calorimetry, it was also found that quercetin and myricetin were able to influence the thermotropic behavior of membrane lipids and, consequently, their packing density. The potentiation of the pore-forming activity of the antifungal polyene macrolide nystatin and antibacterial lipopeptide polymyxin B was shown with introduction of an EBLs. These data indicate a possible synergism of the antimicrobial action of the tested antibiotics and EBLs, which can be used to generate combined broad-spectrum antimicrobial agents.

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2023 – Transmembrane signaling by a synthetic receptor in artificial cells
Vesicle Prep Pro Publication in Nat. Commun (2023) Authors: Bretschneider Søgaard A., Bøtker Pedersen A., Borup Løvschall K., Monge P., Hammer Jakobsen J., Džabbarova L., Friis Nielsen L., Stevanovic S., Walther R., Zelikin A.

Signal transduction across biological membranes is among the most important evolutionary achievements. Herein, for the design of artificial cells, we engineer fully synthetic receptors with the capacity of transmembrane signaling, using tools of chemistry. Our receptors exhibit similarity with their natural counterparts in having an exofacial ligand for signal capture, being membrane anchored, and featuring a releasable messenger molecule that performs enzyme activation as a downstream signaling event. The main difference from natural receptors is the mechanism of signal transduction, which is achieved using a self-immolative linker. The receptor scaffold is modular and can readily be re-designed to respond to diverse activation signals including biological or chemical stimuli. We demonstrate an artificial signaling cascade that achieves transmembrane enzyme activation, a hallmark of natural signaling receptors. Results of this work are relevant for engineering responsive artificial cells and interfacing them and/or biological counterparts in co-cultures.

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2023 – CNS4 causes subtype-specific changes in agonist efficacy and reversal potential of permeant cations in NMDA receptors
Port-a-Patch Publication in Pharmacol. Res. Perspect. (2023) Boehringer S.C., Johnston T.V., Kwapisz L.C., VandeVord P.J., Costa B.M.

The NMDA subtype of glutamate receptor serves as an attractive drug target for the treatment of disorders evolving from hyper- or hypoglutamatergic conditions. Compounds that optimize the function of NMDA receptors are of great clinical sig -nificance. Here, we present the pharmacological characterization of a biased allosteric modulator, CNS4. Results indicate that CNS4 sensitizes ambient levels of agonists and reduces higher- concentration glycine & glutamate efficacy in 1/2AB receptors, but minimally alters these parameters in diheteromeric 1/2A or 1/2B receptors. Glycine efficacy is increased in both 1/2C and 1/2D, while glutamate efficacy is decreased in 1/2C and unaltered in 1/2D. CNS4 does not affect the activity of competitive antago-nist binding at glycine (DCKA) and glutamate (DL- AP5) sites; however, it decreases memantine potency in 1/2A receptors but not in 1/2D receptors. Current–voltage (I- V) relationship studies indicate that CNS4 potentiates 1/2A inward currents, a phe -nomenon that was reversed in the absence of permeable Na+ ions. In 1/2D recep-tors, CNS4 blocks inward currents based on extracellular Ca2+ concentration. Further, CNS4 positively modulates glutamate potency on E781A_1/2A mutant receptors, indicating its role at the distal end of the 1/2A agonist binding domain interface. Together, these findings reveal that CNS4 sensitizes ambient agonists and allosteri-cally modulates agonist efficacy by altering Na+ permeability based on the GluN2 subunit composition. Overall, the pharmacology of CNS4 aligns with the need for drug candidates to treat hypoglutamatergic neuropsychiatric conditions such as loss function GRIN disorders and anti-NMDA receptor encephalitis.

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2023 – Electrophysiological Techniques on the Study of Endolysosomal Ion Channels
Port-a-Patch Publication in Handbook of Experimental Pharmacology (2023) Authors: Cheng-Chan Chen

Endolysosomal ion channels are a group of ion channel proteins that are functionally expressed on the membrane of endolysosomal vesicles. The electrophysiological properties of these ion channels in the intracellular organelle membrane cannot be observed using conventional electrophysiological techniques. This section compiles the different electrophysiological techniques utilized in recent years to study endolysosomal ion channels and describes their methodological characteristics, emphasizing the most widely used technique for whole endolysosome recordings to date. This includes the use of different pharmacological tools and genetic tools for the application of patch-clamping techniques for specific stages of endolysosomes, allowing the recording of ion channel activity in different organelles, such as recycling endosomes, early endosomes, late endosomes, and lysosomes. These electrophysiological techniques are not only cutting-edge technologies that help to investigate the biophysical properties of known and unknown intracellular ion channels but also help us to investigate the physiopathological role of these ion channels in the distribution of dynamic vesicles and to identify new therapeutic targets for precision medicine and drug screening.

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2023 – Voltage-gated sodium channels, potential targets of Tripterygium wilfordii Hook. f. to exert activity and produce toxicity
Port-a-Patch Publication in J. Ethnopharmacol. (2023). Authors: Xu Y., Li W., Wen R., Sun J., Liu X., Zhao S., Zhang J., Liu Y., Zhao M.

Ethnopharmacology relevance
Tripterygium wilfordii Hook. f. has been widely used in clinical practice due to its good anti-inflammatory and analgesic activities. However, its application is limited by potential toxicity and side effects.

Aim of the study
The study aimed to identify the mechanisms responsible for the pharmacological activity and cardiotoxicity of the main monomers of Tripterygium wilfordii.

Materials and methods
Database analysis predicted that ion channels may be potential targets of Tripterygium wilfordii. The regulatory effects of monomers (triptolide, celastrol, demethylzeylasteral, and wilforgine) on protein Nav1.5 and Nav1.7 were predicted and detected by Autodock and patch clamping. Then, we used the formalin-induced pain model and evaluated heart rate and myocardial zymograms to investigate the analgesic activity and cardiotoxicity of each monomer in vivo.

Results
All four monomers were able to bind to Nav1.7 and Nav1.5 with different binding energies and subsequently inhibited the peak currents of both Nav1.7 and Nav1.5. The monomers all exhibited analgesic effects on formalin-induced pain; therefore, we hypothesized that Nav1.7 is one of the key analgesic targets. Demethylzeylasteral reduced heart rate and increased the level of creatine kinase-MB, thus suggesting a potential cardiac risk; data suggested that the inhibitory effect on Nav1.5 might be an important factor underlying its cardiotoxicity.

Conclusion
Our findings provide an important theoretical basis for the further screening of active monomers with higher levels of activity and lower levels of toxicity.

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2023 – Adeno-Associated virus 8 delivers an immunomodulatory peptide to mouse liver more efficiently than to rat liver
Port-a-Patch publicaation in PLoS ONE (2023) Authors: Wang Y., Hurley A., De Giorgi M., Tanner M., Hu R., Pennington M., Lagor W., Beeton C.

Targeting the Kv1.3 potassium channel has proven effective in reducing obesity and the severity of animal models of autoimmune disease. Stichodactyla toxin (ShK), isolated from the sea anemone Stichodactyla helianthus, is a potent blocker of Kv1.3. Several of its analogs are some of the most potent and selective blockers of this channel. However, like most biologics, ShK and its analogs require injections for their delivery, and repeated injections reduce patient compliance during the treatment of chronic diseases. We hypothesized that inducing the expression of an ShK analog by hepatocytes would remove the requirement for frequent injections and lead to a sustained level of Kv1.3 blocker in the circulation. To this goal, we tested the ability of Adeno-Associated Virus (AAV)8 vectors to target hepatocytes for expressing the ShK analog, ShK-235 (AAV-ShK-235) in rodents. We designed AAV8 vectors expressing the target transgene, ShK-235, or Enhanced Green fluorescent protein (EGFP). Transduction of mouse livers led to the production of sufficient levels of functional ShK-235 in the serum from AAV-ShK-235 single-injected mice to block Kv1.3 channels. However, AAV-ShK-235 therapy was not effective in reducing high-fat diet-induced obesity in mice. In addition, injection of even high doses of AAV8-ShK-235 to rats resulted in a very low liver transduction efficiency and failed to reduce inflammation in a well-established rat model of delayed-type hypersensitivity. In conclusion, the AAV8-based delivery of ShK-235 was highly effective in inducing the secretion of functional Kv1.3-blocking peptide in mouse, but not rat, hepatocytes yet did not reduce obesity in mice fed a high-fat diet.

Read more about the article here.

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2023 – Enriched cell-free and cell-based native membrane derived vesicles (nMV) enabling rapid in-vitro electrophysiological analysis of the voltage-gated sodium channel 1.5
Port-a-Patch and Orbit 16 Publication in BBA - Biomembranes (2023) Authors: Pandey Y, Dondapati S.K., Kubick S.

Here, we demonstrate the utility of native membrane derived vesicles (nMVs) as tools for expeditious electrophysiological analysis of membrane proteins. We used a cell-free (CF) and a cell-based (CB) approach for preparing protein-enriched nMVs. We utilized the Chinese Hamster Ovary (CHO) lysate-based cell-free protein synthesis (CFPS) system to enrich ER-derived microsomes in the lysate with the primary human cardiac voltage-gated sodium channel 1.5 (hNaV1.5; SCN5A) in 3 h. Subsequently, CB-nMVs were isolated from fractions of nitrogen-cavitated CHO cells overexpressing the hNaV1.5. In an integrative approach, nMVs were micro-transplanted into  Xenopus laevis oocytes. CB-nMVs expressed native lidocaine-sensitive hNaV1.5 currents within 24 h; CF-nMVs did not elicit any response. Both the CB- and CF-nMV preparations evoked single-channel activity on the planar lipid bilayer while retaining sensitivity to lidocaine application. Our findings suggest a high usability of the quick-synthesis CF-nMVs and maintenance-free CB-nMVs as ready-to-use tools for in-vitro analysis of electrogenic membrane proteins and large, voltage-gated ion channels.

Application note
TMEM175 – “TMEM 175 activity measurement in lysosomes on Nanion’s SURFE2R N1″
SURFE2R N1 application note:

TMEM175 is a lysosomal cation leak channel, which impairment has been linked to Parkinson’s disease and other neurodegenerative disorders; thereby making it an interesting drug target. The presented recordings illustrate SURFE2R’s potential as means for target validation and compound screening against TMEM175 channels residing in lysosomal membranes.

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2023 – Predicting hERG repolarization power at 37°C from recordings at room temperature
SyncroPatch 384 Publication in Clinical and Translational Medicine (2023) Authors: Oliveira-Mendes B.B.R., Alameh M., Montnach J., Ollivier B., Gibaud S., Feliciangeli S., Lesage F., Charpentier F., Loussouarn G., De Waard M., Baró I.

Loss-of-function and gain-of-function mutations in the KCNH2 gene cause long and short-QT syndromes (LQTS or SQTS), respectively, predisposing to life-threatening cardiac arrhythmias. KCNH2 encodes the voltage-gated K+ channel hERG that generates the delayed rectifier K+ current IKr controlling the action potential (AP) duration. Prolonged or shortened ventricular AP durations are visualized as abnormal QT interval duration on the electrocardiogram. The occurrence and severity of KCNH2-related arrhythmias are determined by the variant functional impact. Sequencing KCNH2 has provided a plethora of variants associated or not with pathological cardiac phenotypes and indexed in the ClinVar NCBI database. Discriminating pathogenic variants from benign ones would clarify the genetic background of patients and relatives, and stratify the risk of adverse events. In the face of a wide spectrum of hERG functional defects, we looked for a way to summarize the net loss or gain of function in a unique index. We defined the repolarization power as the time integral of the K+ current (IhERG) developed during an AP clamp.

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2023 – G-Quadruplex-Filtered Selective Ion-to-Ion Current Amplification for Non-Invasive Ion Monitoring in Real Time
Orbit mini Publication in Advanced Materials (2023) Authors: Yoo H., Lee H-R, Kang S-B, Lee J., Park K., Yoo H., Kim J., Chung T.D., Lee K-M., Lim H-H., Son C.Y., Sun J-Y, Oh S.S.

Living cells efflux intracellular ions for maintaining cellular life, so intravital measurements of specific ion signals are of significant importance for studying cellular functions and pharmacokinetics. In this work, de novo synthesis of artificial K+-selective membrane and its integration with polyelectrolyte hydrogel-based open-junction ionic diode (OJID) is demonstrated, achieving a real-time K+-selective ion-to-ion current amplification in complex bioenvironments. By mimicking biological K+ channels and nerve impulse transmitters, in-line K+-binding G-quartets are introduced across freestanding lipid bilayers by G-specific hexylation of monolithic G-quadruplex, and the pre-filtered K+ flow is directly converted to amplified ionic currents by the OJID with a fast response time at 100 ms intervals. By the synergistic combination of charge repulsion, sieving, and ion recognition, the synthetic membrane allows K+ transport exclusively without water leakage; it is 250× and 17× more permeable toward K+ than monovalent anion, Cl, and polyatomic cation, N-methyl-d-glucamine+, respectively. The molecular recognition-mediated ion channeling provides a 500% larger signal for K+ as compared to Li+ (0.6× smaller than K+) despite the same valence. Using the miniaturized device, non-invasive, direct, and real-time K+ efflux monitoring from living cell spheroids is achieved with minimal crosstalk, specifically in identifying osmotic shock-induced necrosis and drug-antidote dynamics.

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2023 – Amphipathic peptide–phospholipid nanofibers: Kinetics of fiber formation and molecular transfer between assemblies
Vesicle Prep Pro Publication in Biophysical Chemistry (2023) Authors: Shimizu C., Ikeda K., Nakao H., Nakano M.

Understanding the kinetics of nano-assembly formation is important to elucidate the biological processes involved and develop novel nanomaterials with biological functions. In the present study, we report the kinetic mechanisms of nanofiber formation from a mixture of phospholipids and the amphipathic peptide 18A[A11C], carrying cysteine substitution of the apolipoprotein A-I-derived peptide 18A at residue 11. 18A[A11C] with acetylated N-terminus and amidated C-terminus can associate with phosphatidylcholine to form fibrous aggregates at neutral pH and lipid-to-peptide molar ratio of ∼1, although the reaction pathways of self-assembly remain unclear. Here, the peptide was added to giant 1-palmitoyl-2-oleoyl phosphatidylcholine vesicles to monitor nanofiber formation under fluorescence microscopy. The peptide initially solubilized the lipid vesicles into particles smaller than the resolution of optical microscope, and fibrous aggregates appeared subsequently. Transmission electron microscopy and dynamic light scattering analyses revealed that the vesicle-solubilized particles were spherical or circular, measuring ∼10–20 nm in diameter. The rate of nanofiber formation of 18A with 1,2-dipalmitoyl phosphatidylcholine from the particles was proportional to the square of lipid–peptide concentration in the system, suggesting that the association of particles, accompanied by conformational changes, was the rate-limiting step. Moreover, molecules in the nanofibers could be transferred between aggregates faster than those in the lipid vesicles. These findings provide useful information for the development and control of nano-assembling structures using peptides and phospholipids.

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2023 – D-galactose causes sinoatrial node dysfunction: from phenotype to mechanism
CardioExcyte 96 Publication in Aging (2023) Authors: Zhang H., Chen C., Liu Y., Chen W., Qi J., Xu Y., Ren Lu., Yang G., Min D., Liu Z., Cai X., Hao M., Xu G., Hou P.

With the population aging, age-related sinoatrial node dysfunction (SND) has been on the rise. Sinoatrial node (SAN) degeneration is an important factor for the age-related SND development. However, there is no suitable animal modeling method in this field. Here, we investigated whether D-galactose could induce SAN degeneration and explored the associated mechanism. In vivo, twelve C57BL/6 mice were divided into Control and D-galactose group to receive corresponding treatments. Senescence was confirmed by analyzing the hair and weight; cardiac function was evaluated through echocardiography, cerebral blood flux and serum-BNP; the SAN function was evaluated by electrocardiogram; fibrotic change was evaluated by Masson's trichrome staining and oxidative stress was assessed through DHE staining and serum indicators. Mechanism was verified through immunofluorescence-staining and Western blotting. In vitro, mouse-atrial-myocytes were treated with D-galactose, and edaravone was utilized as the ROS scavenger. Senescence, oxidative stress, proliferation ability and mechanism were verified through various methods, and intuitive evidence was obtained through electrophysiological assay. Finally, we concluded that D-galactose can be used to induce age-related SND, in which oxidative stress plays a key role, causing PITX2 ectopic expression and downregulates SHOX2 expression, then through the downstream GATA4/NKX2-5 axis, results in pacing-related ion channels dysfunction, and hence SND development.

 

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2023 – PTX3 from vascular endothelial cells contributes to trastuzumab-induced cardiac complications
CardioExcyte 96 Publication in Cardivasc. Res. (2023) Authors: Xu Z., Gao Z., Fu H., Zeng Y., Jin Y., Xu B., Zhang Y., Pan Z., Chen X., Zhang X., Wang X., Yan H., Yang X., Yang B., He Q., Luo P.
Aims

Trastuzumab, the first humanized monoclonal antibody that targets human epidermal growth factor receptor 2 (ERBB2/HER2), is currently used as a first-line treatment for HER2 (+) tumours. However, trastuzumab increases the risk of cardiac complications without affecting myocardial structure, suggesting a distinct mechanism of cardiotoxicity.

Methods and results

We used medium from trastuzumab-treated human umbilical vein endothelial cells (HUVECs) to treat CCC-HEH-2 cells, the human embryonic cardiac tissue-derived cell lines, and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to assess the crosstalk between vascular endothelial cells (VECs) and cardiomyocytes. Protein mass spectrometry analysis was used to identify the key factors from VECs that regulate the function of cardiomyocytes. We applied RNA-sequencing to clarify the mechanism, by which PTX3 causes cardiac dysfunction. We used an anti-human/rat HER2 (neu) monoclonal antibody to generate a rat model that was used to evaluate the effects of trastuzumab on cardiac structure and function and the rescue effects of lapatinib on trastuzumab-induced cardiac side effects. Medium from trastuzumab-treated HUVECs apparently impaired the contractility of CCC-HEH-2 cells and iPSC-CMs. PTX3 from VECs caused defective cardiomyocyte contractility and cardiac dysfunction in mice, phenocopying trastuzumab treatment. PTX3 affected calcium homoeostasis in cardiomyocytes, which led to defective contractile properties. EGFR/STAT3 signalling in VECs contributed to the increased expression and release of PTX3. Notably, lapatinib, a dual inhibitor of EGFR/HER2, could rescue the cardiac complications caused by trastuzumab by blocking the release of PTX3.

Conclusion

We identified a distinct mode of cardiotoxicity, wherein the activation of EGFR/STAT3 signalling by trastuzumab in VECs promotes PTX3 excretion, which contributes to the impaired contractility of cardiomyocytes by inhibiting cellular calcium signalling. We confirmed that lapatinib could be a feasible preventive agent against trastuzumab-induced cardiac complications and provided the rationale for the combined application of lapatinib and trastuzumab in cancer therapy.

 

Publication Link
2023 – Symport and antiport mechanisms of human glutamate transporters
SURFE2R N1 Publication in Nature Communications (2023) Qiu B., Boudker O.

Excitatory amino acid transporters (EAATs) uptake glutamate into glial cells and neurons. EAATs achieve million-fold transmitter gradients by symporting it with three sodium ions and a proton, and countertransporting a potassium ion via an elevator mechanism. Despite the availability of structures, the symport and antiport mechanisms still need to be clarified. We report high-resolution cryo-EM structures of human EAAT3 bound to the neurotransmitter glutamate with symported ions, potassium ions, sodium ions alone, or without ligands. We show that an evolutionarily conserved occluded translocation intermediate has a dramatically higher affinity for the neurotransmitter and the countertransported potassium ion than outward- or inward-facing transporters and plays a crucial role in ion coupling. We propose a comprehensive ion coupling mechanism involving a choreographed interplay between bound solutes, conformations of conserved amino acid motifs, and movements of the gating hairpin and the substrate-binding domain.

Read more in the paper here.

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2023 – A bioelectrochemical approach based on a solid supported membrane to evaluate the effect of natural products on Ca2+-ATPase: The case of 6-gingerol
SURFE2R ONE (a predecessor of the SURFE2R N1) Publication in Biochimica Acta (2023) Authors: Sfragano P.S.., Palchetti I., Tadini-Buoninsegni F.

The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) plays an essential role in maintaining the low cytosolic Ca2+ level that enables a variety of cellular processes. SERCA couples ATP hydrolysis to the transport of two Ca2+ ions against their electrochemical potential gradient from the cytoplasm into the lumen of the sarco/endoplasmic reticulum (SR/ER). Because of its central role in regulating cytoplasmic Ca2+ concentration, SERCA dysfunction has been associated with several pathological conditions. Stimulation of SERCA activity may represent a potential therapeutic strategy in various disease states connected with dysfunctional SERCA. The natural phenolic compound 6-gingerol, the most abundant and the major biologically active compound of ginger, was reported to activate the SERCA enzyme. The present study aimed at investigating the effect of 6-gingerol on SERCA transport activity using a bioelectrochemical approach based on a solid supported membrane (SSM). We first performed a voltammetric characterization of 6-gingerol to better understand its electrochemical behavior. We then studied the interaction of 6-gingerol with SR vesicles containing SERCA adsorbed on the SSM electrode. The measured current signals indicated that ATP-dependent Ca2+ translocation by SERCA was remarkably increased in the presence of 6-gingerol at low micromolar concentration. We also found that 6-gingerol has a rather high affinity for SERCA (EC50 of 1.8 ± 0.3 µM), and SERCA activation by 6-gingerol is reversible. The observed stimulatory effect of 6-gingerol on SERCA Ca2+-translocating activity may be beneficial in the prevention and/or treatment of pathological conditions related to SERCA dysfunction.

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2023 – Mutation in glutamate transporter homologue GltTk provides insights into pathologic mechanism of episodic ataxia 6
SURFE2R N1 Publication in Nature Communications (2023) Colucci E., Anshari Z., Patiño-Ruiz M., Nemchinova M., Whittaker J., Slotboom D., Guskov A.

Episodic ataxias (EAs) are rare neurological conditions affecting the nervous system and typically leading to motor impairment. EA6 is linked to the mutation of a highly conserved proline into an arginine in the glutamate transporter EAAT1. In vitro studies showed that this mutation leads to a reduction in the substrates transport and an increase in the anion conductance. It was hypothesised that the structural basis of these opposed functional effects might be the straightening of transmembrane helix 5, which is kinked in the wild-type protein. In this study, we present the functional and structural implications of the mutation P208R in the archaeal homologue of glutamate transporters GltTk. We show that also in GltTk the P208R mutation leads to reduced aspartate transport activity and increased anion conductance, however a cryo-EM structure reveals that the kink is preserved. The arginine side chain of the mutant points towards the lipidic environment, where it may engage in interactions with the phospholipids, thereby potentially interfering with the transport cycle and contributing to stabilisation of an anion conducting state.

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2023 – Antidepressant-like activity of a brain penetrant HCN channel inhibitor in mice
Patchliner Publication in Front. Pharmacol. (2023) Authors: Pinares-Garcia P., Spyrou J., McKenzie C.E., Forster I.C., Soh M.S., Syazwan E.M., Atif M. Reid C.A.

Changes in Hyperpolarization-Activated Cyclic Nucleotide-Gated (HCN) channel function have been linked to depressive-like traits, making them potential drug targets. However, there is currently no peer-reviewed data supporting the use of a small molecule modulator of HCN channels in depression treatment. Org 34167, a benzisoxazole derivative, has been patented for the treatment of depression and progressed to Phase I trials. In the current study, we analysed the biophysical effects of Org 34167 on HCN channels in stably transfected human embryonic kidney 293 (HEK293) cells and mouse layer V neurons using patch-clamp electrophysiology, and we utilised three high-throughput screens for depressive-like behaviour to assess the activity of Org 34167 in mice. The impact of Org 34167 on locomotion and coordination were measured by performing rotarod and ledged beam tests. Org 34167 is a broad-spectrum inhibitor of HCN channels, slowing activation and causing a hyperpolarising shift in voltage-dependence of activation. It also reduced Ih-mediated sag in mouse neurons. Org 34167 (0.5 mg/kg) reduced marble burying and increased the time spent mobile in the Porsolt swim and tail suspension tests in both male and female BALB/c mice, suggesting reduced depressive-like behaviour. Although no adverse effects were seen at 0.5 mg/kg, an increase in dose to 1 mg/kg resulted in visible tremors and impaired locomotion and coordination. These data support the premise that HCN channels are valid targets for anti-depressive drugs albeit with a narrow therapeutic index. Drugs with higher HCN subtype selectivity are needed to establish if a wider therapeutic window can be obtained.

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2023 – Design, synthesis, and biological evaluation of novel dihydropteridone derivatives possessing oxadiazoles moiety as potent inhibitors of PLK1
SyncroPatch 384 Publication in Eur. J. Med. Chem. (2023) Authors: Li Z., Mei S., Liu J., Huang J., Yue H., Ge T., Wang K., He X., Gu Y-C., Hu C., Tong M., Shi X., Zhao Y., Liu Y., Qin M., Gong P., Hou Y.

Polo like kinase 1 (PLK1) is a serine/threonine kinase that is widely distributed in eukaryotic cells and plays an important role in multiple phases of the cell cycle. Its importance in tumorigenesis has been increasingly recognized in recent years. Herein, we describe the optimization of a series of novel dihydropteridone derivatives (13a-13v and 21g-21l) possessing oxadiazoles moiety as potent inhibitors of PLK1. Compound 21g exhibited improved PLK1 inhibitory capability with an IC50 value of 0.45 nM and significant anti-proliferative activities against four tumor-derived cell lines (MCF-7 IC50 = 8.64 nM, HCT-116 IC50 = 26.0 nM, MDA-MB-231 IC50 = 14.8 nM and MV4-11 IC50 = 47.4 nM) with better pharmacokinetic characteristics than BI2536 in mice (AUC0-t = 11 227 ng h mL−1 vs 556 ng h mL−1). Moreover, 21g exhibited moderate liver microsomal stability and excellent pharmacokinetic profile (AUC0-t = 11227 ng h mL−1, oral bioavailability of 77.4%) in Balb/c mice, acceptable PPB, improved PLK1 inhibitory selectivity, and no apparent toxicity was observed in the acute toxicity assay (20 mg/kg). Further investigation showed that 21 g could arrest HCT-116 cells in G2 phase and induce apoptosis in a dose-dependent manner. These results indicate that 21g is a promising PLK1 inhibitor.

Application Note PDF
Lot-to-lot Robustness Study of Commercial Human iPSC-derived Cardiomyocytes
Case study PDF
Urease-positive bacteria – “Quantification of Urease Activity via High Throughput Conductivity Measurements”
Flyer PDF
Investigating PIEZO1 using the Patchliner
Publication Link
2023 – The Degree of Hydroxylation of Phenolic Rings Determines the Ability of Flavonoids and Stilbenes to Inhibit Calcium-Mediated Membrane Fusion
Vesicle Prep Pro Publication in Nutrients (2023) Authors: Zlodeeva P.D., Shekunov E.V., Ostroumova O.S., Efimova S.S.

This paper discusses the possibility of using plant polyphenols as viral fusion inhibitors with a lipid-mediated mechanism of action. The studied agents are promising candidates for the role of antiviral compounds due to their high lipophilicity, low toxicity, bioavailability, and relative cheapness. Fluorimetry of calcein release at the calcium-mediated fusion of liposomes, composed of a ternary mixture of dioleoyl phosphatidylcholine, dioleoyl phosphatidylglycerol, and cholesterol, in the presence of 4′-hydroxychalcone, cardamonin, isoliquiritigenin, phloretin, resveratrol, piceatannol, daidzein, biochanin A, genistein, genistin, liquiritigenin, naringenin, catechin, taxifolin, and honokiol, was performed. It was found that piceatannol significantly inhibited the calcium-induced fusion of negatively charged vesicles, while taxifolin and catechin showed medium and low antifusogenic activity, respectively. As a rule, polyphenols containing at least two OH-groups in both phenolic rings were able to inhibit the calcium-mediated fusion of liposomes. In addition, there was a correlation between the ability of the tested compounds to inhibit vesicle fusions and to perturb lipid packing. We suggest that the antifusogenic action of polyphenols was determined by the depth of immersion and the orientation of the molecules in the membrane.

Publication Link
2023 – GABRG2 Variants Associated with Febrile Seizures
SyncroPatch 384 Publication in Biomolecules (2023) Authors: Hernandez C.C., Shen Y., Hu N., Shen W., Narayanan V., Ramsey K., He W., Zou L., Macdonald R.L.

Febrile seizures (FS) are the most common form of epilepsy in children between six months and five years of age. FS is a self-limited type of fever-related seizure. However, complicated prolonged FS can lead to complex partial epilepsy. We found that among the GABAA receptor subunit (GABR) genes, most variants associated with FS are harbored in the γ2 subunit (GABRG2). Here, we characterized the effects of eight variants in the GABAA receptor γ2 subunit on receptor biogenesis and channel function. Two-thirds of the GABRG2 variants followed the expected autosomal dominant inheritance in FS and occurred as missense and nonsense variants. The remaining one-third appeared as de novo in the affected probands and occurred only as missense variants. The loss of GABAA receptor function and dominant negative effect on GABAA receptor biogenesis likely caused the FS phenotype. In general, variants in the GABRG2 result in a broad spectrum of phenotypic severity, ranging from asymptomatic, FS, genetic epilepsy with febrile seizures plus (GEFS+), and Dravet syndrome individuals. The data presented here support the link between FS, epilepsy, and GABRG2 variants, shedding light on the relationship between the variant topological occurrence and disease severity.

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2023 – KI04 an Aminoglycosides-Derived Molecule Acts as an Inhibitor of Human Connexin46 Hemichannels Expressed in HeLa Cells
Patchliner Publication in Biomolecules (2023) Authors: Chang C.W.T., Poudyal N., Verdugo D.A., Peña F., Stehberg J., Retamal M.A.

Background: Connexins (Cxs) are proteins that help cells to communicate with the extracellular media and with the cytoplasm of neighboring cells. Despite their importance in several human physiological and pathological conditions, their pharmacology is very poor. In the last decade, some molecules derived from aminoglycosides have been developed as inhibitors of Cxs hemichannels. However, these studies have been performed in E. coli, which is a very simple model. Therefore, our main goal is to test whether these molecules have similar effects in mammalian cells. Methods: We transfected HeLa cells with the human Cx46tGFP and characterized the effect of a kanamycin-derived molecule (KI04) on Cx46 hemichannel activity by time-lapse recordings, changes in phosphorylation by Western blot, localization by epifluorescence, and possible binding sites by molecular dynamics (MD). Results: We observed that kanamycin and KI04 were the most potent inhibitors of Cx46 hemichannels among several aminoglycosides, presenting an IC50 close to 10 μM. The inhibitory effect was not associated with changes in Cx46 electrophoretic mobility or its intracellular localization. Interestingly, 5 mM DTT did not reverse KI04 inhibition, but the KI04 effect completely disappeared after washing out KI04 from the recording media. MD analysis revealed two putative binding sites of KI04 in the Cx46 hemichannel. Results: These results demonstrate that KI04 could be used as a Cx46 inhibitor and could help to develop future selective Cx46 inhibitors.

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2023 – Validation of TREK1 ion channel activators as an immunomodulatory and neuroprotective strategy in neuroinflammation
Port-a-Patch mini Publication in Biol. Chem. (2023) Authors: Schroeter C.B., Nelke C., Schewe M., Spohler L., Herrmann A.M., Müntefering T., Huntemann N., Kuzikov M., Gribbon P., Albrecht S., Bock S., Hundehege P., Neelsen L.C., Baukrowitz T., Seebohm G., Wünsch B., Bittner S., Ruck T., Budde T. Meuth S.G.

Modulation of two-pore domain potassium (K2P) channels has emerged as a novel field of therapeutic strategies as they may regulate immune cell activation and metabolism, inflammatory signals, or barrier integrity. One of these ion channels is the TWIK-related potassium channel 1 (TREK1). In the current study, we report the identification and validation of new TREK1 activators. Firstly, we used a modified potassium ion channel assay to perform high-throughput-screening of new TREK1 activators. Dose-response studies helped to identify compounds with a high separation between effectiveness and toxicity. Inside-out patch-clamp measurements of Xenopus laevis oocytes expressing TREK1 were used for further validation of these activators regarding specificity and activity. These approaches yielded three substances, E1, B3 and A2 that robustly activate TREK1. Functionally, we demonstrated that these compounds reduce levels of adhesion molecules on primary human brain and muscle endothelial cells without affecting cell viability. Finally, we studied compound A2 via voltage-clamp recordings as this activator displayed the strongest effect on adhesion molecules. Interestingly, A2 lacked TREK1 activation in the tested neuronal cell type. Taken together, this study provides data on novel TREK1 activators that might be employed to pharmacologically modulate TREK1 activity.

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2023 – Dimerization mechanism of an inverted-topology ion channel in membranes
Orbit mini Pre-Print Publication in BioRxiv (2023) Authors: Ernst M., Orabi E.A., Stockbridge R.B., Faraldo-Gómez J.D., Robertson J.L.

Many ion channels are multi-subunit complexes with a polar permeation pathway at the oligomeric interface, but their mechanisms of assembly into functional, thermodynamically stable units within the membrane are largely unknown. Here we characterize the assembly of the inverted-topology, homodimeric fluoride channel Fluc, leveraging a known mutation, N43S, that weakens Na+ binding to the dimer interface, thereby unlocking the complex. While single-channel recordings show Na+ is required for activation, single-molecule photobleaching and bulk Förster Resonance Energy Transfer experiments in lipid bilayers demonstrate that N43S Fluc monomers and dimers exist in dynamic equilibrium, even without Na+. Molecular dynamics simulations indicate this equilibrium is dominated by a differential in the lipid-solvation energetics of monomer and dimer, which stems from hydrophobic exposure of the polar ion pathway in the monomer. These results suggest a model wherein membrane-associated forces induce channel assembly while subsequent factors, in this case Na+ binding, result in channel activation.

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2023 – Novel neuroactive steroids as positive allosteric modulators of NMDA receptors: mechanism, site of action, and rescue pharmacology on GRIN variants associated with neurological conditions
SyncroPatch 384 Publication in Cell. Mol. Life Sci. (2023) Authors: Tang W., Beckley J.T., Zhang J., Song R., Xu Y., Kim S., Quirk M.C., Robichaud A.J., Diaz E.S., Myers S.J., Doherty J.J., Ackley M.A., Traynelis S.F., Yuan H.

N-methyl-D-aspartate receptors (NMDARs) play vital roles in normal brain functions (i.e., learning, memory, and neuronal development) and various neuropathological conditions, such as epilepsy, autism, Parkinson’s disease, Alzheimer’s disease, and traumatic brain injury. Endogenous neuroactive steroids such as 24(S)-hydroxycholesterol (24(S)-HC) have been shown to influence NMDAR activity, and positive allosteric modulators (PAMs) derived from 24(S)-hydroxycholesterol scaffold can also enhance NMDAR function. This study describes the structural determinants and mechanism of action for 24(S)-hydroxycholesterol and two novel synthetic analogs (SGE-550 and SGE-301) on NMDAR function. We also show that these agents can mitigate the altered function caused by a set of loss-of-function missense variants in NMDAR GluN subunit-encoding GRIN genes associated with neurological and neuropsychiatric disorders. We anticipate that the evaluation of novel neuroactive steroid NMDAR PAMs may catalyze the development of new treatment strategies for GRIN-related neuropsychiatric conditions.

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2023 – TolC-AcrA complex formation monitored by time dependent single-channel electrophysiology
Orbit mini Publication in Biochimie (2023) Authors: Bodrenko I.V., Zewdie T.A., Wang J., Paul E., Witt S., Winterhalter M.

Characterizing protein-protein interaction on a single molecular level is a challenge, experimentally as well as interpretation of the data. For example, Gram-negative bacteria contain protein complexes spanning the outer and inner cell wall devoted to efflux effectively cell toxic substances. Recent seminal work revealed the high-resolution structure of such a tripartic composition TolC-AcrA-AcrB suggesting to design inhibitors preventing efflux of antibiotics. To show that electrophysiology can provide supporting information here, we reconstitute single TolC homotrimer into a planar lipid membrane, apply a transmembrane voltage and follow the assembly of AcrA to TolC using the modulation of the ion current through TolC channel during binding. In particular, the presence of AcrA in solution increases the average ionic current through TolC and, moreover, reduces the ion-current fluctuations caused by flickering of TolC. Here, we show that statistical properties of ion-current fluctuations (the power spectral density) provide a complementary measure of the interaction of the TolC-AcrA complex in presence of putative efflux pump inhibitors. Both characteristics, the average ion current across TolC and the current noise, taken into consideration together, point to a stiffening of the tip of TolC which might reduce the formation of the complex.

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2023 – Evaluation of Cell-Free Synthesized Human Channel Proteins for In Vitro Channel Research
Orbit mini Publication in Membranes (2023) Authors: Nishiguchi R., Tanaka T., Hayashida J., Nakagita T., Zhou W., Takeda H.

Despite channel proteins being important drug targets, studies on channel proteins remain limited, as the proteins are difficult to express and require correct complex formation within membranes. Although several in vitro synthesized recombinant channels have been reported, considering the vast diversity of the structures and functions of channel proteins, it remains unclear which classes of channels cell-free synthesis can be applied to. In this study, we synthesized 250 clones of human channels, including ion channel pore-forming subunits, gap junction proteins, porins, and regulatory subunits, using a wheat cell-free membrane protein production system, and evaluated their synthetic efficiency and function. Western blotting confirmed that 95% of the channels were successfully synthesized, including very large channels with molecular weights of over 200 kDa. A subset of 47 voltage-gated potassium ion channels was further analyzed using a planar lipid bilayer assay, out of which 80% displayed a voltage-dependent opening in the assay. We co-synthesized KCNB1 and KCNS3, a known heteromeric complex pair, and demonstrated that these channels interact on a liposome. These results indicate that cell-free protein synthesis provides a promising solution for channel studies to overcome the bottleneck of in vitro protein production.

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2023 – Inhibition of the hERG potassium ion channel by different non-nucleoside human cytomegalovirus polymerase antiviral inhibitor series and the exploration of variations on a pyrroloquinoline core to reduce cardiotoxicity potential
SyncroPatch 384 Publication in Bioorganic & Medicinal Chemistry (2023) Authors: Kandadai AS., Bai B., Rahim M., Lin F., Gu Z., Qi X., Zhang X., Dong H., Chen Y., Shen J., Nieman JA.

Many non-nucleoside human cytomegalovirus (HCMV) inhibitors have been reported in patent and scientific literature, however, none have reached commercialization despite the urgent need for new HCMV treatments. Herein we report select compounds from different templates that all had low micromolar human ether-à-go-go (hERG) ion channel IC50 values. We also describe a series of pyrroloquinoline derivatives that were designed and synthesized to understand the effect of various substitution on human cytomegalovirus (HCMV) polymerase activity, antiviral activity, and hERG inhibition. These results demonstrated that hERG inhibition can be significantly altered based on the substitution on this template. An HCMV inhibitor with low hERG inhibition and reduced cytotoxicity is also described. The results suggest substitution can be fine tuned for the non-nucleoside polymerase inhibitors to reduce hERG inhibition and maintain HCMV antiviral potency.

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2023 – Rational design of magnetoliposomes for enhanced interaction with bacterial membrane models
Vesicle Prep Pro Publication in Biochimica & Biophysica Acta (BBA) - Biomembranes (2023) Authors: Soares F.A. Costa P., Sousa C.T., Horta M., Pereira-Leite C., Seabra C.L. Lima S.A.C., Reis S., Nunes C.

There is a growing need for alternatives to target and treat bacterial infection. Thus, the present work aims to develop and optimize the production of PEGylated magnetoliposomes (MLPs@PEG), by encapsulating superparamagnetic iron oxide nanoparticles (SPIONs) within fusogenic liposomes. A Box–Behnken design was applied to modulate size distribution variables, using lipid concentration, SPIONs amount and ultrasonication time as independent variables. As a result of the optimization, it was possible to obtain MLPs@PEG with a mean size of 182 nm, with polydispersity index (PDI) of 0.19, and SPIONs encapsulation efficiency (%EE) around 76%. Cytocompatibility assays showed that no toxicity was observed in fibroblasts, for iron concentrations up to 400 μg/ml. Also, for safe lipid and iron concentrations, no hemolytic effect was detected. The fusogenicity of the nanosystems was first evaluated through lipid mixing assays, based on Förster resonance energy transfer (FRET), using liposomal membrane models, mimicking bacterial cytoplasmic membrane and eukaryotic plasma membrane. It was shown that the hybrid nanosystems preferentially interact with the bacterial membrane model. Confocal microscopy and fluorescence lifetime measurements, using giant unilamellar vesicles (GUVs), validated these results. Overall, the developed hybrid nanosystem may represent an efficient drug delivery system with improved targetability for bacterial membrane.

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2022 – Wastewater bacteria remediating the pharmaceutical metformin: Genomes, plasmids and products
SURFE2R N1 Publication in Front. Physiol. (2022) Authors: Martinez-Vaz B.M., Dodge A.G., Lucero R.M., Stockbridge R.B., Robinson A.A., Tassoulas L.J., Wackett L.P.
1Department of Biolog

Metformin is used globally to treat type II diabetes, has demonstrated anti-ageing and COVID mitigation effects and is a major anthropogenic pollutant to be bioremediated by wastewater treatment plants (WWTPs). Metformin is not adsorbed well by activated carbon and toxic N-chloro derivatives can form in chlorinated water. Most earlier studies on metformin biodegradation have used wastewater consortia and details of the genomes, relevant genes, metabolic products, and potential for horizontal gene transfer are lacking. Here, two metformin-biodegrading bacteria from a WWTP were isolated and their biodegradation characterized. Aminobacter sp. MET metabolized metformin stoichiometrically to guanylurea, an intermediate known to accumulate in some environments including WWTPs. Pseudomonas mendocina MET completely metabolized metformin and utilized all the nitrogen atoms for growth. Pseudomonas mendocina MET also metabolized metformin breakdown products sometimes observed in WWTPs: 1-N-methylbiguanide, biguanide, guanylurea, and guanidine. The genome of each bacterium was obtained. Genes involved in the transport of guanylurea in Aminobacter sp. MET were expressed heterologously and shown to serve as an antiporter to expel the toxic guanidinium compound. A novel guanylurea hydrolase enzyme was identified in Pseudomonas mendocina MET, purified, and characterized. The Aminobacter and Pseudomonas each contained one plasmid of 160 kb and 90 kb, respectively. In total, these studies are significant for the bioremediation of a major pollutant in WWTPs today.

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2022 – High throughput measurement of hERG drug block kinetics using the CiPA dynamic protocol
SyncroPatch 384 Publication in J. Pharm. & Tox. Meth. (2022) Authors: Windley M.J., Farr J., TeBay C., Vandenberg J.I., Hill A.P.

The Comprehensive in vitro Proarrhythmic Assay (CiPA) has promoted use of in silico models of drug effects on cardiac repolarization to improve proarrhythmic risk prediction. These models contain a pharmacodynamic component describing drug binding to hERG channels that required in vitro data for kinetics of block, in addition to potency, to constrain them. To date, development and validation has been undertaken using data from manual patch-clamp. The application of this approach at scale requires the development of a high-throughput, automated patch-clamp (APC) implementation. Here, we present a comprehensive analysis of the implementation of the Milnes, or CiPA dynamic protocol, on an APC platform, including quality control and data analysis. Kinetics and potency of block were assessed for bepridil, cisapride, terfenadine and verapamil with data retention/QC pass rate of 21.8% overall, or as high as 50.4% when only appropriate sweep lengths were considered for drugs with faster kinetics. The variability in IC50 and kinetics between manual and APC was comparable to that seen between sites/platforms in previous APC studies of potency. Whilst the experimental success is less than observed in screens of potency alone, it is still significantly greater than manual patch. With the modifications to protocol design, including sweep length, number of repetitions, and leak correction recommended in this study, this protocol can be applied on APC to acquire data comparable to manual patch clamp.

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2022 – An efficient and scalable data analysis solution for automated electrophysiology platforms
SyncroPatch 768PE (a predecessor of SyncroPatch 384) Publication in SLAS Discovery (2022) Authors: Li T., Ginkel M., Yee A.X., Foster L., Chen J., Heyse S., Steigele S.

Ion channels are drug targets for neurologic, cardiac, and immunologic diseases. Many disease-associated mutations and drugs modulate voltage-gated ion channel activation and inactivation, suggesting that characterizing state-dependent effects of test compounds at an early stage of drug development can be of great benefit.

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2022 – Development of an automated screen for Kv7.2 potassium channels and discovery of a new agonist chemotype
SyncroPatch 384PE (a predecessor model of SyncroPatch 384) Publication in Bioorg. & Med. Chem. Lett. (2022) Authors: Hernandez C.C., Tarfa R.A., Miguel I. Limcaoco J., Liu R., Mondal P., Hill C., Duncan R.K., Tzounopoulos T., Stephenson C.R.J., O'Meara M.J., Wipf P.

To identify pore domain ligands on Kv7.2 potassium ion channels, we compared wild-type (WT) and W236L mutant Kv7.2 channels in a series of assays with previously validated and novel agonist chemotypes. Positive controls were retigabine, flupirtine, and RL-81; i.e. Kv7.2 channel activators that significantly shift voltage-dependent activation to more negative potentials (ΔV50) at 5 µM. We identified 6 new compounds that exhibited differential enhancing activity between WT and W236L mutant channels. Whole cell patch-clamp electrophysiology studies were conducted to identify Kv7.2. Kv7.2/3, Kv7.4, and Kv7.5 selectivity. Our results validate the SyncroPatch platform and establish new structure activity relationships (SAR). Specifically, in addition to selective Kv7.2, Kv7.2/3, Kv7.4. and Kv7.5 agonists, we identified a novel chemotype, ZK-21, a 4-aminotetrahydroquinoline that is distinct from any of the previously described Kv7 channel modifiers. Using flexible receptor docking, ZK-21 was predicted to be stabilized by W236 and bind perpendicular to retigabine, burying the benzyl carbamate group into a tunnel reaching the core of the pore domain.

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2017 – Distribution and kinetics of the Kv1.3-blocking peptide HsTX1[R14A] in experimental rats
Port-a-Patch Publication in Scientific Reports (2017) Authors: Bergmann R., Kubeil M., Zarschler K., Chhabra S., Tajhya R.B., Beeton C., Pennington M.W., Bachmann M., Norton R.S., Stephan H.

The peptide HsTX1[R14A] is a potent and selective blocker of the voltage-gated potassium channel Kv1.3, which is a highly promising target for the treatment of autoimmune diseases and other conditions. In order to assess the biodistribution of this peptide, it was conjugated with NOTA and radiolabelled with copper-64. [64Cu]Cu-NOTA-HsTX1[R14A] was synthesised in high radiochemical purity and yield. The radiotracer was evaluated in vitro and in vivo. The biodistribution and PET studies after intravenous and subcutaneous injections showed similar patterns and kinetics. The hydrophilic peptide was rapidly distributed, showed low accumulation in most of the organs and tissues, and demonstrated high molecular stability in vitro and in vivo. The most prominent accumulation occurred in the epiphyseal plates of trabecular bones. The high stability and bioavailability, low normal-tissue uptake of [64Cu]Cu-NOTA-HsTX1[R14A], and accumulation in regions of up-regulated Kv channels both in vitro and in vivo demonstrate that HsTX1[R14A] represents a valuable lead for conditions treatable by blockade of the voltage-gated potassium channel Kv1.3. The pharmacokinetics shows that both intravenous and subcutaneous applications are viable routes for the delivery of this potent peptide.

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2022 – Reclassification of a likely pathogenic Dutch founder variant in KCNH2; implications of reduced penetrance
SyncroPatch 384PE (a predecessor model of SyncroPatch 384) Publication in Human Molecular Genetics (2022) Authors: Copier J.S., Bootsma M., Ng C.A., Wilde A.A.M., Bertels R.A., Bikker H., Christiaans I., van der Crabben S. N., Hol J.A., Koopmann T.T., Knijnenburg J., Lommerse A.A.J., van der Smagt J.J., Bezzina C.R., Vandenberg J.I., Verkerk A.O, Barge-Schaapveld D.Q.C.M, Lodder E.M.

Due to challenges with historical data and the diversity of assay formats, in silico models for safety-related endpoints are often based on discretized data instead of the data on a natural continuous scale. Models for discretized endpoints have limitations in usage and interpretation that can impact compound design. Here, we present a consistent data inference approach, exemplified on two data sets of Ether-à-go-go-Related Gene (hERG) K+ inhibition data, for dose–response and screening experiments that are generally applicable for in vitro assays. hERG inhibition has been associated with severe cardiac effects and is one of the more prominent safety targets assessed in drug development, using a wide array of in vitro and in silico screening methods. In this study, the IC50 for hERG inhibition is estimated from diverse historical proprietary data. The IC50 derived from a two-point proprietary screening data set demonstrated high correlation (R = 0.98, MAE = 0.08) with IC50s derived from six-point dose–response curves. Similar IC50 estimation accuracy was obtained on a public thallium flux assay data set (R = 0.90, MAE = 0.2). The IC50 data were used to develop a robust quantitative model. The model’s MAE (0.47) and R2 (0.46) were on par with literature statistics and approached assay reproducibility. Using a continuous model has high value for pharmaceutical projects, as it enables rank ordering of compounds and evaluation of compounds against project-specific inhibition thresholds. This data inference approach can be widely applicable to assays with quantitative readouts and has the potential to impact experimental design and improve model performance, interpretation, and acceptance across many standard safety endpoints.

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2023 – Toward Quantitative Models in Safety Assessment: A Case Study to Show Impact of Dose–Response Inference on hERG Inhibition Models
SyncroPatch 384 Publication in Int. J. Mol. Sci. (2023). Authors: Melnikov F., Anger L.T., Hasselgren C.

Due to challenges with historical data and the diversity of assay formats, in silico models for safety-related endpoints are often based on discretized data instead of the data on a natural continuous scale. Models for discretized endpoints have limitations in usage and interpretation that can impact compound design. Here, we present a consistent data inference approach, exemplified on two data sets of Ether-à-go-go-Related Gene (hERG) K+ inhibition data, for dose–response and screening experiments that are generally applicable for in vitro assays. hERG inhibition has been associated with severe cardiac effects and is one of the more prominent safety targets assessed in drug development, using a wide array of in vitro and in silico screening methods. In this study, the IC50 for hERG inhibition is estimated from diverse historical proprietary data.

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2022 – Discovery of Novel Orally Bioavailable Triazoles with Potent and Broad-Spectrum Antifungal Activity In Vitro and In Vivo
SyncroPatch 384i (a predecessor model of the SyncroPatch 384) Publication in Journal of Medicinal Chemistry (2022) Authors: Ni T., Xie F., Hao Y., Li L., Zhu S., Wu H., Chi X., Yan L., Jiang Y., Zhang D.

In our continuing efforts to discover novel triazoles with improved antifungal activity in vitro and in vivo, a series of 41 novel compounds containing 1,2,3-triazole side chains were designed and synthesized via a click reaction based on our previous work. Most of the compounds showed moderate to excellent broad-spectrum antifungal activity in vitro. Among them, the most promising compound 9A16 displayed excellent antifungal and anti-drug-resistant fungal ability (MIC80 = 0.0156–8 μg/mL). In addition, compound 9A16 showed powerful in vivo efficacy on mice systematically infected with Candida albicans SC5314, Cryptococcus neoformans H99, fluconazole-resistant C. albicans 100, and Aspergillus fumigatus 7544. Moreover, compared to fluconazole, compound 9A16 showed better in vitro anti-biofilm activity and was more difficult to induce drug resistance in a 1 month induction of resistance assay in C. albicans. With favorable pharmacokinetics, an acceptable safety profile, and high potency in vitro and in vivo, compound 9A16 is currently under preclinical investigation.

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2022 – Improved PIEZO1 agonism through 4-benzoic acid modification of Yoda1
SyncroPatch 384 Publication in Br. J. Pharmacol. (2022) Authors: Parsonage G., Cuthbertson K., Endesh N., Murciano N., Hyman A.J., Revill C.H., Povstyan O.V., Chuntharpursat-Bon E., Debant M., Ludlow M.J., Futers T.S., Lichtenstein L., Kinsella J.A., Bartoli F., Rotordam M.G., Becker N., Brüggemann A., Foster R., Beech D.J.

4-Benzoic acid modification of Yoda1 improves PIEZO1 agonist activity at PIEZO1 channels. We suggest naming this new modulator Yoda2. It should be a useful tool compound in physiological assays and facilitate efforts to identify a binding site. Such compounds may have therapeutic potential, for example, in diseases linked genetically to PIEZO1 such as lymphatic dysplasia.

2023- Solid-Supported Membrane (SSM)-Based Electrophysiology Assays Using Surface Electrogenic Event Reader Technology (SURFE2R) in Early Drug Discovery
SURFE2R-technology (custom-built system) Publication in Current Protocols (2023) Authors: Pommereau A., Licher T., Bärenz F.

 

This article presents detailed descriptions of procedures and troubleshooting tips for solid-supported membrane (SSM)-based electrophysiology assays (SURFE2R) to measure electrogenic solute carrier transporter proteins (SLCs) and assess the effects of compounds that modulate their activity. SURFE2R allows the use of the standard 96-well format, making it an ideal platform for tertiary assays in a drug-discovery campaign. The assays are performed with cell-line-derived membrane fractions or proteoliposomes containing the transporter of interest. Three main protocols are described for the isolation of membrane fractions from cell culture and the generation of proteoliposomes containing the transporter of interest. Additionally, detailed protocols for SURFE2R single concentration and dose-response experiments are included to measure the potencies of test compounds in stimulating or inhibiting transporter function (EC50 or IC50 values, respectively) and kinetic functional assays to calculate apparent affinity (kM) and maximal velocity (Vmax) of substrate uptake.

Find the article here.

2023 – Monitoring the Reversibility of GPCR Signaling by Combining Photochromic Ligands with Label-free Impedance Analysis
CardioExcyte 96 technology (custom-built system) Publication in Angewandte Chemie (2023) Authors: Wirth U., Erl J., Azzam S., Höring C., Skiba M., Singh R., Hochmuth K., Keller M., Wegener J., König B.

G protein-coupled cell surface receptors (GPCR) trigger complex intracellular signaling cascades upon agonist binding. Classic pharmacological assays provide information about binding affinities, activation or blockade at different stages of the signaling cascade, but real time dynamics and reversibility of these processes remain often disguised. We show that combining photochromic NPY receptor ligands, which can be toggled in their receptor activation ability by irradiation with light of different wavelengths, with whole cell label-free impedance assays allows observing the cell response to receptor activation and its reversibility over time. The concept demonstrated on NPY receptors may be well applicable to many other GPCRs providing a deeper insight into the time course of intracellular signaling processes.

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2023 – Multi-Stimuli-Responsive and Mechano-Actuated Biomimetic Membrane Nanopores Self-Assembled from DNA
Orbit mini Publication in Advanced Materials (2023) Authors: Xing Y., Dorey A., Howorka S.

In bioinspired design, biological templates are mimicked in structure and function by highly controllable synthetic means. Of interest are static barrel-like nanopores that enable molecular transport across membranes for use in biosensing, sequencing, and biotechnology. However, biological ion channels offer additional functions such as dynamic changes of the entire pore shape between open and closed states, and triggering of dynamic processes with biochemical and physical stimuli. To better capture this complexity, this report presents multi-stimuli and mechano-responsive biomimetic nanopores which are created with DNA nanotechnology. The nanopores switch between open and closed states, whereby specific binding of DNA and protein molecules as stimuli locks the pores in the open state. Furthermore, the physical stimulus of high transmembrane voltage switches the pores into a closed state. In addition, the pore diameters are larger and more tunable than those of natural templates. These multi-stimuli-responsive and mechanically actuated nanopores mimic several aspects of complex biological channels yet offer easier control over pore size, shape and stimulus response. The designer pores are expected to be applied in biosensing and synthetic biology.

2023 – Human Induced Pluripotent Stem Cell Derived Nociceptors suitable for Automated Patch Clamp High Throughput Pain Drug Discovery
Application Note PDF
Nav, Cav, Kv – High throughput APC recordings from RealDRGTM iPSC sensory neurons for analgesia drug discovery
Patchliner and SyncroPatch 384 application note: RealDRGs kindly provided by Anatomic Inc.
Conference poster
2023- Pharmacology of transient receptor potential cation (TRP) channels using different activation stimuli
Poster presented at the 7th RSC-BMCS / SCI Symposium on Ion Channels as Therapeutic Targets
2023- Identification of novel TMEM175 modulators using high-throughput automated patch-clamp and solid supported membrane- (SSM-) based electrophysiology platforms
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2023 – Automated Patch-Clamp and Induced Pluripotent Stem Cell-Derived Cardiomyocytes: A Synergistic Approach in the Study of Brugada Syndrome
Patchliner and SyncroPatch 384 Publication (review) in Int. J. Mol. Sci (2023) Authors: Melgari D., Calamaio S., Frosio A., Prevostini R., Anastasia L., Pappone C., Rivolta I.

The development of high-throughput automated patch-clamp technology is a recent breakthrough in the field of Brugada syndrome research. Brugada syndrome is a heart disorder marked by abnormal electrocardiographic readings and an elevated risk of sudden cardiac death due to arrhythmias. Various experimental models, developed either in animals, cell lines, human tissue or computational simulation, play a crucial role in advancing our understanding of this condition, and developing effective treatments. In the perspective of the pathophysiological role of ion channels and their pharmacology, automated patch-clamp involves a robotic system that enables the simultaneous recording of electrical activity from multiple single cells at once, greatly improving the speed and efficiency of data collection. By combining this approach with the use of patient-derived cardiomyocytes, researchers are gaining a more comprehensive view of the underlying mechanisms of heart disease. This has led to the development of more effective treatments for those affected by cardiovascular conditions.

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2023 – Energetics and kinetics of membrane permeation of photoresists for bioprinting
Vesicle Prep Pro Pre-Print Publication in BioRxiv (2023) Authors: Diedrich L., Brosz M., Abele T., Steinke S,, Gräter F., Göpfrich K. Aponte-Santamaría C.

Three-dimensional (3D) bioprinting is a promising technology which typically uses bioinks to pattern cells and their scaffolds. The selection of cytocompatible inks is critical for the printing success. In laserbased 3D bioprinting, photoresist molecules are used as bioinks. We propose that cytotoxicity can be a consequence of the interaction of photoresists with lipid membranes and their permeation into the cell. Here, molecular dynamics simulations and in vitro assays address this issue, retrieving partition coefficients, free energies, and permeabilities for eight commonly-used photoresists in model lipid bilayers. Crossing the hydrophobic center of the membrane constitutes the rate limiting step during permeation. In addition, three photoresists feature a preferential localization site at the acyl chain headgroup interface. Photoresist permeabilities range over eight orders of magnitude, with some molecules being membrane-permeable on bioprinting timescales. Moreover, permeation correlates well with the oil-water partition coefficients and is severely hampered by the lipid ordering imposed by the lipid saturation. Overall, the mechanism of interaction of photoresists with model lipid bilayers is provided here, helping to classify them according to their residence in the membrane and permeation through it. This is useful information to guide the selection of cytocompatible photoresists for 3D bioprinting.

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2023 – Cryo-EM reveals an unprecedented binding site for NaV1.7 inhibitors enabling rational design of potent hybrid inhibitors
SyncroPatch 384 Publication in eLife (2023) Authors: Kschonsak M., Jao C.C., Arthur C.P., Rohou A.L., Bergeron P., Ortwine D.F., McKerrall S.J., Hackos D.H., Deng L., Chen J., Li T., Dragovich P.S., Volgraf M., Wright M.R., Payandeh J., Ciferri C., Tellis J.C.

The voltage-gated sodium (NaV) channel NaV1.7 has been identified as a potential novel analgesic target due to its involvement in human pain syndromes. However, clinically available NaV channel-blocking drugs are not selective among the nine NaV channel subtypes, NaV1.1–NaV1.9. Moreover, the two currently known classes of NaV1.7 subtype-selective inhibitors (aryl- and acylsulfonamides) have undesirable characteristics that may limit their development. To this point understanding of the structure–activity relationships of the acylsulfonamide class of NaV1.7 inhibitors, exemplified by the clinical development candidate GDC-0310, has been based solely on a single co-crystal structure of an arylsulfonamide inhibitor bound to voltage-sensing domain 4 (VSD4). To advance inhibitor design targeting the NaV1.7 channel, we pursued high-resolution ligand-bound NaV1.7-VSD4 structures using cryogenic electron microscopy (cryo-EM). Here, we report that GDC-0310 engages the NaV1.7-VSD4 through an unexpected binding mode orthogonal to the arylsulfonamide inhibitor class binding pose, which identifies a previously unknown ligand binding site in NaV channels. This finding enabled the design of a novel hybrid inhibitor series that bridges the aryl- and acylsulfonamide binding pockets and allows for the generation of molecules with substantially differentiated structures and properties. Overall, our study highlights the power of cryo-EM methods to pursue challenging drug targets using iterative and high-resolution structure-guided inhibitor design. This work also underscores an important role of the membrane bilayer in the optimization of selective NaV channel modulators targeting VSD4.

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2023 – Protein 14-3-3 Influences the Response of the Cardiac Sodium Channel Nav1.5 to Antiarrhythmic Drugs
SyncroPatch 384 Publication in J. Pharmacol. Exp. Ther. (2023) Authors: Zheng Y., Deschênes I.

The cardiac sodium channel Nav1.5 is a key contributor to the cardiac action potential, and dysregulations in Nav1.5 can lead to cardiac arrhythmias. Nav1.5 is a target of numerous antiarrhythmic drugs (AADs). Previous studies identified the protein 14-3-3 as a regulator of Nav1.5 biophysical coupling. Inhibition of 14-3-3 can remove the Nav1.5 functional coupling and has been shown to inhibit the dominant-negative effect of Brugada syndrome mutations. However, it is unknown whether the coupling regulation is involved with AADs’ modulation of Nav1.5. Indeed, AADs could reveal important structural and functional information about Nav1.5 coupling. Here, we investigated the modulation of Nav1.5 by four classic AADs, quinidine, lidocaine, mexiletine, and flecainide, in the presence of 14-3-3 inhibition. The experiments were carried out by high-throughput patch-clamp experiments in an HEK293 Nav1.5 stable cell line. We found that 14-3-3 inhibition can enhance acute block by quinidine, whereas the block by other drugs was not affected. We also saw changes in the use- and dose-dependency of quinidine, lidocaine, and mexiletine when inhibiting 14-3-3. Inhibiting 14-3-3 also shifted the channel activation toward hyperpolarized voltages in the presence of the four drugs studied and slowed the recovery of inactivation in the presence of quinidine. Our results demonstrated that the protein 14-3-3 and Nav1.5 coupling could impact the effects of AADs. Therefore, 14-3-3 and Nav1.5 coupling are new mechanisms to consider in the development of drugs targeting Nav1.5.

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2023 – ANXA11 biomolecular condensates facilitate protein-lipid phase coupling on lysosomal membranes
Vesicle Prep Pro Pre-Print Publication in BioRxiv (2023) Authors: Nixon-Abell J., Ruggeri F.S., Qamar S., Herling T.W., Czekalska M.A., Shen Yi., Wang G., King C., Fernandopulle M.S., Sneideris T., Watson J.L., Pillai V.V.S., Meadows W., Henderson J.W., Chambers J.E., Wagstaff J.L., Williams S.H., Coyle H., Lu Y., Zhang S., Marciniak S.J., Freund S.M.V., Derivery E., Ward M.E., Vendruscolo M., Knowles T.P.J. St George-Hyslop P.

Phase transitions of cellular proteins and lipids play a key role in governing the organisation and coordination of intracellular biology. The frequent juxtaposition of proteinaceous biomolecular condensates to cellular membranes raises the intriguing prospect that phase transitions in proteins and lipids could be co-regulated. Here we investigate this possibility in the ribonucleoprotein (RNP) granule-ANXA11-lysosome ensemble, where ANXA11 tethers RNP granule condensates to lysosomal membranes to enable their co-trafficking. We show that changes to the protein phase state within this system, driven by the low complexity ANXA11 N-terminus, induce a coupled phase state change in the lipids of the underlying membrane. We identify the ANXA11 interacting proteins ALG2 and CALC as potent regulators of ANXA11-based phase coupling and demonstrate their influence on the nanomechanical properties of the ANXA11-lysosome ensemble and its capacity to engage RNP granules. The phenomenon of protein-lipid phase coupling we observe within this system offers an important template to understand the numerous other examples across the cell whereby biomolecular condensates closely juxtapose cell membranes.

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2023- Functional characterization of SGLT1 using SSM-based electrophysiology: Kinetics of sugar binding and translocation
SURFE2R- N1 Publication in Front. Physiol. (2023) Authors: Bazzone A., Zerlotti R., Barthmes M., Fertig N.

Beside the ongoing efforts to determine structural information, detailed functional studies on transporters are essential to entirely understand the underlying transport mechanisms. We recently found that solid supported membrane-based electrophysiology (SSME) enables the measurement of both sugar binding and transport in the Na+/sugar cotransporter SGLT1 (Bazzone et al, 2022a). Here, we continued with a detailed kinetic characterization of SGLT1 using SSME, determining KM and KDapp for different sugars, kobs values for sugar-induced conformational transitions and the effects of Na+, Li+, H+ and Cl on sugar binding and transport. We found that the sugar-induced pre-steady-state (PSS) charge translocation varies with the bound ion (Na+, Li+, H+ or Cl), but not with the sugar species, indicating that the conformational state upon sugar binding depends on the ion. Rate constants for the sugar-induced conformational transitions upon binding to the Na+-bound carrier range from 208 s−1 for D-glucose to 95 s−1 for 3-OMG. In the absence of Na+, rate constants are decreased, but all sugars bind to the empty carrier. From the steady-state transport current, we found a sequence for sugar specificity (Vmax/KM): D-glucose > MDG > D-galactose > 3-OMG > D-xylose. While KM differs 160-fold across tested substrates and plays a major role in substrate specificity, Vmax only varies by a factor of 1.9. Interestingly, D-glucose has the lowest Vmax across all tested substrates, indicating a rate limiting step in the sugar translocation pathway following the fast sugar-induced electrogenic conformational transition. SGLT1 specificity for D-glucose is achieved by optimizing two ratios: the sugar affinity of the empty carrier for D-glucose is similarly low as for all tested sugars (KD,Kapp = 210 mM). Affinity for D-glucose increases 14-fold (KD,Naapp = 15 mM) in the presence of sodium as a result of cooperativity. Apparent affinity for D-glucose during transport increases 8-fold (KM = 1.9 mM) compared to KD,Naapp due to optimized kinetics. In contrast, KM and KDapp values for 3-OMG and D-xylose are of similar magnitude. Based on our findings we propose an 11-state kinetic model, introducing a random binding order and intermediate states corresponding to the electrogenic transitions detected via SSME upon substrate binding.

 

Read more in the paper and find out more about other Solute Carriers in the article collection here.

 

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2023 – Nav1.2 and BK channel interaction shapes the action potential in the axon initial segment
SyncroPatch 384 Publication in J. Physiol. (2023) Authors: Filipis L., Blömer L.A., Montnach J., Loussouarn G., De Waard M., Canepari M.

In neocortical layer-5 pyramidal neurons, the action potential (AP) is generated in the axon initial segment (AIS) when the membrane potential (Vm) reaches the threshold for activation of the voltage-gated Na+ channels (VGNCs) Nav1.2 and Nav1.6. Yet, whereas these VGNCs are known to differ in spatial distribution along the AIS and in biophysical properties, our understanding of the functional differences between the two channels remains elusive. Here, using ultrafast Na+Vm and Ca2+ imaging in combination with partial block of Nav1.2 by the peptide G1G4-huwentoxin-IV, we demonstrate an exclusive role of Nav1.2 in shaping the generating AP. Precisely, we show that selective block of ∼30% of Nav1.2 widens the AP in the distal part of the AIS and we demonstrate that this effect is due to a loss of activation of BK Ca2+-activated K+ channels (CAKCs). Indeed, Ca2+ influx via Nav1.2 activates BK CAKCs, determining the amplitude and the early phase of repolarization of the AP in the AIS. By using control experiments using 4,9-anhydrotetrodotoxin, a moderately selective inhibitor of Nav1.6, we concluded that the Ca2+ influx shaping the early phase of the AP is exclusive of Nav1.2. Hence, we mimicked this result with a neuron model in which the role of the different ion channels tested reproduced the experimental evidence. The exclusive role of Nav1.2 reported here is important for understanding the physiology and pathology of neuronal excitability.

2022 – The suitability of high throughput automated patch clamp for physiological applications
SyncroPatch 384 Publication in The Journal of Physiology (2022). Authors: Obergrussberger A., Rinke-Weiß I., Goetze T.A., Rapedius M., Brinkwirth N., Becker N., Rotordam M.G., Hutchison L., Madau P., Pau D., Dalrymple D., Braun N., Friis S., Pless S.A., Fertig N.

Although automated patch clamp (APC) devices have been around for many years and have become an integral part of many aspects of drug discovery, high throughput instruments with GΩ seal data quality are relatively new. Experiments where a large number of compounds are screened against ion channels are ideally suited to high throughput APC, particularly when the amount of compound available is low. Here we evaluate different APC approaches using a variety of ion channels and screening settings. We have performed a screen of 1,920 compounds on GluN1/GluN2A NMDA receptors for negative allosteric modulation using both the SyncroPatch 384 and FLIPRTM. Additionally, we tested the effect of 36 arthropod venoms on NaV1.9 using a single 384-well plate on the SyncroPatch 384. As an example for mutant screening, a range of acid-sensing ion channel variants were tested and the success rate increased through FACS prior to APC experiments. GΩ seal data quality makes the 384- format accessible to recording of primary and stem cell-derived cells on the SyncroPatch 384. We show recordings in voltage and current clamp modes of stem cell-derived cardiomyocytes. In addition, the option of intracellular solution exchange enabled investigations into the effects of intracellular Ca2+ and cAMP on TRPC5 and HCN2 currents, respectively. Together, this highlights the broad applicability and versatility of APC platforms and also outlines some limitations of the approach.

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2023 – Beyond CBD: Inhibitory effects of lesser studied phytocannabinoids on human voltage-gated sodium channels
Patchliner Publication in Front. Physiol. (2023) Authors: Milligan C.J., Anderson L.L., McGregor I.S., Arnold J.C., Petrou S.

Introduction: Cannabis contains cannabidiol (CBD), the main non-psychoactive phytocannabinoid, but also many other phytocannabinoids that have therapeutic potential in the treatment of epilepsy. Indeed, the phytocannabinoids cannabigerolic acid (CBGA), cannabidivarinic acid (CBDVA), cannabichromenic acid (CBCA) and cannabichromene (CBC) have recently been shown to have anti-convulsant effects in a mouse model of Dravet syndrome (DS), an intractable form of epilepsy. Recent studies demonstrate that CBD inhibits voltage-gated sodium channel function, however, whether these other anti-convulsant phytocannabinoids affect these classic epilepsy drug-targets is unknown. Voltage-gated sodium (NaV) channels play a pivotal role in initiation and propagation of the neuronal action potential and NaV1.1, NaV1.2, NaV1.6 and NaV1.7 are associated with the intractable epilepsies and pain conditions.

Methods: In this study, using automated-planar patch-clamp technology, we assessed the profile of the phytocannabinoids CBGA, CBDVA, cannabigerol (CBG), CBCA and CBC against these human voltage-gated sodium channels subtypes expressed in mammalian cells and compared the effects to CBD.

Results: CBD and CBGA inhibited peak current amplitude in the low micromolar range in a concentration-dependent manner, while CBG, CBCA and CBC revealed only modest inhibition for this subset of sodium channels. CBDVA inhibited NaV1.6 peak currents in the low micromolar range in a concentration-dependent fashion, while only exhibiting modest inhibitory effects on NaV1.1, NaV1.2, and NaV1.7 channels. CBD and CBGA non-selectively inhibited all channel subtypes examined, whereas CBDVA was selective for NaV1.6. In addition, to better understand the mechanism of this inhibition, we examined the biophysical properties of these channels in the presence of each cannabinoid. CBD reduced NaV1.1 and NaV1.7 channel availability by modulating the voltage-dependence of steady-state fast inactivation (SSFI, V0.5 inact), and for NaV1.7 channel conductance was reduced. CBGA also reduced NaV1.1 and NaV1.7 channel availability by shifting the voltage-dependence of activation (V0.5 act) to a more depolarized potential, and for NaV1.7 SSFI was shifted to a more hyperpolarized potential. CBDVA reduced channel availability by modifying conductance, SSFI and recovery from SSFI for all four channels, except for NaV1.2, where V0.5 inact was unaffected.

Discussion: Collectively, these data advance our understanding of the molecular actions of lesser studied phytocannabinoids on voltage-gated sodium channel proteins.

Webinar
07.03.2013 | Webinar: Top gear ion channel screening
Authors: Nanion Experts
Webinar
24.10.2013 | Webinar: SyncroPatch 384PE – the PatchEngine. Superior Ion Channel Drug Screening
Authors: Nanion Experts
Podcast
spotl]i[ght – Podcast – Trong Shen (University of Toronto)
Guest: Trong Shen (University of Toronto)

In this edition of the podcast - we speak to Tron Shen and his unique experiences working in an academic lab (Hinz Lab). Listen to his unique story, which encompases a stop in the world of finance.

Podcast
spotl]i[ght – Podcast – Wandi Zhu (Harvard)
Guest: Wandi Zhu (Harvard)

Bacterial and mammalian NaV channels provide insights into the molecular basis of channel gating and will facilitate organism-specific drug discovery.

Podcast
spotl]i[ght – Podcast – Nasreen Choudhury (Rush University)
Guest: Nasreen Choudhury (Rush University)

In this edition of the podcast - we spoke to Postdoctoral Research Fellow Nasreen Choudhury about her research of Alziehmer's disease and the role that ion channels play. Nasreen completed her PhD at the Indian Institute of Science where she published a paper about TREK1 channel, read it here. Nasreen went on to working at the University of Leicester where she published her investigation into Kv3 channels.

When Nasreen finishes her postdoctoral stint at Rush University Medical Center, she plans to switch to an industry role where she can work with automated patch clamp platforms

Podcast
spotl]i[ght – Podcast – Shashank Pant (University Of Illinois Urbana-Champaign)
Guest: Shashank Pant (University Of Illinois Urbana-Champaign)

On this episode of the podcast we spoke with Shashank Pant (University of Illinois, Urbana-Champaign). We talked about how he started his journey as a researcher and how his curiosity of the scientific process continues to serve as a motivation to publish in high impact journals such as Nature (where he was recently part of an internationally collaboration with with Renae Ryan's Lab, University of Sydney).
Here they looked at Glutamate, specifically providing insight into the mechanism by which glutamate transporters support their dual function

Podcast
spotl]i[ght – Podcast – Katherine Henzler-Wildman (University of Wisconsin-Madison) & Nathan Thomas c
Guest: Katherine Henzler-Wildman (University of Wisconsin-Madison) & Nathan Thomas (University of California - San Diego)

On this episode of the podcast we spoke with Katie and Nathan about their work (specifically around developing a method to provide a fast, easy, general method for measuring transport stoichiometry via solid supported membrane electrophysiology) which will facilitate future mechanistic and functional studies of ion-coupled transporters. You can read the full publication titled: A solid-supported membrane electrophysiology assay for efficient characterization of ion-coupled transport here.

Katie's Lab uses NMR as a primary technique to monitor protein structure and dynamics, taking advantage of the extensive resources available through NMRFAM. By comparing NMR data with biochemical and functional assays, insight into the mechanisms of secondary active transport, multidrug recognition, ion channel gating and ion selectivity is gained.

Nathan is presentlt a Postdoctoral Researchers at Tezcan Lab - UCSD (University of California San Diego,) Department of Chemistry and Biochemistry.

Podcast
spotl]i[ght – Podcast – Kerstin Göpfrich (Max Planck Institute for Medical Research)
Guest: Kerstin Göpfrich (Max Planck Institute for Medical Research)

In this edition of the podcast - we speak to Kerstin Göpfrich Ph.D. who is currently attempting how to construct a cell with a bottom up approach and exploring how life could be different with DNA origami.

Podcast
spotl]i[ght – Podcast – Dr. Filip Van Petegem (University of British Columbia)
Guest: Dr. Filip Van Petegem (University of British Columbia)

On this episode of the podcast we spoke with Dr. Filip Van Petegem about his journey starting as a structural biologist, to the work his lab is currently focused on. Van Petegem Lab studies the structure and function of ion channels, membrane protein responsible for electrical signaling in excitable cells.

Podcast
spotl]i[ght – Podcast – Dr. Yohei Ohashi (Medical Research Council – UK)
Guest: Dr. Yohei Ohashi (Medical Research Council - UK)

On this episode of the podcast we spoke with Dr. Yohei Ohashi about his unique journey from the University of Kyoto where he was working on his PhD specifically within plant molecular biology. Dr. Yohei Ohashi is a multidisciplinary researcher with over 15 years of experience in basic/translational biology.

Podcast
spotl]i[ght – Podcast – Dr. Carlos Pardo-Pastor (King’s College London)
Guest: Dr. Carlos Pardo-Pastor (King's College London)

Dr Carlos Pardo-Pastor (King's College London) is a Research Fellow in the Randall Centre for Cell & Molecular Biophysics. He started his scientific journey studying Human Biology, an MSc in Biomedical Research and then completed his PhD in Biomedicine at Universitat Pompeu Fabra.

On this episode of the podcast we spoke with Carlos about his most recent publication (Piezo2 Channel), you can read the full publication here. He highlights the importance of funding in science, his personal experiences in applying for grants, and some helpful tips for staying motivated as a researcher and connecting with peers in the field.

Podcast
spotl]i[ght – Podcast – Dr. Fernanda C Cardoso (University of Queensland)
Guest: Dr. Fernanda C Cardoso (University of Queensland)

Dr. Fernanda C Cardoso joined the Institute for Molecular Bioscience at University of Queensland to develop therapies to treat complex neurological diseases. In this episode, she tells us about her passion from a young age to study biology and arachnids. Find out more about Fernanda here.

Here she describes the benefits of working with an Automated Patch Clamp set-up (in comparison to the more traditional manual patch clamp set-up). Her work focuses on Sodium and Calcium Channels, specifically dealing with disorders associated with chronic pain.

Podcast
spotl]i[ght – Podcast – Catherine Webley (University College London)
Guest: Catherine Webley (University College London)

In this edition of the podcast - we speak to Catherine Webley, Master of Chemistry, who completed a placement in the academic research lab in the Solar Centre at King Abdullah University of Science and Technology under the supervision of Prof. Iain McCulloch. She describes her 'love affair' with how the natural world works and the impact that the recent Nobel Prize winners have had on her.

Podcast
spotl]i[ght – Podcast – Claudia Weidling (University of Copenhagen)
Guest: Claudia Weidling (University of Copenhagen)

In this edition of the podcast - we speak to Claudia Weidling Ph.D. who tells us about her recent publication in Nature, her experience being a Ph.D student during a pandemic and what advice she would offer to prospective Ph.D students.

Podcast
spotl]i[ght – Podcast – Beatrice Badone (University of Milano-Bicocca)
Guest: Beatrice Badone (University of Milano-Bicocca)

Beatrice Badone, a Ph.D. student at the University of Milano-Bicocca. Beatrice works with Cardiomyocytes and explains how drug screening can be improved and can be made more accessible and ethical for scientists.

Podcast
spotl]i[ght – Podcast – Benjamin Lane (University of Leeds)
Guest: Benjamin Lane (University of Leeds)

Benjamin is a Wellcome trust PhD student, who is working under the supervision of Dr Christos Pliotas and Dr Stephen Muench. He recently joined their groups to work on his project combining Cryo-EM and PELDOR with electrophysiology, to structurally and functionally characterise novel mechanosensitive ion channels.

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2018 – Structures of monomeric and oligomeric forms of the Toxoplasma gondii perforin-like protein 1
Vesicle Prep Pro Publication in Science Advances (2018) Authors: Ni T., Williams S.I., Rezelj S., Anderluh G., Harlos K., Stansfeld P.J., Gilbert R.J.C.

Toxoplasma and Plasmodium are the parasitic agents of toxoplasmosis and malaria, respectively, and use perforin-like proteins (PLPs) to invade host organisms and complete their life cycles. The Toxoplasma gondii PLP1 (TgPLP1) is required for efficient exit from parasitophorous vacuoles in which proliferation occurs. We report structures of the membrane attack complex/perforin (MACPF) and Apicomplexan PLP C-terminal β-pleated sheet (APCβ) domains of TgPLP1. The MACPF domain forms hexameric assemblies, with ring and helix geometries, and the APCβ domain has a novel β-prism fold joined to the MACPF domain by a short linker. Molecular dynamics simulations suggest that the helical MACPF oligomer preserves a biologically important interface, whereas the APCβ domain binds preferentially through a hydrophobic loop to membrane phosphatidylethanolamine, enhanced by the additional presence of inositol phosphate lipids. This mode of membrane binding is supported by site-directed mutagenesis data from a liposome-based assay. Together, these structural and biophysical findings provide insights into the molecular mechanism of membrane targeting by TgPLP1.

Podcast
spotl]i[ght – Podcast – Professor Lars Kaestner (Saarland University)
Guest: Professor Lars Kaestner (Saarland University)

In this edition of the podcast - we speak to Prof. Lars Kaestner and his work with Red Blood Cells; specifically looking at advances in diagnostic tools for Neuroacanthocytosis.

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2022 – Unraveling the host-selective toxic interaction of cassiicolin with lipid membranes and its cytotoxicity
Vesicle Prep Pro Publication in Phytopathology (2022) Authors: Ngo K.X., Quo N.B., Nguyen P.D.N., Furusho H., Miyata M., Shimonaka T., Chau N.N.B.C., Vinh N.P., Nghia N.A., Mohammed T.O., Ichikawa T., Kodera N., Konno H., Fukuma T., Ando T.

Corynespora cassiicola is the pathogen that causes Corynespora leaf fall (CLF) disease. Cassiicolin (Cas), a toxin produced by C. cassiicola, is responsible for CLF disease in rubber trees (Hevea brasiliensis). Currently, the molecular mechanism of the cytotoxicity of Cas and its host selectivity have not been fully elucidated. To gain insight into these issues, we analyzed the binding of Cas1 and Cas2 to membranes consisting of different plant lipids and their membrane-disruption activities. Our real-time observations with high-speed atomic force microscopy (HS-AFM) and confocal microscopy reveal that the binding and disruption activities of Cas1 and Cas2 are strongly dependent on the types of membrane lipids. The mixtures of DPPC with DPPA, MGDG, DGDG, and stigmasterol are more susceptible to membrane damage caused by Cas1 and Cas2 than DPPC alone or its mixtures with sitosterol, DGTS-d9, and DGTS. This difference derives from the stronger binding of the toxins to membranes with the former lipid composition. Cytotoxicity tests on rubber leaves of RRIV 1, RRIV 4, and PB 255 clones suggest that the toxins cause necrosis of rubber leaves, except for the strong resistance of PB 255 against Cas2. Cryo-SEM analyses of necrotic leaf tissues exposed to Cas1 confirm that cytoplasmic membranes are vulnerable to the toxin. Thus, the host selectivity of Cas toxin in CLF disease is attained by the lipid-dependent binding activity of Cas to the membrane, and the cytotoxicity of Cas arises from its ability to disrupt membranes.

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2022 – The Small Heat Shock Protein, HSPB1, Interacts with and Modulates the Physical Structure of Membranes
Vesicle Prep Pro Publication in International Journal of Molecular Science (2022) Authors: Csoboz B., Gombos I., Kóta Z., Dukic B., Klement E., Varga-Zsíros V., Lipinszki Z., Páli T., László Vígh L., Török Z.

Small heat shock proteins (sHSPs) have been demonstrated to interact with lipids and modulate the physical state of membranes across species. Through these interactions, sHSPs contribute to the maintenance of membrane integrity. HSPB1 is a major sHSP in mammals, but its lipid interaction profile has so far been unexplored. In this study, we characterized the interaction between HSPB1 and phospholipids. HSPB1 not only associated with membranes via membrane-forming lipids, but also showed a strong affinity towards highly fluid membranes. It participated in the modulation of the physical properties of the interacting membranes by altering rotational and lateral lipid mobility. In addition, the in vivo expression of HSPB1 greatly affected the phase behavior of the plasma membrane under membrane fluidizing stress conditions. In light of our current findings, we propose a new function for HSPB1 as a membrane chaperone.

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2022 – The Staphylococcus aureus CidA and LrgA Proteins Are Functional Holins Involved in the Transport of By-Products of Carbohydrate Metabolism
Vesicle Prep Pro Publication in mBio (2022) Authors: Endres J. L., Chaudhari S. S., Zhang X., Prahlad J., Wang S-Q., Foley L. A., Luca S., Bose J. L., Thomas V. C., Bayles K. W.

The Staphylococcus aureus cidABC and lrgAB operons encode members of a well-conserved family of proteins thought to be involved in programmed cell death (PCD). Based on the structural similarities that CidA and LrgA share with bacteriophage holins, we have hypothesized that these proteins function by forming pores within the cytoplasmic membrane. To test this, we utilized a "lysis cassette" system that demonstrated the abilities of the cidA and lrgA genes to support bacteriophage endolysin-induced cell lysis. Typical of holins, CidA- and LrgA-induced lysis was dependent on the coexpression of endolysin, consistent with the proposed holin-like functions of these proteins. In addition, the CidA and LrgA proteins were shown to localize to the surface of membrane vesicles and cause leakage of small molecules, providing direct evidence of their hole-forming potential. Consistent with recent reports demonstrating a role for the lrgAB homologues in other bacterial and plant species in the transport of by-products of carbohydrate metabolism, we also show that lrgAB is important for S. aureus to utilize pyruvate during microaerobic and anaerobic growth, by promoting the uptake of pyruvate under these conditions. Combined, these data reveal that the CidA and LrgA membrane proteins possess holin-like properties that play an important role in the transport of small by-products of carbohydrate metabolism. IMPORTANCE The Staphylococcus aureus cidABC and lrgAB operons represent the founding members of a large, highly conserved family of genes that span multiple kingdoms of life. Despite the fact that they have been shown to be involved in bacterial PCD, very little is known about the molecular/biochemical functions of the proteins they encode. The results presented in this study reveal that the cidA and lrgA genes encode proteins with bacteriophage holin-like functions, consistent with their roles in cell death. However, these studies also demonstrate that these operons are involved in the transport of small metabolic by-products of carbohydrate metabolism, suggesting an intriguing link between these two seemingly disparate processes.

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2022 – Soybean meal peptides regulated membrane phase of giant unilamellar vesicles: A key role for bilayer amphipathic region localization
Vesicle Prep Pro Publication in Food Research International (2022) Authors: Jiang F., Liu J., Du Z., Liu X., Shang X., Yu Y., Zhang T.

Membrane phase separation forms liquid-ordered (Lo) and liquid-disordered (Ld) phases and is involved in cellular processes and functions. Our previous study has confirmed that peptides can regulate phase separation by increasing the Lo phase. However, the specific mechanisms underlying the phase separation regulation of peptides remain poorly understood. This study aimed to explore the effect of soybean meal peptides on phase separation and illustrate the correlation between phase regulation and membrane localization of the peptides. Phase separation was studied by giant unilamellar vesicles (GUVs), and membrane localization of the peptides was detected by steady-state fluorescence quenching. Our results revealed that peptides YYK, CLA, and SLW enhanced the Lo phase while WLQ decreased the Lo phase. The localization in the membrane amphiphilic region of the peptides played a crucial role in their regulation of phase separation. The more localization of the peptides (YYK, CLA, and SLW) in the membrane amphiphilic region, the stronger the capacity to increase the Lo phase.

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2022 – The nuclear egress complex of Epstein-Barr virus buds membranes through an oligomerization-driven mechanism
Vesicle Prep Pro Publication in PLOS Pathogens (2022) Authors: Thorsen M., Draganova E., Heldwein E.

During replication, herpesviral capsids are translocated from the nucleus into the cytoplasm by an unusual mechanism, termed nuclear egress, that involves capsid budding at the inner nuclear membrane. This process is mediated by the viral nuclear egress complex (NEC) that deforms the membrane around the capsid. Although the NEC is essential for capsid nuclear egress across all three subfamilies of the Herpesviridae, most studies to date have focused on the NEC homologs from alpha- and beta- but not gammaherpesviruses. Here, we report the crystal structure of the NEC from Epstein-Barr virus (EBV), a prototypical gammaherpesvirus. The structure resembles known structures of NEC homologs yet is conformationally dynamic. We also show that purified, recombinant EBV NEC buds synthetic membranes in vitro and forms membrane-bound coats of unknown geometry. However, unlike other NEC homologs, EBV NEC forms dimers in the crystals instead of hexamers. The dimeric interfaces observed in the EBV NEC crystals are similar to the hexameric interfaces observed in other NEC homologs. Moreover, mutations engineered to disrupt the dimeric interface reduce budding. Putting together these data, we propose that EBV NEC-mediated budding is driven by oligomerization into membrane-bound coats.

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2022 – On the assembly of zwitterionic block copolymers with phospholipids
Vesicle Prep Pro Publication in European Polymer Journal (2022) Authors: Spanjers J., Brodszkij E., Gal N., Pedersen J., Städler B.

Materials containing zwitterionic polymers are interesting candidates for diverse applications due to their versatile properties. The assembly of the amphiphilic block copolymer poly(cholesteryl methacrylate)-block-poly(2-methacryloyloxyethyl phosphorylcholine) with three different phospholipids (1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)) into small and giant vesicles is reported focusing on their morphology, size, and membrane properties. Giant hybrid vesicles were obtained for all types of lipids, but DOPC was more suitable to assemble small hybrid vesicles without a large fraction of hybrid micelles present. Further, the permeability of the small vesicle membranes towards 5(6)-carboxyfluorescein is very similar to comparable sized liposomes. In contrast, the permeability of the giant hybrid vesicle membranes towards 5(6)-carboxy-X-rhodamine is higher compared to only cholesterol-containing lipid giant vesicles. DOPS-containing vesicles showed pH-dependent morphology changes. Hybrid vesicles containing DOPS and DOPE in addition to the block copolymer have the highest association with HepG2 cells. In contrast, only DOPC-containing hybrid vesicles can be incorporated into alginate beads. Taken together, using these block copolymers with a zwitterionic hydrophilic extension of the chosen architecture offers fundamental insight into the possibility to assemble hybrid vesicles and their potential in bottom-up synthetic biology.

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2022 – Polymer Micelles vs Polymer–Lipid Hybrid Vesicles: A Comparison Using RAW 264.7 Cells
Vesicle Prep Pro Publication in Frontiers in Biomacromolecules (2022) Authors: Ade C., Qian X., Brodszkij E., Andres P.D.D., Spanjers J., Westensee I.N., Städler B.

Bottom-up synthetic biology aims to integrate artificial moieties with living cells and tissues. Here, two types of structural scaffolds for artificial organelles were compared in terms of their ability to interact with macrophage-like murine RAW 264.7 cells. The amphiphilic block copolymer poly(cholesteryl methacrylate)-block-poly(2-carboxyethyl acrylate) was used to assemble micelles and polymer–lipid hybrid vesicles together with 1,2-dioleoyl-sn-glycero-3-phosphocholine or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) lipids in the latter case. In addition, the pH-sensitive fusogenic peptide GALA was conjugated to the carriers to improve their lysosomal escape ability. All assemblies had low short-term toxicity toward macrophage-like murine RAW 264.7 cells, and the cells internalized both the micelles and hybrid vesicles within 24 h. Assemblies containing DOPE lipids or GALA in their building blocks could escape the lysosomes. However, the intracellular retention of the building blocks was only a few hours in all the cases. Taken together, the provided comparison between two types of potential scaffolds for artificial organelles lays out the fundamental understanding required to advance soft material-based assemblies as intracellular nanoreactors.

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2022 – Membrane composition of polymer-lipid hybrid vesicles
Vesicle Prep Pro Publication in Frontiers in Applied Materials Today (2022) Authors: Brodszkij E., Westensee I.N., Holleufer S.F., Ade C., De Dios Andres P., Pedersen J.S., Städler B.

Hybrid vesicles (HVs) assembled from phospholipids and amphiphilic block copolymers (BCPs) are a more recent alternative to liposomes and polymersomes. We aim to change the properties of the HV membranes by varying the chemical composition of the hydrophobic block in the BCPs that have poly(carboxyethyl acrylate) (PCEA) as the hydrophilic part. To this end, statistical copolymers of cholesteryl methacrylate and either butyl methacrylate (BuMA) or 2-hydroxyethyl methacrylate (HEMA) as well as the corresponding homopolymers were synthesized and used as macroinitiator for the extension with PCEA. All the BCPs allowed for the assembly of small and giant HVs with soybean L-α-phosphatidylcholine. The extend of the co-extisting micellar populations varied as shown by transmission electron microscopy and small-angle X-ray spectroscopy. Although the membrane packings derived from spectra when using Laurdan as an environmentally sensitive fluorescent probe were comparable between the different HVs, their permeability towards 5(6)-carboxy-X-rhodamine or carboxyfluorescein depended on the membrane composition, i.e., HEMA-containing membranes had higher permeability than membranes containing the other tested BCPs for small and giant HVs. Further, membranes with BuMA offered the most suitable environment for the association with β-galactosidase illustrated by the efficient substrate conversion. Taken together, the hydrophobic block is a relevant mean to control the morphologies and membrane properties of HVs.

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2022 – Membrane lipid organization and nicotinic acetylcholine receptor function: A two-way physiological relationship
Vesicle Prep Pro Publication in Archives of Biochemistry and Biophysics (2022) Authors: Fabiani C., Georgiev V., Peñalva D., Sigaut L., Pietrasanta L., Corradi J., Dimova R., Antollini S.

Nicotinic acetylcholine receptors (nAChRs) are involved in a great range of physiological and pathological conditions. Since they are transmembrane proteins, they interact strongly with the lipids surrounding them. Thus, the plasma membrane composition and heterogeneity play an essential role for the correct nAChR function, on the one hand, and the nAChR influences its immediate lipid environment, on the other hand. The aim of this work was to investigate in more detail the role of the biophysical properties of the membrane in nAChR function and vice versa, focusing on the relationship between Chol and nAChRs. To this end, we worked with different model systems which were treated either with (i) more Chol, (ii) cholesteryl hemisuccinate, or (iii) the enzyme cholesterol oxidase to generate different membrane sterol conditions and in the absence and presence of γTM4 peptide as a representative model of the nAChR. Fluorescence measurements with crystal violet and patch-clamp recordings were used to study nAChR conformation and function, respectively. Using confocal microscopy of giant unilamellar vesicles we probed the membrane phase state/order and organization (coexistence of lipid domains) and lipid-nAChR interaction. Our results show a feedback relationship between membrane organization and nAChR function, i.e. whereas the presence of a model of nAChRs conditions membrane organization, changing its lipid microenvironment, membrane organization and composition perturb nAChRs function. We postulate that nAChRs have a gain of function in disordered membrane environments but a loss of function in ordered ones, and that Chol molecules at the outer leaflet in annular sites and at the inner leaflet in non-annular sites are related to nAChR gating and desensitization, respectively. Thus, depending on the membrane composition, organization, and/or order, the nAChR adopts different conformations and locates in distinct lipid domains and this has a direct effect on its function.

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2022 – Evaluation of Polyoxazolines Insertion into the Epidermis: From Membrane Models to in Vivo Studies
Vesicle Prep Pro Pre-Print Publication in European Journal of Pharmaceutics and Biopharmaceutics (2022) Authors: Simon L., E. Bellard E., B. Jouanmiqueou B., V. Lapinte V., N. Marcotte N., J.M. Devoisselle J.M., Rols M.P., Golzio M., Bégu S.

In this study, we evaluated the potential of amphiphilic polyoxazolines (POx) to interact with biological membranes thanks to models of increasing complexity, from a simple lipid bilayer using giant unilamellar vesicles (GUV), to plasma membranes of three different cell types, fibroblasts, keratinocytes and melanocytes, which are found in human skin. Upon assessing an excellent penetration into GUV membranes and cultured cells, we addressed POx’s potential to penetrate the murine skin within an in vivo model. Exposure studies were made with native POx and with POx encapsulated within lipid nanocapsules (LNC). Our findings indicate that POx’s interactions with membranes tightly depend on the nature of the alkyl chain constituting the POx. Saturated C 16 POx insert rapidly and efficiently into GUV and plasma membranes, while unsaturated C 18:2 POx insert to a smaller extent. The high amount of membrane-inserted saturated C 16 POx impacts cell viability to a greater extent than the unsaturated C 18:2 POx. The in vivo study, performed on mice, showed an efficient accumulation of both POx types in the stratum corneum barrier, reaching the upper epidermis, independently of POx’s degree of saturation. Furthermore, the formulation of POx into lipid nanocapsules allowed delivering an encapsulated molecule, the quercetin, in the upper epidermis layers of murine skin, proving POx’s efficacy for topical delivery of active molecules. Overall, POx proved to be an excellent choice for topical delivery, which might in turn offer new possibilities for skin treatments in diseases such as psoriasis or melanomas.

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2022 – Lipid Microenvironment Modulates the Pore-Forming Ability of Polymyxin B
Vesicle Prep Pro Publication in Antibiotics (2022) Authors: Zakharova A., Efimova S., Ostroumova O.

The ability of polymyxin B, an antibiotic used to treat infections caused by multidrug-resistant Gram-negative bacteria as a last-line therapeutic option, to form ion pores in model membranes composed of various phospholipids and lipopolysaccharides was studied. Our data demonstrate that polymyxin B predominantly interacts with negatively charged lipids. Susceptibility decreases as follows: Kdo2-Lipid A >> DOPG ≈ DOPS >> DPhPG ≈ TOCL ≈ Lipid A. The dimer and hexamer of polymyxin B are involved in the pore formation in DOPG(DOPS)- and Kdo2-Lipid A-enriched bilayers, respectively. The pore-forming ability of polymyxin B significantly depends on the shape of membrane lipids, which indicates that the antibiotic produces toroidal lipopeptide-lipid pores. Small amphiphilic molecules diminishing the membrane dipole potential and inducing positive curvature stress were shown to be agonists of pore formation by polymyxin B and might be used to develop innovative lipopeptide-based formulations.

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2022 – Chromone-Containing Allylmorpholines Influence Ion Channels in Lipid Membranes via Dipole Potential and Packing Stress
Vesicle Prep Pro Publication in International Journal of Molecular Science (2022) Authors: Efimova S., Martynyuk V., Zakharova A., Yudintceva N., Chernov N., Yakovlev I., Ostroumova O.

Herein, we report that chromone-containing allylmorpholines can affect ion channels formed by pore-forming antibiotics in model lipid membranes, which correlates with their ability to influence membrane boundary potential and lipid-packing stress. At 100 µg/mL, allylmorpholines 1, 6, 7, and 8 decrease the boundary potential of the bilayers composed of palmitoyloleoylphosphocholine (POPC) by about 100 mV. At the same time, the compounds do not affect the zeta-potential of POPC liposomes, but reduce the membrane dipole potential by 80–120 mV. The allylmorpholine-induced drop in the dipole potential produce 10–30% enhancement in the conductance of gramicidin A channels. Chromone-containing allylmorpholines also affect the thermotropic behavior of dipalmytoylphosphocholine (DPPC), abolishing the pretransition, lowering melting cooperativity, and turning the main phase transition peak into a multicomponent profile. Compounds 4, 6, 7, and 8 are able to decrease DPPC’s melting temperature by about 0.5–1.9 °C. Moreover, derivative 7 is shown to increase the temperature of transition of palmitoyloleoylphosphoethanolamine from lamellar to inverted hexagonal phase. The effects on lipid-phase transitions are attributed to the changes in the spontaneous curvature stress. Alterations in lipid packing induced by allylmorpholines are believed to potentiate the pore-forming ability of amphotericin B and gramicidin A by several times.

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2022 – Egg White Peptides Increased the Membrane Liquid-Ordered Phase of Giant Unilamellar Vesicles: Visualization, Localization, and Phase Regulation Mechanism
Vesicle Prep Pro Publication in Journal of Agricultural and Food Chemistry (2022) Authors: Jiang F., Liu J., Niu X., Zhang D., Wang E., Zhang T.

Cell membranes are heterogeneous and consist of liquid-ordered (Lo) and liquid-disordered (Ld) phases due to phase separation. Membrane regulation of egg white peptides (LCAY and QVPLW) was confirmed in our previous study. However, the underlying mechanism of phase regulation by the peptides has not been elucidated. This study aimed to explore the effect of LCAY and QVPLW on the membrane phase separation and illustrate their mechanism by giant unilamellar vesicles (GUVs). Based on phase separation visualization, LCAY and QVPLW were found to increase the Lo phase by rearranging lipids and ordering the Ld phase. LCAY and QVPLW can bind to the GUVs and localize in the amphiphilic region of the membrane. By hydrogen bonds and hydrophobic interactions, LCAY and QVPLW may play a cholesterol-like role in regulating phase separation.

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2021 – Unexpected Gating Behaviour of an Engineered Potassium Channel Kir
Vesicle Prep Pro Publication in Frontiers in Molecular Biosciences (2021) Authors: Fagnen, C., Bannwarth, L., Zuniga, D., Oubella, I., De Zorzi, R., Forest, E., Scala, R., Guilbault, S., Bendahhou, S., Perahia, D., & Vénien-Bryan, C.

In this study, we investigated the dynamics and functional characteristics of the KirBac3.1 S129R, a mutated bacterial potassium channel for which the inner pore-lining helix (TM2) was engineered so that the bundle crossing is trapped in an open conformation. The structure of this channel has been previously determined at high atomic resolution. We explored the dynamical characteristics of this open state channel using an in silico method MDeNM that combines molecular dynamics simulations and normal modes. We captured the global and local motions at the mutation level and compared these data with HDX-MS experiments. MDeNM provided also an estimation of the probability of the different opening states that are in agreement with our electrophysiological experiments. In the S129R mutant, the Arg129 mutation releases the two constriction points in the channel that existed in the wild type but interestingly creates another restriction point.

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2022 – Astrocytes in Paper Chips and Their Interaction with Hybrid Vesicles
Vesicle Prep Pro Publication in Advanced Biology (2022) Authors: Meyer C., De Dios Andres P., Brodszkij E., Westensee I., Lyons J., Vaz S., Städler B.

The role of astrocytes in brain function has received increased attention lately due to their critical role in brain development and function under physiological and pathophysiological conditions. However, the biological evaluation of soft material nanoparticles in astrocytes remains unexplored. Here, the interaction of crosslinked hybrid vesicles (HVs) and either C8-D1A astrocytes or primary astrocytes cultured in polystyrene tissue culture or floatable paper-based chips is investigated. The amphiphilic block copolymer poly(cholesteryl methacrylate)-block-poly(2-carboxyethyl acrylate) (P1) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine lipids are used for the assembly of HVs with crosslinked membranes. The assemblies show no short-term toxicity towards the C8-D1A astrocytes and the primary astrocytes, and both cell types internalize the HVs when cultured in 2D cell culture. Further, it is demonstrated that both the C8-D1A astrocytes and the primary astrocytes could mature in paper-based chips with preserved calcium signaling and glial fibrillary acidic protein expression. Last, it is confirmed that both types of astrocytes could internalize the HVs when cultured in paper-based chips. These findings lay out a fundamental understanding of the interaction between soft material nanoparticles and astrocytes, even when primary astrocytes are cultured in paper-based chips offering a 3D environment.

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2021 – Proton gradients from light-harvesting E. coli control DNA assemblies for synthetic cells
Vesicle Prep Pro Publication in Nature Communications (2021) Authors: Jahnke K., Ritzmann N., Fichtler J., Nitschke A., Dreher Y., Abele T., Hofhaus G., Platzman I., Schröder R. R., Müller D. J., Spatz J. P., Göpfrich K.

Bottom-up and top-down approaches to synthetic biology each employ distinct methodologies with the common aim to harness living systems. Here, we realize a strategic merger of both approaches to convert light into proton gradients for the actuation of synthetic cellular systems. We genetically engineer E. coli to overexpress the light-driven inward-directed proton pump xenorhodopsin and encapsulate them in artificial cell-sized compartments. Exposing the compartments to light-dark cycles, we reversibly switch the pH by almost one pH unit and employ these pH gradients to trigger the attachment of DNA structures to the compartment periphery. For this purpose, a DNA triplex motif serves as a nanomechanical switch responding to the pH-trigger of the E. coli. When DNA origami plates are modified with the pH-sensitive triplex motif, the proton-pumping E. coli can trigger their attachment to giant unilamellar lipid vesicles (GUVs) upon illumination. A DNA cortex is formed upon DNA origami polymerization, which sculpts and deforms the GUVs. We foresee that the combination of bottom-up and top down approaches is an efficient way to engineer synthetic cells.

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2021 – Structural basis for VPS34 kinase activation by Rab1 and Rab5 on membranes
Vesicle Prep Pro Publication in Nature Communications (2021) Authors: Tremel S., Ohashi Y., Morado D.R., Bertram J., Perisic O., Brandt L.T.L., von Wrisberg M-K., Chen Z.A., Maslen S.L., Kovtun O., Skehel M., Rappsilber J., Lang K., Munro S., Briggs J.A.G., Williams R.L.

The lipid phosphatidylinositol-3-phosphate (PI3P) is a regulator of two fundamental but distinct cellular processes, endocytosis and autophagy, so its generation needs to be under precise temporal and spatial control. PI3P is generated by two complexes that both contain the lipid kinase VPS34: complex II on endosomes (VPS34/VPS15/Beclin 1/UVRAG), and complex I on autophagosomes (VPS34/VPS15/Beclin 1/ATG14L). The endosomal GTPase Rab5 binds complex II, but the mechanism of VPS34 activation by Rab5 has remained elusive, and no GTPase is known to bind complex I. Here we show that Rab5a–GTP recruits endocytic complex II to membranes and activates it by binding between the VPS34 C2 and VPS15 WD40 domains. Electron cryotomography of complex II on Rab5a-decorated vesicles shows that the VPS34 kinase domain is released from inhibition by VPS15 and hovers over the lipid bilayer, poised for catalysis. We also show that the GTPase Rab1a, which is known to be involved in autophagy, recruits and activates the autophagy-specific complex I, but not complex II. Both Rabs bind to the same VPS34 interface but in a manner unique for each. These findings reveal how VPS34 complexes are activated on membranes by specific Rab GTPases and how they are recruited to unique cellular locations.

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2021 – Phosphodiesterase Type 5 Inhibitors Greatly Affect Physicochemical Properties of Model Lipid Membranes
Vesicle Prep Pro Publication in Membranes (2021) Authors: Zakharova A.A., Efimova S.S.,Ostroumova O.S.

Although phosphodiesterase type 5 inhibitors are widely used and well-studied drugs, the potential benefits of their application in the treatment of various diseases and new drug delivery systems, including liposome forms, are still being discussed. In this regard, the role of the lipid matrix of cell membranes in the pharmacological action of the inhibitors is of special interest. It was shown that sildenafil, vardenafil, and tadalafil caused a significant decrease in the boundary potential of model membranes composed of palmitoyloleoylphosphatidylcholine or its mixture with cholesterol, by 70–80 mV. The reduction in the membrane dipole potential induced by inhibitors led to a 20–25% increase in the conductance of cation-selective pores formed by the antimicrobial peptide gramicidin A. The addition of sildenafil or vardenafil also led to a significant decrease in the temperature of the main phase transition of dipalmytoylphosphatidylcholine, by about 1.5 °C, while tadalafil did not change the melting temperature. Sildenafil, vardenafil, and tadalafil enhanced the pore-forming activity of the antifungal polyene antibiotic nystatin by 11, 13, and 2 times, respectively. This fact might indicate the induction of membrane curvature stress by the inhibitors. The data obtained might be of special interest for the development of lipid-mediated forms of drugs.

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2021 – Pore-forming Esx proteins mediate toxin secretion by Mycobacterium tuberculosis
Vesicle Prep Pro Publication in Nature Communications (2021) Authors: Tak U., Dokland T. and Niederweis M.

Mycobacterium tuberculosis secretes the tuberculosis necrotizing toxin (TNT) to kill host cells. Here, we show that the WXG100 proteins EsxE and EsxF are essential for TNT secretion. EsxE and EsxF form a water-soluble heterodimer (EsxEF) that assembles into oligomers and long filaments, binds to membranes, and forms stable membrane-spanning channels. Electron microscopy of EsxEF reveals mainly pentameric structures with a central pore. Mutations of both WXG motifs and of a GXW motif do not affect dimerization, but abolish pore formation, membrane deformation and TNT secretion. The WXG/GXW mutants are locked in conformations with altered thermostability and solvent exposure, indicating that the WXG/GXW motifs are molecular switches controlling membrane interaction and pore formation. EsxF is accessible on the bacterial cell surface, suggesting that EsxEF form an outer membrane channel for toxin export. Thus, our study reveals a protein secretion mechanism in bacteria that relies on pore formation by small WXG proteins.

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2021 – Is the Membrane Lipid Matrix a Key Target for Action of Pharmacologically Active Plant Saponins?
Vesicle Prep Pro Publication in International Journal of Molecular Sciences  (2021) Authors: Efimova S.S., Ostroumova O.S.

This study was focused on the molecular mechanisms of action of saponins and related compounds (sapogenins and alkaloids) on model lipid membranes. Steroids and triterpenes were tested. A systematic analysis of the effects of these chemicals on the physicochemical properties of the lipid bilayers and on the formation and functionality of the reconstituted ion channels induced by antimicrobial agents was performed. It was found that digitonin, tribulosin, and dioscin substantially reduced the boundary potential of the phosphatidylcholine membranes. We concluded that saponins might affect the membrane boundary potential by restructuring the membrane hydration layer. Moreover, an increase in the conductance and lifetime of gramicidin A channels in the presence of tribulosin was due to an alteration in the membrane dipole potential. Differential scanning microcalorimetry data indicated the key role of the sapogenin core structure (steroid or triterpenic) in affecting lipid melting and disordering. We showed that an alteration in pore forming activity of syringomycin E by dioscin might be due to amendments in the lipid packing. We also found that the ability of saponins to disengage the fluorescent marker calcein from lipid vesicles might be also determined by their ability to induce a positive curvature stress.

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2021 – Membrane interaction and disulphide-bridge formation in the unconventional secretion of Tau
Vesicle Prep Pro Publication in Bioscience Reports (2021) Authors: Hellen M., Bhattacharjee A., Uronen R-L., Huttunen H.J.

Misfolded, pathological tau protein propagates from cell to cell causing neuronal degeneration in Alzheimer’s disease and other tauopathies. The molecular mechanisms of this process have remained elusive. Unconventional secretion of tau takes place via several different routes, including direct penetration through the plasma membrane. Here, we show that tau secretion requires membrane interaction via disulphide bridge formation. Mutating residues that reduce tau interaction with membranes or formation of disulphide bridges decrease both tau secretion from cells, and penetration through artificial lipid membranes. Our results demonstrate that tau is indeed able to penetrate protein-free membranes in a process independent of active cellular processes and that both membrane interaction and disulphide bridge formation are needed for this process. QUARK-based de novo modelling of the second and third microtubule-binding repeat domains (MTBDs), in which the two cysteine residues of 4R isoforms of tau are located, supports the concept that this region of tau could form transient amphipathic helices for membrane interaction.

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2021 – EPA and DHA differentially modulate membrane elasticity in the presence of cholesterol
Vesicle Prep Pro Publication in Biophysical Journal (2021) Authors: Jacobs M.L., Faizi H.A., Peruzzi J.A., Vlahovska P.M., Kamat N.P.

Polyunsaturated fatty acids (PUFAs) modify the activity of a wide range of membrane proteins and are increasingly hypothesized to modulate protein activity by indirectly altering membrane physical properties. Among the various physical properties affected by PUFAs, the membrane area expansion modulus (Ka), which measures membrane strain in response to applied force, is expected to be a significant controller of channel activity. Yet the impact of PUFAs on membrane Ka has not been measured previously. Through a series of micropipette aspiration studies, we measured the apparent Ka (Kapp) of phospholipid model membranes containing non-esterified fatty acids. First, we measured membrane Kapp as a function of the location of the unsaturated bond and degree of unsaturation in the incorporated fatty acids and found that Kapp generally decreases in the presence of fatty acids with three or more unsaturated bonds. Next, we assessed how select ω-3 PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), affect the Kapp of membranes containing cholesterol. In vesicles prepared with high amounts of cholesterol, which should increase the propensity of the membrane to phase segregate, we found that inclusion of DHA decreases the Kapp in comparison to EPA. We also measured how these ω-3 PUFAs affect membrane fluidity and bending rigidity to determine how membrane Kapp changes in relation to these other physical properties. Our study shows that PUFAs generally decrease Kapp of membranes and that EPA and DHA have differential effects on Kapp when membranes contain higher levels of cholesterol. Our results suggest membrane phase behavior, and the distribution of membrane elasticizing amphiphiles, impacts the ability of a membrane to stretch.

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2021 – Highly Basic Clusters in the Herpes Simplex Virus 1 Nuclear Egress Complex Drive Membrane Budding by Inducing Lipid Ordering
Vesicle Prep Pro Publication in mBio (2021) Authors: Thorsen M.K., Lai A., Lee M.W., Hoogerheide D.P., Wong G.C.L., Freed J.H., Heldweina E.E

During replication of herpesviruses, capsids escape from the nucleus into the cytoplasm by budding at the inner nuclear membrane. This unusual process is mediated by the viral nuclear egress complex (NEC) that deforms the membrane around the capsid by oligomerizing into a hexagonal, membrane-bound scaffold. Here, we found that highly basic membrane-proximal regions (MPRs) of the NEC alter lipid order by inserting into the lipid headgroups and promote negative Gaussian curvature. We also find that the electrostatic interactions between the MPRs and the membranes are essential for membrane deformation. One of the MPRs is phosphorylated by a viral kinase during infection, and the corresponding phosphomimicking mutations block capsid nuclear egress. We show that the same phosphomimicking mutations disrupt the NEC-membrane interactions and inhibit NEC-mediated budding in vitro, providing a biophysical explanation for the in vivo phenomenon. Our data suggest that the NEC generates negative membrane curvature by both lipid ordering and protein scaffolding and that phosphorylation acts as an off switch that inhibits the membrane-budding activity of the NEC to prevent capsid-less budding.

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2021 – Dynamic remodeling of giant unilamellar vesicles induced by monoglyceride nano-micelles: Insights into supramolecular organization
Vesicle Prep Pro Publication in Applied Materials Today (2021) Authors: Yoon B.K., Kim M.C., Jackman J.A., Cho N-J

There is broad interest in developing nanostructured assemblies composed of fatty acids and monoglycerides to inhibit membrane-enveloped pathogens and modulate immune cell behavior. Herein, we investigated the interactions of micellar nanostructures composed of a biologically active monoglyceride, glycerol monolaurate (GML), or its ether-bonded equivalent, 1-O-dodecyl-rac-glycerol (DDG), with cell-membrane-mimicking giant unilamellar vesicles (GUVs). Our findings revealed that GML nanostructures induced fission or fusion depending on the GML concentration and corresponding degree of supramolecular organization, while DDG nanostructures only caused aggregation-like disruption of the GUV outer surface. In specific conditions, the GML nanostructures also triggered pearling instability, which led to dynamic membrane remodeling behavior and the pattern of GML interactions was consistent across simplified and complex membrane compositions. Notably, the spectrum of membrane morphological changes induced by GML nanostructures, including fission, fusion, and pearling behaviors, is appreciably wider than the fission behavior exhibited by fatty acid nanostructures in past studies. Collectively, these findings demonstrate how controlling the supramolecular organization of monoglycerides within nanostructured assemblies can be useful to modulate the type and degree of membrane interactions relevant to biophysical and nanomedicine applications.

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2021 – Electroformation of Giant Unilamellar Vesicles and A Case Study of the Specific Host-Toxin Interaction of Lipid Membranes with Cassiicolin
Vesicle Prep Pro Protocol in Bio-101 (2021) Authors: Ngo KX., Weichbrodt C., Quoc NB

An alternating electric field is applied to induce swelling of thin lipid films and generation of giant unilamellar vesicles (GUVs) on an indium tin oxide (ITO)-coated glass surface. The process is, hereafter, referred to as the electroformation of GUVs. Several important parameters such as lipid manipulation, temperature, osmolarity and ionic strength of the solutions involved, and the electric field (current (DC, AC), amplitude, frequency) should be optimal for the successful electroformation of GUVs. In our case study, GUVs composed of lipid mixtures available in plant cells provide many benefits for studying the lipid-dependent pathogenicity of cassiicolin (Cas) toxins and thereby deciphering the host-selective toxin interaction of Cas toxins with the specific lipid membranes of plant cells. GUVs gently maintained in the solution furnish perfectly suspended and intact lipid membranes similar to cytoplasmic membranes enabling us to examine the selective binding of GFP-Cas1 and GFP-Cas2 to the specific lipid membranes. In this protocol, we briefly explain the principle of electroformation method and provide the experimental conditions and the manipulation for successfully making GUVs composed of plant lipids (DPPC, DPPC/DPPA, DPPC/MGDG, DPPC/DGDG, DPPC/stigmasterol, DPPC/sitosterol, DPPC/DGTS-d9, and DPPC/DGTS). 

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2021 – Binding of DNA origami to lipids: maximizing yield and switching via strand displacement
Vesicle Prep Pro Publication in Nucleic Acids Research (2021) Authors: Daljit Singh J. K., Darley E., Ridone P., Gaston J. P., Abbas A., Wickham S. F J., Baker M. A B

Liposomes are widely used as synthetic analogues of cell membranes and for drug delivery. Lipid-binding DNA nanostructures can modify the shape, porosity and reactivity of liposomes, mediated by cholesterol modifications. DNA nanostructures can also be designed to switch conformations by DNA strand displacement. However, the optimal conditions to facilitate stable, high-yield DNA–lipid binding while allowing controlled switching by strand displacement are not known. Here, we characterized the effect of cholesterol arrangement, DNA structure, buffer and lipid composition on DNA–lipid binding and strand displacement. We observed that binding was inhibited below pH 4, and above 200 mM NaCl or 40 mM MgCl2, was independent of lipid type, and increased with membrane cholesterol content. For simple motifs, binding yield was slightly higher for double-stranded DNA than single-stranded DNA. For larger DNA origami tiles, four to eight cholesterol modifications were optimal, while edge positions and longer spacers increased yield of lipid binding. Strand displacement achieved controlled removal of DNA tiles from membranes, but was inhibited by overhang domains, which are used to prevent cholesterol aggregation. These findings provide design guidelines for integrating strand displacement switching with lipid-binding DNA nanostructures. This paves the way for achieving dynamic control of membrane morphology, enabling broader applications in nanomedicine and biophysics.

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2021 – Design of biologically active binary protein 2D materials
Vesicle Prep Pro Publication in Nature (2021) Authors: Ben-Sasson A.J., Watson J.L., Sheffler W., Camp Johnson M., Bittleston A., Somasundaram L., Decarreau J., Jiao F., Chen J., Mela I., Drabek A.A., Jarrett S.M., Blacklow S.C., Kaminski C.F., Hura G.L., De Yoreo J.J., Kollman J.M., Ruohola-Baker H., Derivery E., Baker D.

Ordered two-dimensional arrays such as S-layers and designed analogues have intrigued bioengineers, but with the exception of a single lattice formed with flexible linkers, they are constituted from just one protein component. Materials composed of two components have considerable potential advantages for modulating assembly dynamics and incorporating more complex functionality. Here we describe a computational method to generate co-assembling binary layers by designing rigid interfaces between pairs of dihedral protein building blocks, and use it to design a p6m lattice. The designed array components are soluble at millimolar concentrations, but when combined at nanomolar concentrations, they rapidly assemble into nearly crystalline micrometre-scale arrays nearly identical to the computational design model in vitro and in cells without the need for a two-dimensional support. Because the material is designed from the ground up, the components can be readily functionalized and their symmetry reconfigured, enabling formation of ligand arrays with distinguishable surfaces, which we demonstrate can drive extensive receptor clustering, downstream protein recruitment and signalling. Using atomic force microscopy on supported bilayers and quantitative microscopy on living cells, we show that arrays assembled on membranes have component stoichiometry and structure similar to arrays formed in vitro, and that our material can therefore impose order onto fundamentally disordered substrates such as cell membranes. In contrast to previously characterized cell surface receptor binding assemblies such as antibodies and nanocages, which are rapidly endocytosed, we find that large arrays assembled at the cell surface suppress endocytosis in a tunable manner, with potential therapeutic relevance for extending receptor engagement and immune evasion. Our work provides a foundation for a synthetic cell biology in which multi-protein macroscale materials are designed to modulate cell responses and reshape synthetic and living systems.

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2021 – Alamethicin Channels as a Signal Transduction Element in an Immobilized Single Giant Unilamellar Vesicle
Vesicle Prep Pro Publication in Sensors and Materials (2021) Authors: Fukuda H., Nitta M., Sakamoto M., Shoji A., Sugawara M.

A single giant unilamellar vesicle (GUV) functionalized with an anti-bovine serum albumin (BSA) antibody was immobilized on an avidin slip, and alamethicin channels were embedded as a signal transduction element for creating a channel-based molecular sensing system. The GUV sensor based on the membrane-bound anti-BSA antibody receptor exhibited alamethicin activities that reflected the binding of BSA (an analyte) at the membrane/solution interface. The normalized integrated current at −60 mV was able to be used as a measure of the amount of BSA in a solution. The quantification of BSA at pg/mL level was demonstrated.

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2021 – Binding of DNA origami to lipids: maximising yield and switching via strand-displacement
Vesicle Prep Pro Publication in Nucleic Acids Research (2021) Authors: Singh J.KD., Darley E., Ridone P., Gaston J. P., Abbas A., Wickham S.FJ., Baker M.AB.

iposomes are widely used as synthetic analogues of cell membranes and for drug delivery. Lipid-binding DNA nanostructures can modify the shape, porosity and reactivity of liposomes, mediated by cholesterol modifications. DNA nanostructures can also be designed to switch conformations by DNA strand displacement. However, the optimal conditions to facilitate stable, high-yield DNA–lipid binding while allowing controlled switching by strand displacement are not known. Here, we characterized the effect of cholesterol arrangement, DNA structure, buffer and lipid composition on DNA–lipid binding and strand displacement. We observed that binding was inhibited below pH 4, and above 200 mM NaCl or 40 mM MgCl2, was independent of lipid type, and increased with membrane cholesterol content. For simple motifs, binding yield was slightly higher for double-stranded DNA than single-stranded DNA. For larger DNA origami tiles, four to eight cholesterol modifications were optimal, while edge positions and longer spacers increased yield of lipid binding. Strand displacement achieved controlled removal of DNA tiles from membranes, but was inhibited by overhang domains, which are used to prevent cholesterol aggregation. These findings provide design guidelines for integrating strand displacement switching with lipid-binding DNA nanostructures. This paves the way for achieving dynamic control of membrane morphology, enabling broader applications in nanomedicine and biophysics.

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2020 – The mechanisms of action of water-soluble aminohexanoic and malonic adducts of fullerene C60 with hexamethonium on model lipid membranes
Vesicle Prep Pro Publication found in Biochimica et Biophysica Acta (BBA) -  Biomembranes (2020) Authors: Efimova S.S., Khaleneva D.A., Litasova E.V., Piotrovskiy L.B., Ostroumova O.S.

In an attempt to understand the possibility of applications of the fullerene-based systems for transporting various polar compounds like hexamethonium through the blood–brain barrier, we studied the influence of a series of derivatives of fullerene C60 in the form of salts with hexamethonium bis-anion, namely the adducts of fullerenols with 6-aminohexanoic acid (IEM-2197), and two bis-adduct malonic acid derivatives of fullerene with addents bound in two hemispheres (IEM-2143) and in equatorial positions (IEM-2144), on model membranes. We showed that IEM-2197 induced the disintegration of the bilayers composed of DOPC at the concentrations more than 2 mg/ml. IEM-2144 and IEM-2143-induced ion-permeable pores at concentrations of 0.3 and 0.02 mg/ml, respectively; herewith, IEM-2143 was characterized by the greater efficiency than IEM-2144. IEM-2197 did not significantly affect the phase behavior of DPPC, while the melting temperature significantly decreased with addition of IEM-2144 and IEM-2143. The increase in the half-width of the main transition peaks by more than 2.0 °C in the presence of IEM-2144 and IEM-2143 was observed, along with the pronounced peak deconvolution. We proposed that the immersion of IEM-2144 and IEM-2143 into the polar region of the DOPC or DPPC bilayers led to an increase in the relative mobility of tails and formation of ion-permeable defects. IEM-2197 demonstrated the more pronounced effects on the melting and ion permeability of PG- and PS-containing bilayers compared to PC-enriched membranes. These results indicated that IEM-2197 preferentially interacts with the negatively charged lipids compared to neutral species.

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2021 – A microfluidic platform for sequential assembly and separation of synthetic cell models
Vesicle Prep Pro Pre-Print Publication in ACS Synthetic Biology (2021) Authors: Tivony R., Fletcher M., Al Nahas K., Keyser U. F.

Cell-sized vesicles like giant unilamellar vesicles (GUVs) are established as a promising biomimetic model for studying cellular phenomena in isolation. However, the presence of residual components and by-products, generated during vesicles preparation and manipulation, severely limits the utility of GUVs in applications like synthetic cells. Therefore, with the rapidly growing field of synthetic biology, there is an emergent demand for techniques that can continuously purify cell-like vesicles from diverse residues, while GUVs are being simultaneously synthesized and manipulated. We developed a microfluidic platform capable of purifying GUVs through stream bifurcation, where a stream of vesicles suspension is partitioned into three fractions - purified GUVs, residual components, and a washing solution. Using our purification approach, we showed that giant vesicles can be separated from various residues – that range in size and chemical composition – with a very high efficiency (e = 0.99), based on size and deformability of the filtered objects. In addition, by incorporating the purification module with a microfluidic-based GUV-formation method, octanol-assisted liposome assembly (OLA), we established an integrated production-purification microfluidic unit that sequentially produces, manipulates, and purifies GUVs. We demonstrate the applicability of the integrated device to synthetic biology through sequentially fusing SUVs with freshly prepared GUVs and separating the fused GUVs from extraneous SUVs and oil droplets at the same time.

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2020 – Soluble cyanobacterial carotenoprotein as a robust antioxidant nanocarrier and delivery module
Vesicle Prep Pro Publication in Antioxidants (Basel) (2020) Authors: Maksimov E.G., Zamaraev A.V., Parshina E.Y., Slonimskiy Y.B., Slastnikova T.A., Abdrakhmanov A.A., Babaev P.A., Efimova, S.S., Ostroumova O.S., Stepanov A.V., Ryabova, A.V., Friedrich T., Sluchanko N.N.

To counteract oxidative stress, antioxidants including carotenoids are highly promising, yet their exploitation is drastically limited by the poor bioavailability and fast photodestruction, whereas current delivery systems are far from being efficient. Here we demonstrate that the recently discovered nanometer-sized water-soluble carotenoprotein from Anabaena (termed CTDH) transiently interacts with liposomes to efficiently extract carotenoids via carotenoid-mediated homodimerization, yielding violet-purple protein samples amenable to lyophilization and long-term storage. We characterize spectroscopic properties of the pigment-protein complexes and thermodynamics of liposome-protein carotenoid transfer and demonstrate the highly efficient delivery of echinenone form CTDH into liposomes. Most importantly, we show carotenoid delivery to membranes of mammalian cells, which provides protection from reactive oxygen species. The described carotenoprotein may be considered as part of modular systems for the targeted antioxidant delivery. Significance statement: Carotenoids are excellent natural antioxidants but their delivery to vulnerable cells is challenging due to their hydrophobic nature and susceptibility to degradation. Thus, systems securing antioxidant stability and facilitating targeted delivery are of great interest for the design of medical agents. In this work, we have demonstrated that soluble cyanobacterial carotenoprotein can deliver echinenone into membranes of liposomes and mammalian cells with almost 70 % efficiency, which alleviates the induced oxidative stress. Our findings warrant the robustness of the protein-based carotenoid delivery for studies of carotenoid activities and effects on cell models.

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2020 – The Disordering Effect of Plant Metabolites on Model Lipid Membranes of Various Thickness
Vesicle Prep Pro Publication in Cell and Tissue Biology (2020) Authors: Efimova S.S., Ostroumova O.S.

This study was focused on the effect of plant metabolites (phloretin, capsaicin, digitonin, diosgeninThis study was focused on the effect of plant metabolites (phloretin, capsaicin, digitonin, diosgeninand betulin) on the model lipid membranes. The methods of assessing the permeability of lipid bilayers basedon measuring the leakage of a fluorescent marker (calcein) from liposomes and differential scanning microcalorimetryof vesicle suspension were used. It was found that the release rate of calcein from 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) liposomes with test compounds added to the suspension at aratio with lipid 1 : 50 decreased in the order capsaicin > phloretin >> betulin ≈ diosgenin ≈ digitonin. In thecase of cholesterol- and ergosterol-containing POPC liposomes, the activity decreased as diosgenin ≈ digitonin> betulin > capsaicin > phloretin. It was demonstrated that phloretin and capsaicin significantly reducethe melting temperature (Tm) and to increase the half-width of the main peak on the endotherm (T1/2) ofdipalmitoylphosphocholine (DPPC), distearoylphosphocholine (DSPC) and diarachidoylphosphocholine(DAPC). These results show that the incorporation of these small molecules between the polar “heads” ofphosphocholine. It was found that the increase in the length of saturated chains of membrane-forming lipids(from 16 to 20 hydrocarbon units), the absolute values of ΔTm and ΔT1/2 decreased in the presence of phloretinand increased with capsaicin. This may be the result of differences in the localization of phloretin and capsaicinin the membrane. Steroid saponins exhibited a weak effect on the thermotropic behavior of phosphocholines.The absolute values of ΔTm and ΔT1/2 decreased in the order DPPC, DSPC, and DAPC and increasedin the order betulin, diosgenin, and digitonin. Steroid saponins are characterized by a more pronouncedeffect on the thermotropic behavior of the sterol–phospholipid mixture. The findings are consistent with theassumption of a high affinity of the tested saponins for sterol-containing membranes.

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2020 – Possible Mechanisms of Toxicity of Local Aminoamide Anesthetics: Lipid-Mediated Action of Ropivacaine
Vesicle Prep Pro Publication in Cell and Tissue Biology (2020) Authors: Zakharova A. A., Efimova S.S., Koryachkin V.A., Zabolotskii D.V., Ostroumova O.S. 

This work is devoted to the identification of molecular mechanisms of action of local anesthetic ropivacaine and other aminoamides (mepivacaine and bupivacaine) on the membrane physicochemical properties and formation and functioning of various ion channels in model lipid bilayers. The boundary membrane potential and its components, permeability for fluorescent markers, and the temperature and cooperativity of the melting of membrane lipid, as well as the mosaic organization of the bilayer, were studied. It was found that ropivacaine, as well as mepivacaine and bupivacaine, changed the surface charge of the bilayer and increased the membrane boundary potential. It was demonstrated that the permeability of lipid vesicles for calcein increased with the introduction of aminoamides, while the temperature and cooperativity of the melting of saturated phosphocholines decreased. The effect of anesthetics on the packing density of lipids in the membrane correlated with the hydrophobicity of their molecules. A comparison of the effects of aminoamides allowed three mechanisms of anesthetics action on the functioning of ion channels to be determined: increasing the surface potential of the membrane, decreasing the packing density of lipids in the membrane, and blocking ion channels.

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2020 – Romo1-Derived Antimicrobial Peptide Is a New Antimicrobial Agent against Multidrug-Resistant Bacteria in a Murine Model of Sepsis
Vesicle Prep Pro Publication in mBio (2020) Authors: Lee H.-R., You D.-G., Kim H.K., Sohn J.W., Kim M.J., Park J.K., Lee G.Y., Yoo Y.D.

To overcome increasing bacterial resistance to conventional antibiotics, many antimicrobial peptides (AMPs) derived from host defense proteins have been developed. However, there are considerable obstacles to their application to systemic infections because of their low bioavailability. In the present study, we developed an AMP derived from Romo1 (AMPR-11) that exhibits a broad spectrum of antimicrobial activity. AMPR-11 showed remarkable efficacy against sepsis-causing bacteria, including multidrug-resistant strains, with low toxicity in a murine model of sepsis after intravenous administration. It seems that AMPR-11 disrupts bacterial membranes by interacting with cardiolipin and lipid A. From the results of this study, we suggest that AMPR-11 is a new class of agent for overcoming low efficacy in the intravenous application of AMPs and is a promising candidate to overcome multidrug resistance.

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2020 – Membrane characteristics tune activities of endosomal and autophagic human VPS34 complexes
Vesicle Prep Pro Publication in eLife (2020) Authors: Ohashi Y., Tremel S., Masson G.R., McGinney L., Boulanger J., Rostislavleva K., Johnson C.M., Niewczas I., Clark J., Williams R.J.

The lipid kinase VPS34 orchestrates diverse processes, including autophagy, endocytic sorting, phagocytosis, anabolic responses and cell division. VPS34 forms various complexes that help adapt it to specific pathways, with complexes I and II being the most prominent ones. We found that physicochemical properties of membranes strongly modulate VPS34 activity. Greater unsaturation of both substrate and non-substrate lipids, negative charge and curvature activate VPS34 complexes, adapting them to their cellular compartments. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) of complexes I and II on membranes elucidated structural determinants that enable them to bind membranes. Among these are the Barkor/ATG14L autophagosome targeting sequence (BATS), which makes autophagy-specific complex I more active than the endocytic complex II, and the Beclin1 BARA domain. Interestingly, even though Beclin1 BARA is common to both complexes, its membrane-interacting loops are critical for complex II, but have only a minor role for complex I.

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2020 – Polymer–Lipid Hybrid Vesicles and Their Interaction with HepG2 Cells
Vesicle Prep Pro Publication in Small (2020) Authors: Brodszkij E., Westensee I.N., Bertelsen M., Gal N., Boesen T.,Städler B.

Polymer–lipid hybrid vesicles are an emerging type of nano‐assemblies that show potential as artificial organelles among others. Phospholipids and poly(cholesteryl methacrylate)‐block‐poly(methionine methacryloyloxyethyl ester (METMA)—random– 2‐carboxyethyl acrylate (CEA)) labeled with a Förster resonance energy transfer (FRET) reporter pair are used for the assembly of small and giant hybrid vesicles with homogenous distribution of both building blocks in the membrane as confirmed by the FRET effect. These hybrid vesicles have no inherent cytotoxicity when incubated with HepG2 cells up to 1.1 × 1011 hybrid vesicles per mL, and they are internalized by the cells. In contrast to the fluorescent signal originating from the block copolymer, the fluorescent signal coming from the lipids is barely detectable in cells incubated with hybrid vesicles for 6 h followed by 24 h in cell media, suggesting that the two building blocks have a different intracellular fate. These findings provide important insight into the design criteria of artificial organelles with potential structural integrity.

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2020 – Hybrid giant lipid vesicles incorporating a PMMA-based copolymer
Vesicle Prep Pro Publication found in Biochimica et Biophysica Acta (BBA) - General Subjects (2020) Authors: Miele Y., Mingotaud A.F., Caruso E., Malacarne M. C., Izzo L., Lonetti B., Rossi F.

Background - In recent years, there has been a growing interest in the formation of copolymer-lipid hybrid self-assemblies, which allow combining and improving the main features of pure lipid-based and copolymer-based systems known for their potential applications in the biomedical field. As the most common method used to obtain giant vesicles is electroformation, most systems so far used low Tg polymers for their flexibility at room temperature.

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2020 – Identification of a Membrane Binding Peptide in the Envelope Protein of MHV CoroNaVirus
Vesicle Prep Pro Publication in Viruses (2020) Authors: Alsaadi E.A.J., Neuman B.W., Jones I.M.

CoroNaViruses (CoVs) are enveloped, positive sense, single strand RNA viruses that cause respiratory, intestinal and neurological diseases in mammals and birds. Following replication, CoVs assemble on intracellular membranes including the endoplasmic reticulum Golgi intermediate compartment (ERGIC) where the envelope protein (E) functions in virus assembly and release. In consequence, E potentially contains membrane-modifying peptides. To search for such peptides, the E coding sequence of Mouse Hepatitis Virus (MHV) was inspected for its amino acid conservation, proximity to the membrane and/or predicted amphipathic helices. Peptides identified in silico were synthesized and tested for membrane-modifying activity in the presence of giant unilamellar vesicles (GUVs) consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), sphingomyelin and cholesterol. To confirm the presence of membrane binding peptides identified in the context of a full-length E protein, the wild type and a number of mutants in the putative membrane binding peptide were expressed in Lenti-X-293T mammalian and insect cells, and the distribution of E antigen within the expressing cell was assessed. Our data identify a role for the post-transmembrane region of MHV E in membrane binding.

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2020 – Exploring the Relationship between BODIPY Structure and Spectroscopic Properties to Design Fluorophores for Bioimaging
Vesicle Prep Pro Publication in Chemistry a European Journal (2020) Authors: Donnelly J.L., Offenbartl-Stiegert D., Marín-Beloqui J.M., Rizzello L., Battaglia G., Clarke T.M., Howorka S., Wilden J.D.

Designing chromophores for biological applications requires a fundamental understanding of how the chemical structure of a chromophore influences its photophysical properties. We here describe the synthesis of a library of BODIPY dyes, exploring diversity at various positions around the BODIPY core. The results show that the nature and position of substituents have a dramatic effect on the spectroscopic properties. Substituting in a heavy atom or adjusting the size and orientation of a conjugated system provides a means of altering the spectroscopic profiles with high precision. The insight from the structure–activity relationship was applied to devise a new BODIPY dye with rationally designed photochemical properties including absorption towards the near‐infrared region. The dye also exhibited switch‐on fluorescence to enable visualisation of cells with high signal‐to‐noise ratio without washing‐out of unbound dye. The BODIPY‐based probe is non‐cytotoxic and compatible with staining procedures including cell fixation and immunofluorescence microscopy.

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2020 – Facile Mixing of Phospholipids Promotes Self-Assembly of Low-Molecular-Weight Biodegradable Block Co-Polymers into Functional Vesicular Architectures
Vesicle Prep Pro Publication in Polymers (2020) Authors: Khan A., Ho J.C.S., Roy S., Bo Liedberg B., Nallani M.

In this work, we have used low-molecular-weight (PEG12-b-PCL6, PEG12-b-PCL9 or PEG16-b-PLA38; MW, 1.25-3.45 kDa) biodegradable block co-polymers to construct nano- and micron-scaled hybrid (polymer/lipid) vesicles, by solvent dispersion and electroformation methods, respectively. The hybrid vesicles exhibit physical properties (size, bilayer thickness and small molecule encapsulation) of a vesicular boundary, confirmed by cryogenic transmission electron microscopy, calcein leakage assay and dynamic light scattering. Importantly, we find that these low MW polymers, on their own, do not self-assemble into polymersomes at nano and micron scales. Using giant unilamellar vesicles (GUVs) model, their surface topographies are homogeneous, independent of cholesterol, suggesting more energetically favorable mixing of lipid and polymer. Despite this mixed topography with a bilayer thickness similar to that of a lipid bilayer, variation in surface topology is demonstrated using the interfacial sensitive phospholipase A2 (sPLA2). The biodegradable hybrid vesicles are less sensitive to the phospholipase digestion, reminiscent of PEGylated vesicles, and the degree of sensitivity is polymer-dependent, implying that the nano-scale surface topology can further be tuned by its chemical composition. Our results reveal and emphasize the role of phospholipids in promoting low MW polymers for spontaneous vesicular self-assembly, generating a functional hybrid lipid-polymer interface.

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2020 – Destructing the Plasma Membrane with Activatable Vesicular DNA Nanopores
Vesicle Prep Pro Publication in ACS Appl. Mater. Interfaces (2020) Authors: Chen L, Liang S., Chen Y., Wu M., Zhang Y.

Pore-forming proteins are an agent for attack or defense in various organisms, and its cytolytic activity has medical potential in cancer therapy. Despite recent advances in mimicking these proteins by amphipathic DNA nanopores, it remains inefficient to incorporate them into lipid bilayers. Here, we present the development of vesicular DNA nanopores that can controllably open a lipid membrane. Different from previously reported DNA nanopores that randomly insert into the planar bilayers, we design on-command fusogenic liposome-incorporated transmembrane DNA nanopores (FLIPs) that bypass the direct insertion process. By steric deshielding of fusogenic liposomal supports under low pH conditions, the embedded FLIPs are transferred and perforate lipid bilayers. We find that FLIPs depolarize the plasma membrane and thereby induce pyroptosis-like cell death. We further demonstrate the use of FLIPs to inhibit tumor growth in murine tumor models, which provides a new route to cancer nanotherapy. 

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2020 – Effect of DMSO on the Mechanical and Structural Properties of Model and Biological Membranes
Vesicle Prep Pro Publication in the Biophysical Journal (2020) Authors: Gironi B., Kahveci Z., McGill B., Lechner B.D., Pagliara S., Metz J., Morresi A., Palombo F., Sassi P., Petrov P.G.

Dimethyl sulfoxide (DMSO) is widely used in a number of biological and biotechnological applications, mainly because of its effects on the cell plasma membrane, but the molecular origins of this action are yet to be fully clarified. In this work, we used two- and three-component synthetic membranes (liposomes) and the plasma membrane of human erythrocytes to investigate the effect of DMSO when added to the membrane-solvating environment. Fourier transform infrared spectroscopy and thermal fluctuation spectroscopy revealed significant differences in the response of the two types of liposome systems to DMSO in terms of the bilayer thermotropic behavior, available free volume of the bilayer, its excess surface area, and bending elasticity. DMSO also alters the mechanical properties of the erythrocyte membrane in a concentration-dependent manner and is capable of increasing membrane permeability to ATP at even relatively low concentrations (3% v/v and above). Taken in its entirety, these results show that DMSO is likely to have a differential effect on heterogeneous biological membranes, depending on their local composition and structure, and could affect membrane-hosted biological functions.

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2020 – Design and Assembly of Membrane-Spanning DNA Nanopores
Vesicle Prep Pro Publication in Nanopore Technology (2020) Authors: Göpfrich K., Ohmann A., Keyser U.F.

Versatile lipid membrane-inserting nanopores have been made by functionalizing DNA nanostructures with hydrophobic tags. Here, we outline design and considerations to obtain DNA nanopores with the desired dimensions and conductance properties. We further provide guidance on their reconstitution into lipid membranes.

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2020 – Design of Protein Logic Gate System Operating on Lipid Membranes
Vesicle Prep Pro Publication in ACS Synth. Biol. (2020) Authors: Omersa N., Aden S., Kisovec M., Podobnik M., Anderluh G.

Lipid membranes are becoming increasingly popular in synthetic biology due to their biophysical properties and crucial role in communication between different compartments. Several alluring protein–membrane sensors have already been developed, whereas protein logic gates designs on membrane-embedded proteins are very limited. Here we demonstrate the construction of a two-level protein–membrane logic gate with an OR-AND logic. The system consists of an engineered pH-dependent pore-forming protein listeriolysin O and its DARPin-based inhibitor, conjugated to a lipid vesicle membrane. The gate responds to low pH and removal of the inhibitor from the membrane either by switching to a reducing environment, protease cleavage, or any other signal depending on the conjugation chemistry used for inhibitor attachment to the membrane. This unique protein logic gate vesicle system advances generic sensing and actuator platforms used in synthetic biology and could be utilized in drug delivery. 

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2020 – Cholesterol and desmosterol incorporation into ram sperm membrane before cryopreservation: Effects on membrane biophysical properties and sperm quality
Vesicle Prep Pro Publication in Biochimica et Biophysica Acta (BBA) - Biomembranes (2020) Authors: de las Mercedes Carro M., Peñalva D.A., Antollini S.S., Hozbora F.A., Buschiazzo J.

Ram sperm are particularly sensitive to freeze-thawing mainly due to their lipid composition, limiting their use in artificial insemination programs. We evaluated the extent of cholesterol and desmosterol incorporation into ram sperm through incubation with increasing concentrations of methyl-β-cyclodextrin (MβCD)-sterol complexes, and its effect on membrane biophysical properties, membrane lateral organization and cryopreservation outcome. Sterols were effectively incorporated into the sperm membrane at 10 and 25 mM MβCD-sterols, similarly increasing membrane lipid order at physiological temperature and during temperature decrease. Differential ordering effect of sterols in ternary-mixture model membranes revealed a reduced tendency of desmosterol of segregating into ordered domains. Live cell imaging of fluorescent cholesterol showed sterol incorporation and evidenced the presence of sperm sub-populations compatible with different sterol contents and a high concentration of sterol rich-ordered domains mainly at the acrosome plasma membrane. Lateral organization of the plasma membrane, assessed by identification of GM1-related rafts, was preserved after sterol incorporation except when high levels of sterols (25 mM MβCD-desmosterol) were incorporated. Ram sperm incubation with 10 mM MβCD-sterols prior to cryopreservation in a cholesterol-free extender improved sperm quality parameters after cooling and freezing. While treatment with 10 mM MβCD-cholesterol increased sperm motility, membrane integrity and tolerance to osmotic stress after thawing, incorporation of desmosterol increased the ability of ram sperm to overcome osmotic stress. Our research provides evidence on the effective incorporation and biophysical behavior of cholesterol and desmosterol in ram sperm membranes and on their consequences in improving functional parameters of sperm after temperature decrease and freezing.

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2020 – Coating and Stabilization of Liposomes by Clathrin-Inspired DNA Self-Assembly
Vesicle Prep Pro Publication found in ACS Nano (2020) Authors: Baumann, K.N., Piantanida L., García-Nafría J., Sobota D., Voïtchovsky K., Knowles T.P.J., Hernández-Ainsa S.

The self-assembly of the protein clathrin on biological membranes facilitates essential processes of endocytosis and has provided a source of inspiration for materials design by the highly ordered structural appearance. By mimicking the architecture of the protein building blocks and clathrin self-assemblies to coat liposomes with biomaterials,advanced hybrid carriers can be derived. Here, we present a method for fabricating DNA-coated liposomes by hydrophobically anchoring and subsequently connecting DNA-based triskelion structures on the liposome surface inspired by the assembly of the protein clathrin. Dynamic light scattering, ζ-potential, confocal microscopy, and cryo-electron microscopy measurements independently demonstrate successful DNA coating. Nanomechanical measurements conducted with atomic force microscopy show that the DNA coating enhances the mechanical stability of the liposomes relative to uncoated ones. Furthermore, we provide the possibility to reverse the coating process by triggering the disassembly of the DNA coats through a toehold-mediated displacement reaction. Our results describe a straightforward, versatile, and reversible approach for coating and stabilizing lipid vesicles through the assembly of rationally designed DNA structures. This method has potential for further development toward the ordered arrangement of tailored functionalities on the surface of liposomes and for applications as hybrid nanocarriers.

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2020 – Atg3 promotes Atg8 lipidation via altering lipid diffusion and rearrangement
Vesicle Prep Pro Publication in Protein Science (2020) Authors: Wang S., Li Y., Ma C.

Atg3‐catalyzed transferring of Atg8 to phosphatidylethanolamine (PE) in the phagophore membrane is essential for autophagy. Previous studies have demonstrated that this process requires Atg3 to interact with the phagophore membrane via its N‐terminal amphipathic helix. In this study, by using combined biochemical and biophysical approaches, our data showed that in addition to binding to the membranes, Atg3 attenuates lipid diffusion and enriches lipid molecules with smaller headgroup. Our data suggest that Atg3 promotes Atg8 lipidation via altering lipid diffusion and rearrangement.

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2020 – Characterization of lipid composition and diffusivity in OLA generated vesicles
Vesicle Prep Pro Publication in Biochimica et Biophysica Acta (BBA) - Biomembranes (2020) Authors: Schaich M., Sobota D., Sleath H., Cama J., Keyser U.F.

Giant Unilamellar Vesicles (GUVs) are a versatile tool in many branches of science, including biophysics and synthetic biology. Octanol-Assisted Liposome Assembly (OLA), a recently developed microfluidic technique enables the production and testing of GUVs within a single device under highly controlled experimental conditions. It is therefore gaining significant interest as a platform for use in drug discovery, the production of artificial cells and more generally for controlled studies of the properties of lipid membranes. In this work, we expand the capabilities of the OLA technique by forming GUVs of tunable binary lipid mixtures of DOPC, DOPG and DOPE. Using fluorescence recovery after photobleaching we investigated the lateral diffusion coefficients of lipids in OLA liposomes and found the expected values in the range of 1 μm2/s for the lipid systems tested. We studied the OLA derived GUVs under a range of conditions and compared the results with electroformed vesicles. Overall, we found the lateral diffusion coefficients of lipids in vesicles obtained with OLA to be quantitatively similar to those in vesicles obtained via traditional electroformation. Our results provide a quantitative biophysical validation of the quality of OLA derived GUVs, which will facilitate the wider use of this versatile platform.

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2019 – The Mechanisms of Action of Triindolylmethane Derivatives on Lipid Membranes
Vesicle Prep Pro Publication in Acta Naturae (2019) Authors: Efimova, S.S. Tertychnaya T.E., Lavrenov S.N., Ostroumova, O.S.

The effects of new synthetic antibacterial agents – tris(1-pentyl-1H-indol-3-yl)methylium chloride (LCTA-1975) and (1-(4-(dimethylamino)-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl)-1H-indol-3-yl)bis(1-propyl- 1H-indol-3-yl)methylium chloride (LCTA-2701 – on model lipid membranes were studied. The ability of the tested agents to form ion-conductive transmembrane pores, influence the electrical stability of lipid bilayers and the phase transition of membrane lipids, and cause the deformation and fusion of lipid vesicles was investigated. It was established that both compounds exert a strong detergent effect on model membranes. The results of differential scanning microcalorimetry and measuring of the threshold transmembrane voltage that caused membrane breakdown before and after adsorption of LCTA-1975 and LCTA-2701 indicated that both agents cause disordering of membrane lipids. Synergism of the uncoupling action of antibiotics and the alkaloid capsaicin on model lipid membranes was shown. The threshold concentration of the antibiotic that caused an increase in the ion permeability of the lipid bilayer depended on the membrane lipid composition. It was lower by an order of magnitude in the case of negatively charged lipid bilayers than for the uncharged membranes. This can be explained by the positive charge of the tested agents. At the same time, LCTA-2701 was characterized by greater efficiency than LCTA-1975. In addition to its detergent action, LCTA-2701 can induce ion-permeable transmembrane pores: step-like current fluctuations corresponding to the opening and closing of individual ion channels were observed. The difference in the mechanisms of action might be related to the structural features of the antibiotic molecules: in the LCTA-1975 molecule, all three substituents at the nitrogen atoms of the indole rings are identical and represent n-alkyl (pentyl) groups, while LCTA-2701 contains a maleimide group, along with two alkyl substituents (n-propyl). The obtained results might be relevant to our understanding of the mechanism of action of new antibacterial agents, explaining the difference in the selectivity of action of the tested agents on the target microorganisms and their toxicity to human cells. Model lipid membranes should be used in further studies of the trends in the modification and improvement of the structures of new antibacterial agents.

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2020 – Actuation of synthetic cells with proton gradients generated by light-harvesting E. coli
Vesicle Prep Pro prePublication in Research Square (2020) Authors: Jahnke K., Ritzmann N., Fichtler J., Nitschke A., Dreher Y., Abele T., Hofhaus G., Platzman I., Schröder R., Muller D., Spatz J., Göpfrich K.

Bottom-up and top-down approaches to synthetic biology each employ distinct methodologies with the common aim to harness new types of living systems. Both approaches, however, face their own challenges towards biotechnological and biomedical applications. Here, we realize a strategic merger to convert light into proton gradients for the actuation of synthetic cellular systems. We genetically engineer E. coli to overexpress the light-driven inward-directed proton pump xenorhodopsin and encapsulate them as organelle mimics in artificial cell-sized compartments. Exposing the compartments to light-dark cycles, we can reversibly switch the pH by almost one pH unit and employ these pH gradients to trigger the attachment of DNA structures to the compartment periphery. For this purpose, a DNA triplex motif serves as a nanomechanical switch responding to the pH-trigger of the E. coli. By attaching a polymerized DNA origami plate to the DNA triplex motif, we obtain a cytoskeleton mimic that considerably deforms lipid vesicles in a pH-responsive manner. We foresee that the combination of bottom-up and top down approaches is an efficient way to engineer synthetic cells as potent microreactors.

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2019 – Protein-free division of giant unilamellar vesicles controlled by enzymatic activity
Vesicle Prep Pro Pre-Publication in BioRxiv (2019) Authors: Dreher Y., Spatz J.P., Göpfrich K.

Cell division is one of the hallmarks of life. Success in the bottom-up assembly of synthetic cells will, no doubt, depend on strategies for the controlled autonomous division of protocellular compartments. Here, we describe the protein-free division of giant unilamellar lipid vesicles (GUVs) based on the combination of two physical principles – phase separation and osmosis. We visualize the division process with confocal fluorescence microscopy and derive a conceptual model based on the vesicle geometry. The model successfully predicts the shape transformations over time as well as the time point of the final pinching of the daughter vesicles. Remarkably, we show that two fundamentally distinct yet highly abundant processes – water evaporation and metabolic activity – can both regulate the autonomous division of GUVs. Our work may hint towards mechanisms that governed the division of protocells and adds to the strategic toolbox of bottom-up synthetic biology with its vision of bringing matter to life.

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2019 – Self‐Assembly of Giant Unilamellar Vesicles by Film Hydration Methodologies
Vesicle Prep Pro Publication in Advanced Biosystems (2019) Authors: Rideau E., Wurm F.R., Landfester K.

Self‐assembly of lipids or polymeric amphiphiles into vesicular structures has been achieved by various methods since the first generation of liposomes in the 1960s. Vesicles can be obtained with diameters from the nanometer to the micrometer regime. From the perspective of cell mimicking, vesicles with diameters of several micrometers are most relevant. These vesicles are called giant unilamellar vesicles (GUVs). Commonly used methods to form GUVs are solvent‐displacement techniques, especially since the development of microfluidics. These methodologies however, trap undesirable organic solvents in their membrane as well as other potentially undesired additives (surfactants, polyelectrolytes, polymers, etc.). In contrast to those strategies, summarized herein are solvent‐free approaches as suitable clean alternatives. The vesicles are formed from a dry thin layer of the lipid or amphiphilic polymers and are hydrated in aqueous media using the entropically favored self‐assembly of amphiphiles into GUVs. The rearrangement of the amphiphilic films into vesicular structures is usually aided by shear forces such as an alternative current (electroformation) or the swelling of water‐soluble polymeric supports (gel‐assisted hydration).

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2019 – Linker length in fluorophore–cholesterol conjugates directs phase selectivity and cellular localisation in GUVs and live cells
Vesicle Prep Pro Publication in RSC Advances (2019) Authors: O' Connor D., Byrnea A., Keyes T.E.

Lipid membrane fluorescent probes that are both domain-selective and compatible with demanding microscopy methods are crucial to elucidate the presence and function of rafts and domains in cells and biophysical models. Whereas targeting fluorescent probes to liquid-disordered (Ld) domains is relatively facile, it is far more difficult to direct probes with high selectivity to liquid-ordered (Lo) domains. Here, a simple, one-pot approach to probe–cholesterol conjugation is described using Steglich esterification to synthesise two identical BODIPY derivatives that differ only in the length of the aliphatic chain between the dye and cholesterol. In the first, BODIPY-Ar-Chol, the probe and cholesterol were directly ester linked and in the second BODIPY-Ahx-Chol, a hexyl linker separated probe from cholesterol. Uptake and distribution of each probe was compared in ternary, phase separated giant unilamellar vesicles (GUVs) using a commercial Ld marker as a reference. BODIPY-Ar-Chol targets almost exclusively the Ld domains with selectivity of >90% whereas by contrast introducing the C6 linker between the probe and cholesterol drove the probe to Lo with excellent selectivity (>80%). The profound impact of the linker length extended also to uptake and distribution in live mammalian cells. BODIPY-Ahx-Chol associates strongly with the plasma membrane where it partitioned preferably into opposing micron dimensioned do-mains to a commercial Ld marker and its concentration at the membrane was reduced by cyclodextrin treatment of the cells. By contrast the BODIPY-Ahx-Chol permeated the membrane and localised strongly to lipid droplets within the cell. The data demonstrates the profound influence of linker length in cholesterol bioconjugates in directing the probe.

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2019 – Mechanisms of Regulation of Amyloid-Induced Permeability of Model Lipid Membranes by Polyphenols
Vesicle Prep Pro Publication in Cell Tiss. Biol. (2019) Authors: Efimova, S.S. & Ostroumova, O.S.

This work is devoted to the study of the processes of formation and functioning of ion channels by amyloidogenic peptides, pathological aggregation and accumulation of which is a cause of neurodegenerative disorders. The effect of the plant polyphenols phloretin, butein, resveratrol, isoliquiritigenin, 4'-hydroxychalcone, and cardamonine on the pore-forming activity of β-amyloid peptide fragment 25–35 in bilayer lipid membranes from palmitoyl-phosphocholine was studied. It was demonstrated that the introduction of phloretin, butein or isoliquiritigenin in membrane-bathing solutions to a concentration of 20 µM leads to the increase of macroscopic transmembrane currents induced by peptide. At the same time, cardamonine, 4'-hydroxychalcone, and resveratrol have no effect on the activity of β-amyloid peptide fragment 25–35. The comparison of the results of studying the effect of tested polyphenols on electric and elastic properties of model membranes and pore-forming ability of β-amyloid peptide fragment 25–35 allowed it to concluded that there is no connection between the potentiating effect of phloretin, butein, or isoliquiritigenin and changes in the physicochemical properties of lipid bilayers. Results obtained by means of a confocal fluorescent microscopy indicate that the domain organization of the lipid bilayer may play a role in the pore-forming activity of amyloidogenic peptide. The results of electrophysiological measurements obtained for α-synuclein (another protein forming ion-permeable pores) do not contradict the hypothesis of binding of polyphenols, hydroxylated in the 7 position of the A cycle and in the 4'-position of the B cycle, with an open propane fragment with β-layers formed by amyloid peptides.

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2019 – Fluorescent Artificial Receptor-Based Membrane Assay (FARMA) for Spatiotemporally Resolved Monitoring of Biomembrane Permeability
Vesicle Prep Pro Pre-Publication in ChemRxiv (2019) Authors: Biedermann F., Ghale F., Hennig A., Nau W.M.

The spatiotemporally resolved monitoring of membrane translocation, e.g., of drugs or toxins, has been a long-standing goal. Herein, we introduce the fluorescent artificial receptor-based membrane assay (FARMA), a facile, label-free method. With FARMA, the permeation of more than hundred organic compounds (drugs, toxins, pesticides, neurotransmitters, peptides, etc.) through vesicular phospholipid bilayer membranes has been monitored in real time (µs-h time scale) and with high sensitivity (nM-µM concentration), affording permeability coefficients across an exceptionally large range from 10-9‑10-3 cm s-1. From a fundamental point of view, FARMA constitutes a powerful tool to assess structure-permeability relationships and to test biophysical models for membrane passage. From an applied perspective, FARMA can be extended to high-throughput screening by adaption of the microplate reader format, to spatial monitoring of membrane permeation by microscopy imaging, and to the compartmentalized monitoring of enzymatic activity.

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2019 – Hepatitis B virus X protein induces size-selective membrane permeabilization through interaction with cardiolipin
Vesicle Prep Pro Publication in Biochimica et Biophysica Acta (BBA) - Biomembranes (2019) Authors: You D.G., Cho Y.Y., Lee H.R., Lee J.H., Yu S.J., Yoon J.H., Yoo Y.D., Kim Y.J., Lee G.Y.

Hepatitis B virus X protein (HBx) functions in a variety of cellular events during the HBV life cycle. In a previous study, we reported that the HBx protein is sufficient to induce mitochondrial membrane permeabilization; however, the exact mechanism of HBx-induced mitochondrial membrane permeabilization has been not proposed. In this study, we report that HBx specifically targets cardiolipin (CL) and induces membrane permeabilization depending on CL concentration in mitochondrial outer membrane-mimic artificial liposomes. Interestingly, HBx-induced membrane permeabilization was enhanced by liposomes containing phosphatidylethanolamine, which plays a crucial role in forming a negative curvature on the membrane. We also show that the 68-117 region of HBx, which interacts with mitochondria, is necessary for membrane permeabilization. We examined the size of the pores formed by HBx and found that HBx permeates fluorescent dyes depending on the hydrodynamic diameter with a pore size of approximately 10 nm. The results of this study suggest that CL is necessary for HBx-induced membrane permeabilization and provide important information that suggests a new strategy for anti-HBV therapy.

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2019 – Barcoding biological reactions with DNA-functionalized vesicles
Vesicle Prep Pro Publication in Angew Chem Int Ed Engl. (2019) Authors: Peruzzi J.A., Jacobs, ML., Vu T.Q., Kamat N.P.

Targeted vesicle fusion is a promising approach to selectively control interactions between vesicle compartments and would enable the initiation of biological reactions in complex aqueous environments. Here, we explore how two features of vesicle membranes, DNA tethers and phase-segregated membranes, promote fusion between specific vesicle populations. We show that membrane phase-segregation provides an energetic driver for membrane fusion that increases the efficiency of DNA-mediated fusion events. Using this system, we show that orthogonality provided by DNA tethers allows us to direct fusion and delivery of DNA cargo to specific vesicle populations. We then demonstrate that vesicle fusion between DNA-tethered vesicles can be used to initiate in vitro protein expression that leads to the synthesis of model soluble and membrane proteins. The ability to engineer orthogonal fusion events between DNA-tethered vesicles will provide a new strategy to control the spatio-temporal dynamics of cell-free reactions, expanding opportunities to engineer artificial cellular systems.

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2019 – Cellular uptake of self-assembled phytantriol-based hexosomes is independent of major endocytic machineries
Vesicle Prep Pro Publication in Journal of Colloid Interface Science (2019) Authors: Rodrigues L., Schneider F. , Zhang X., Larsson E., Moodie L., Dietz H., Papadakis C., Winter G., Lundmark R., Hubert M.

Despite increasing interests in non-lamellar liquid crystalline dispersions, such as hexosomes, for drug delivery, little is known about their interactions with cells and mechanism of cell entry. Here we examine the cellular uptake of hexosomes based on phytantriol and mannide monooleate by HeLa cells using live cell microscopy in comparison to conventional liposomes. To investigate the importance of specific endocytosis pathways upon particle internalization, we silenced regulatory proteins of major endocytosis pathways using short interfering RNA. While endocytosis plays a significant role in liposome internalization, hexosomes are not taken up via endocytosis but through a mechanism that is dependent on cell membrane tension. Biophysical studies using biomembrane models highlighted that hexosomes have a high affinity for membranes and an ability to disrupt lipid layers. Our data suggest that direct biomechanical interactions of hexosomes with membrane lipids play a crucial role and that the unique morphology of hexosomes is vital for their membrane activity. Based on these results, we propose a mechanism, where hexosomes destabilize the bilayer, allowing them to “phase through” the membrane. Understanding parameters that influence the uptake of hexosomes is critical to establish them as carrier systems that can potentially deliver therapeutics efficiently to intracellular sites of action.

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2019 – A trifunctional linker for palmitoylation and peptide and protein localization in biological membranes
Vesicle Prep Pro Publication in ChemBioChem (2019) Authors: Syga L., De Vries R.H., van Oosterhout H., Bartelds R., Boersma A.J., Roelfes G., Poolman B.

Attachment of lipophilic groups is an important post‐translational modification of proteins, which involves the coupling of one or more anchors such as fatty acids, isoprenoids, phospholipids or glycosylphosphatidyl inositols. To study its impact on the membrane partitioning of hydrophobic peptides or proteins, we designed a tyrosine‐based trifunctional linker. The linker allows in a single step facile incorporation of two different functionalities at a cysteine. We determined the effect of the lipid modification on the membrane partitioning of the synthetic α‐helical model peptide WALP w/wo palmitoyl groups in giant unilamellar vesicles that contain a liquid‐ordered (Lo) and liquid‐disordered (Ld) phase. Introduction of two palmitoyl groups did not alter the localization of the membrane peptides, nor did the membrane thickness or lipid composition. In all cases, the peptide was retained in the Ld phase. These data demonstrate that the Lo domain in model membranes is highly unfavorable for a single membrane‐spanning peptide.

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2019 – An Intrinsically Disordered Region in OSBP Acts as an Entropic Barrier to Control Protein Dynamics and Orientation at Membrane Contact Sites
Vesicle Prep Pro Publication in Developmental Cell (2019) Authors: Jamecna D., Polidori J., Mesmin B., Dezi M., Levy D., Bigay J., Antonny B.

Lipid transfer proteins (LTPs) acting at membrane contact sites (MCS) between the ER and other organelles contain domains involved in heterotypic (e.g., ER to Golgi) membrane tethering as well as domains involved in lipid transfer. Here, we show that a long ≈90 aa intrinsically unfolded sequence at the N terminus of oxysterol-binding protein (OSBP) controls OSBP orientation and dynamics at MCS. This Gly-Pro-Ala-rich sequence, whose hydrodynamic radius is twice as that of folded domains, prevents the two PH domains of the OSBP dimer from homotypically tethering two Golgi-like membranes and considerably facilitates OSBP in-plane diffusion and recycling at MCS. Although quite distant in sequence, the N terminus of OSBP-related protein-4 (ORP4) has similar effects. We propose that N-terminal sequences of low complexity in ORPs form an entropic barrier that restrains protein orientation, limits protein density, and facilitates protein mobility in the narrow and crowded MCS environment.

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2018 – Visualizing Tension and Growth in Model Membranes Using Optical Dyes
Vesicle Prep Pro Publication in Biophysical Journal (2018) Authors: Boyd M.A., Kamat N.P.

Cells dynamically regulate their membrane surface area during a variety of processes critical to their survival. Recent studies with model membranes have pointed to a general mechanism for surface area regulation under tension in which cell membranes unfold or take up lipid to accommodate membrane strain. Yet we lack robust methods to simultaneously measure membrane tension and surface area changes in real time. Using lipid vesicles that contain two dyes isolated to spatially distinct parts of the membrane, we introduce, to our knowledge, a new method to monitor the processes of membrane stretching and lipid uptake in model membranes. Laurdan, located within the bilayer membrane, and Förster resonance energy transfer dyes, localized to the membrane exterior, act in concert to report changes in membrane tension and lipid uptake during osmotic stress. We use these dyes to show that membranes under tension take up lipid more quickly and in greater amounts compared to their nontensed counterparts. Finally, we show that this technique is compatible with microscopy, enabling real-time analysis of membrane dynamics on a single vesicle level. Ultimately, the combinatorial use of these probes offers a more complete picture of changing membrane morphology. Our optical method allows us to remotely track changes in membrane tension and surface area with model membranes, offering new opportunities to track morphological changes in artificial and biological membranes and providing new opportunities in fields ranging from mechanobiology to drug delivery.

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2019 – A Fusion Peptide in the Spike Protein of MERS CoroNaVirus
Vesicle Prep Pro Publication in Viruses (2019) Authors: Alsaadi E.A.J., Neuman B.W., and Jones I.M.

CoroNaViruses represent current and emerging threats for many species, including humans. Middle East respiratory syndrome-related coroNaVirus (MERS-CoV) is responsible for sporadic infections in mostly Middle Eastern countries, with occasional transfer elsewhere. A key step in the MERS-CoV replication cycle is the fusion of the virus and host cell membranes mediated by the virus spike protein, S. The location of the fusion peptide within the MERS S protein has not been precisely mapped. We used isolated peptides and giant unilamellar vesicles (GUV) to demonstrate membrane binding for a peptide located near the N-terminus of the S2 domain. Key residues required for activity were mapped by amino acid replacement and their relevance in vitro tested by their introduction into recombinant MERS S protein expressed in mammalian cells. Mutations preventing membrane binding in vitro also abolished S-mediated syncytium formation consistent with the identified peptide acting as the fusion peptide for the S protein of MERS-CoV.

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2018 – Probing Interactions between AuNPs/AgNPs and Giant Unilamellar Vesicles (GUVs) Using Hyperspectral Dark-field Microscopy
Vesicle Prep Pro Publication in the International Journal of Molecular Sciences (2018) Authors: Bhat A., Huan K., Cooks T., Boukari H., Lu Q.

Noble metallic nanoparticles (NPs) such as gold and silver nanoparticles (AuNPs and AgNPs) have been shown to exhibit anti-tumor effect in anti-angiogenesis, photothermal and radio therapeutics. On the other hand, cell membranes are critical locales for specific targeting of cancerous cells. Therefore, NP-membrane interactions need be studied at molecular level to help better understand the underlying physicochemical mechanisms for future applications in cancer nanotechnology. Herein, we report our study on the interactions between citrate stabilized colloidal AuNPs/AgNPs (10 nm in size) and giant unilamellar vesicles (GUVs) using hyperspectral dark-field microscopy. GUVs are large model vesicle systems well established for the study of membrane dynamics. GUVs used in this study were prepared with dimyristoyl phosphatidylcholine (DMPC) and doped with cholesterol at various molar concentrations. Both imaging and spectral results support that AuNPs and AgNPs interact very differently with GUVs, i.e., AuNPs tend to integrate in between the lipid bilayer and form a uniform golden-brown crust on vesicles, whereas AgNPs are bejeweled on the vesicle surface as isolated particles or clusters with much varied configurations. The more disruptive capability of AuNPs is hypothesized to be responsible for the formation of golden brown crusts in AuNP-GUV interaction. GUVs of 20 mol% CHOL:DMPC were found to be a most economical concentration for GUVs to achieve the best integrity and the least permeability, consistent with the finding from other phase studies of lipid mixture that the liquid-ordered domains have the largest area fraction of the entire membrane at around 20 mol% of cholesterol.

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2018 – Regulation of the Pore-Forming Activity of Cecropin A by Local Anesthetics
Vesicle Prep Pro Publication in Cell and Tissue Biology (2018) Authors: Efimova S.S., Medvedev R.Ya., Chulkov E.G., Schagina L.V., Ostroumova O.S.

The influence of local anesthetics on the regulation of the channel-forming activity of the antimicrobial peptide cecropin A has been investigated. The mean current flowing through the single cecropin channels isc was determined, and steady-state transmembrane current induced by cecropin AI∞ was measured. It has been shown that the introduction of 1 mM of bupivacaine, benzocaine or 0.3 mM of tetracaine into the membrane bathing solution results in a decrease in isc and I∞. At the same time, the addition of 1 mM lidocaine or procaine to the membrane-bathing solutions does not lead to a significant change in isc and I∞. Comparison of the absolute values and the sign of the change in the boundary potential of negatively charged membranes and relative changes of isc and I∞ after addition of local anesthetics shows that neither parameter correlates with the membrane boundary potential. The results of studying the effect of tested local anesthetics on the phase transition of membrane lipids allow us to conclude that the observed changes of isc and I∞ are due to modulation of the elastic properties of the membrane.

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2018 – Methods for Single-Molecule Sensing and Detection Using Bacteriophage Phi29 DNA Packaging Motor
Vesicle Prep Pro book chapter in Molecular Motors (2018) Authors: Haque F, Zhang H, Wang S, Chang C-L, Savran C, Guo P.

Bacteriophage phi29 DNA packaging motor consists of a dodecameric portal channel protein complex termed connector that allows transportation of genomic dsDNA and a hexameric packaging RNA (pRNA) ring to gear the motor. The elegant design of the portal protein has facilitated its applications for real-time single-molecule detection of biopolymers and chemicals with high sensitivity and selectivity. The robust self-assembly property of the pRNA has enabled biophysical studies of the motor complex to determine the stoichiometry and structure/folding of the pRNA at single-molecule level. This chapter focuses on biophysical and analytical methods for studying the phi29 motor components at the single-molecule level, such as single channel conductance assays of membrane-embedded connectors; single molecule photobleaching (SMPB) assay for determining the stoichiometry of phi29 motor components; fluorescence resonance energy transfer (FRET) assay for determining the structure and folding of pRNA; atomic force microscopy (AFM) for imaging pRNA nanoparticles of various size, shape, and stoichiometry; and bright-field microscopy with magnetomechanical system for direct visualization of viral DNA packaging process. The phi29 system with explicit engineering capability has incredible potentials for diverse applications in nanotechnology and nanomedicine including, but not limited to, DNA sequencing, drug delivery to diseased cells, environmental surveillance, and early disease diagnosis.

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2018 – Phase partitioning, solvent-switchable BODIPY probes for high contrast cellular imaging and FCS
Vesicle Prep Pro Publication in New Journal of Chemistry (2018) Authors: O’Connor D. Byrne A., Dolana C., Keyes T.E.

Lipophilic BODIPY fluorphores, in which the BODIPY core bears pendant dipyrido[3,2-a:2′,3′-c]phenazine (Dppz) or naphthyridyl and cholesterol substituents were designed and prepared as lipid probes for both liposomes and live cell imaging. The probes are non-emissive in water but permeate both GUV and live cell membranes and provide high contrast fluorescence and lifetime imaging of membranous structures and lipid droplets in cells and are suitable for FCS measurements on lipid membranes.

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2018 – Lipid-mediated regulation of pore-forming activity of syringomycin E by thyroid hormones and xanthene dyes
Vesicle Prep Pro Publication in Biochimica et Biophysica Acta (BBA) - Biomembranes (2018) Authors: Efimova S.S., Zakharova A.A., Ismagilov A.A., Schagina L.V., Malev V.V., Bashkirov P.V., Ostroumova O.S.

The effects of dipole modifiers, thyroid hormones (thyroxine and triiodothyronine) and xanthene dyes (Rose Bengal, phloxineB, erythrosin, eosinY and fluorescein) on the pore-forming activity of the lipopeptide syringomycin E (SRE) produced by Pseudomonas syringae were studied in a model bilayer. Thyroxine does not noticeably influence the steady-state number of open SRE channels (Nop), whereas triiodothyronine decreases it 10-fold at − 50 mV. Rose Bengal, phloxine B and erythrosin significantly increase Nop by 350, 100 and 70 times, respectively. Eosin Y and fluorescein do not practically affect the pore-forming activity of SRE. Recently, we showed that hormones decrease the dipole potential of lipid bilayers by approximately 60 mV at 50 μM, while Rose Bengal, phloxine B and erythrosin at 2.5 μM reduce the membrane dipole potential by 120, 80 and 50 mV, respectively. In the present study using differential scanning microcalorimetry, confocal fluorescence microscopy, the calcein release technique and measurements of membrane curvature elasticity, we show that triiodothyronine strongly affects the fluidity of model membranes: its addition leads to a significant decrease in the temperature and cooperativity of the main phase transition of DPPC, calcein leakage from DOPC vesicles, fluidization of solid domains in DOPC/DPPC liposomes, and promotion of lipid curvature stress. Thyroxine exerts a weaker effect. Xanthene dyes do not influence the phase transition of DPPC. Despite the decrease in the dipole potential, thyroid hormones modulate SRE channels predominantly via the elastic properties of the membrane, whereas the xanthene dyes Rose Bengal, phloxine B and erythrosine affect SRE channels via bilayer electrostatics.

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2018 – Method for immobilization of living and synthetic cells for high-resolution imaging and single-particle tracking
Vesicle Prep Pro Publication in Scientific Reports (2018) Authors: Syga Ł., Spakman D., Punter C.M., Poolman B.

Super-resolution imaging and single-particle tracking require cells to be immobile as any movement reduces the resolution of the measurements. Here, we present a method based on APTES-glutaraldehyde coating of glass surfaces to immobilize cells without compromising their growth. Our method of immobilization is compatible with Saccharomyces cerevisiae, Escherichia coli, and synthetic cells (here, giant-unilamellar vesicles). The method introduces minimal background fluorescence and is suitable for imaging of single particles at high resolution. With S. cerevisiae we benchmarked the method against the commonly used concaNaValin A approach. We show by total internal reflection fluorescence microscopy that modifying surfaces with ConA introduces artifacts close to the glass surface, which are not present when immobilizing with the APTES-glutaraldehyde method. We demonstrate validity of the method by measuring the diffusion of membrane proteins in yeast with single-particle tracking and of lipids in giant-unilamellar vesicles with fluorescence recovery after photobleaching. Importantly, the physical properties and shape of the fragile GUVs are not affected upon binding to APTES-glutaraldehyde coated glass. The APTES-glutaraldehyde is a generic method of immobilization that should work with any cell or synthetic system that has primary amines on the surface.

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2018 – High contrast imaging and flexible photomanipulation for quantitative in vivo multiphoton imaging with polygon scanning microscope
Vesicle Prep Pro Publication in the Journal of Biophotonics (2018) Authors: Li Y., Montague S.J., Brüstle A., He X., Gillespie C., Gaus K., Gardiner E.E., Lee W.M.

In this study, we introduce two key improvements that overcome limitations of existing polygon scanning microscopes while maintaining high spatial and temporal imaging resolution over large field of view (FOV). First, we proposed a simple and straightforward means to control the scanning angle of the polygon mirror to carry out photomanipulation without resorting to high speed optical modulators. Second, we devised a flexible data sampling method directly leading to higher image contrast by over 2-fold and digital images with 100 megapixels (10 240 × 10 240) per frame at 0.25 Hz. This generates sub-diffraction limited pixels (60 nm per pixels over the FOV of 512 μm) which increases the degrees of freedom to extract signals computationally. The unique combined optical and digital control recorded fine fluorescence recovery after localized photobleaching (r ~10 μm) within fluorescent giant unilamellar vesicles and micro-vascular dynamics after laser-induced injury during thrombus formation in vivo. These new improvements expand the quantitative biological-imaging capacity of any polygon scanning microscope system.

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2018 – Hybrid vesicles from lipids and block copolymers: Phase behavior from the micro- to the nano-scale
Vesicle Prep Pro Publication in Colloids and Surfaces B: Biointerfaces (2018) Authors: Magnani C., Montis C., Mangiapia G., Mingotaud A.-F., Mingotaud C., Roux C., Joseph P., Berti D., Lonetti B.

In recent years, there has been a growing interest in the formation of copolymers-lipids hybrid self-assemblies, which allow combining and improving the main features of pure lipids-based and copolymer-based systems known for their potential applications in the biomedical field. In this contribution we investigate the self-assembly behavior of dipalmitoylphosphatidylcholine (DPPC) mixed with poly(butadiene-b-ethyleneoxide) (PBD-PEO), both at the micro- and at the nano-length scale. Epifluorescence microscopy and Laser Scanning Confocal microscopy are employed to characterize the morphology of micron-sized hybrid vesicles. The presence of fluid-like inhomogeneities in their membrane has been evidenced in all the investigated range of compositions. Furthermore, a microfluidic set-up characterizes the mechanical properties of the prepared assemblies by measuring their deformation upon flow: hybrids with low lipid content behave like pure polymer vesicles, whereas objects mainly composed of lipids show more variability from one vesicle to the other. Finally, the structure of the nanosized assemblies is characterized through a combination of Dynamic Light Scattering, Small Angle Neutron Scattering and Transmission Electron Microscopy. A vesicles-to-wormlike transition has been evidenced due to the intimate mixing of DPPC and PBD-PEO at the nanoscale. Combining experimental results at the micron and at the nanoscale improves the fundamental understanding on the phase behavior of copolymer-lipid hybrid assemblies, which is a necessary prerequisite to tailor efficient copolymer-lipid hybrid devices.

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2018 – Development of an on-chip antibiotic permeability assay with single molecule detection capability
Vesicle Prep Pro Publication in IEEE Transactions on NanoBioscience (2018) Authors: Guzel F.D., Citak F.

Electrophysiology is the method of choice to characterize membrane channels. In this study, we demonstrate a patch pipette based simple miniaturization that allows performing conductance measurements on a planar lipid bilayer in a microfluidic channel. Membrane proteins were reconstituted into Giant Unilamellar Vesicles (GUVs) by electroswelling, and GUVs with a single channel insertion were patched at the tip of pipette. We applied this approach to investigate the interactions of porins from E. coli with single antibiotics, and this will potentially provide information on the permeability rates. The results of this study suggest that this approach can be extended to the integration of several pipettes into the microfluidic channel from different positions, allowing the multiplexed recordings and also reducing the substrate consumption below μL volumes.

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2018 – Differential interactions of bacterial lipopolysaccharides with lipid membranes: implications for TRPA1-mediated chemosensation
Vesicle Prep Pro Publication in Scientific Reports (2018) Authors: Startek J.B., Talavera K., Voets T., Alpizar Y.A.

Bacterial lipopolysaccharides (LPS) activate the TRPA1 cation channels in sensory neurons, leading to acute pain and inflammation in mice and to aversive behaviors in fruit flies. However, the precise mechanisms underlying this effect remain elusive. Here we assessed the hypothesis that TRPA1 is activated by mechanical perturbations induced upon LPS insertion in the plasma membrane. We asked whether the effects of different LPS on TRPA1 relate to their ability to induce mechanical alterations in artificial and cellular membranes. We found that LPS from E. coli, but not from S. minnesota, activates TRPA1. We then assessed the effects of these LPS on lipid membranes using dyes whose fluorescence properties change upon alteration of the local lipid environment. E. coli LPS was more effective than S. minnesota LPS in shifting Laurdan’s emission spectrum towards lower wavelengths, increasing the fluorescence anisotropy of diphenylhexatriene and reducing the fluorescence intensity of merocyanine 540. These data indicate that E. coli LPS induces stronger changes in the local lipid environment than S. minnesota LPS, paralleling its distinct ability to activate TRPA1. Our findings indicate that LPS activate TRPA1 by producing mechanical perturbations in the plasma membrane and suggest that TRPA1-mediated chemosensation may result from primary mechanosensory mechanisms.

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2018 – Cargo induces retromer-mediated membrane remodeling on membranes
Vesicle Prep Pro Publication in ascb Molecular Biology of the Cell (2018) Authors: Purushothaman L.K., Ungermann C.

Endosomes serve as a central sorting station of lipids and proteins that arrive via vesicular carrier from the plasma membrane and the Golgi complex. At the endosome, retromer complexes sort selected receptors and membrane proteins into tubules or vesicles that bud off the endosome. The mature endosome finally fuses with the lysosome. Retromer complexes consist of a cargo selection complex (CSC) and a membrane remodeling part (SNX-BAR or Snx3 in yeast), and different assemblies of retromer mediate recycling of different cargoes. Due to this complexity, the exact order of events that results in carrier formation is not yet understood. Here, we reconstituted this process on giant unilamellar vesicles together with purified retromer complexes from yeast and selected cargoes. Our data reveal that the membrane remodeling activity of both Snx3 and the SNX-BAR complex is strongly reduced at low concentrations, which can be reactivated by CSC. At even lower concentrations, these complexes still associate with membranes, but only remodel membranes in the presence of their specific cargoes. Our data thus favor a simple model, where cargo functions as a specific trigger of retromer-mediated sorting on endosomes.

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2018 – Cholesterol Enriched Archaeosomes as a Molecular System for Studying Interactions of Cholesterol-Dependent Cytolysins with Membranes
Vesicle Prep Pro Publication in The Journal of Membrane Biology (2018) Authors: Rezelj S., Kozorog M, Švigelj T., Ulrih N.P., Žnidaršič N., Podobnik M., Anderluh G.

Archaeosomes are vesicles made of lipids from archaea. They possess many unique features in comparison to other lipid systems, with their high stability being the most prominent one, making them a promising system for biotechnological applications. Here, we report a preparation protocol of large unilamellar vesicles, giant unilamellar vesicles (GUVs), and nanodiscs from archaeal lipids with incorporated cholesterol. Incorporation of cholesterol led to additional increase in thermal stability of vesicles. Surface plasmon resonance, sedimentation assays, intrinsic tryptophan fluorescence measurements, calcein release experiments, and GUVs experiments showed that members of cholesterol-dependent cytolysins, listeriolysin O (LLO), and perfringolysin O (PFO), bind to cholesterol-rich archaeosomes and thereby retain their pore-forming activity. Interestingly, we observed specific binding of LLO, but not PFO, to archaeosomes even in the absence of cholesterol. This suggests a new capacity of LLO to bind to carbohydrate headgroups of archaeal lipids. Furthermore, we were able to express LLO inside GUVs by cell-free expression. GUVs made from archaeal lipids were highly stable, which could be beneficial for synthetic biology applications. In summary, our results describe novel model membrane systems for studying membrane interactions of proteins and their potential use in biotechnology.

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2018 – A synthetic enzyme built from DNA flips 10e7 lipids per second in biological membranes
Vesicle Prep Pro Publication in the Nature Communications (2018) Authors: Ohmann A, Li C-Y, Maffeo C, Al Nahas K, Baumann K.N, Göpfrich K, Yoo J, Keyser U.F, & Aksimentiev A.

Mimicking enzyme function and increasing performance of naturally evolved proteins is one of the most challenging and intriguing aims of nanoscience. Here, we employ DNA nanotechnology to design a synthetic enzyme that substantially outperforms its biological archetypes. Consisting of only eight strands, our DNA nanostructure spontaneously inserts into biological membranes by forming a toroidal pore that connects the membrane’s inner and outer leaflets. The membrane insertion catalyzes spontaneous transport of lipid molecules between the bilayer leaflets, rapidly equilibrating the lipid composition. Through a combination of microscopic simulations and fluorescence microscopy we find the lipid transport rate catalyzed by the DNA nanostructure exceeds 107 molecules per second, which is three orders of magnitude higher than the rate of lipid transport catalyzed by biological enzymes. Furthermore, we show that our DNA-based enzyme can control the composition of human cell membranes, which opens new avenues for applications of membrane-interacting DNA systems in medicine.

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2018 – Acyl chain asymmetry and polyunsaturation of brain phospholipids facilitate membrane vesiculation without leakage
Vesicle Prep Pro Publication in eLife (2018) Authors: Manni M.M., Tiberti M.L., Pagnotta S., Barelli H., Gautier R., Antonny B.

Phospholipid membranes form cellular barriers but need to be flexible enough to divide by fission. Phospholipids generally contain a saturated fatty acid (FA) at position sn1 whereas the sn2-FA is saturated, monounsaturated or polyunsaturated. Our understanding of the impact of phospholipid unsaturation on membrane flexibility and fission is fragmentary. Here, we provide a comprehensive view of the effects of the FA profile of phospholipids on membrane vesiculation by dynamin and endophilin. Coupled to simulations, this analysis indicates that: (i) phospholipids with two polyunsaturated FAs make membranes prone to vesiculation but highly permeable; (ii) asymmetric sn1-saturated-sn2-polyunsaturated phospholipids provide a tradeoff between efficient membrane vesiculation and low membrane permeability; (iii) When incorporated into phospholipids, docosahexaenoic acid (DHA; omega-3) makes membranes more deformable than arachidonic acid (omega-6). These results suggest an explanation for the abundance of sn1-saturated-sn2-DHA phospholipids in synaptic membranes and for the importance of the omega-6/omega-3 ratio on neuronal functions.

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2017 – Lipid phase separation in the presence of hydrocarbons in giant unilamellar vesicles
Vesicle Prep Pro Publication in AIMS Biophysics (2017) Authors: Bartelds R., Barnoud J., Boersma A.J., Marrink S.J., Poolman B.

Hydrophobic hydrocarbons are absorbed by cell membranes. The effects of hydrocarbons on biological membranes have been studied extensively, but less is known how these compounds affect lipid phase separation. Here, we show that pyrene and pyrene-like hydrocarbons can dissipate lipid domains in phase separating giant unilamellar vesicles at room temperature. In contrast, related aromatic compounds left the phase separation intact, even at high concentration. We hypothesize that this behavior is because pyrene and related compounds lack preference for either the liquid-ordered (Lo) or liquid-disordered (Ld) phase, while larger molecules prefer Lo, and smaller, less hydrophobic molecules prefer Ld. In addition, our data suggest that localization in the bilayer (depth) and the shape of the molecules might contribute to the effects of the aromatic compounds. Localization and shape of pyrene and related compounds are similar to cholesterol and therefore these molecules could behave as such.

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2018 – A direct role for hepatitis B virus X protein in inducing mitochondrial membrane permeabilization
Vesicle Prep Pro Publication in Journal of Viral Hepatitis (2018) Authors: Lee H‐R., Cho Y.Y., Lee G.Y., You D‐g., Yoo Y.D., Kim Y.J.

Hepatitis B virus X protein (HBx) acts as a multifunctional protein that regulates intracellular signalling pathways during HBV infection. It has mainly been studied in terms of its interaction with cellular proteins. Here, we show that HBx induces membrane permeabilization independently of the mitochondrial permeability transition pore complex. We generated mitochondrial outer membrane‐mimic liposomes to observe the direct effects of HBx on membranes. We found that HBx induced membrane permeabilization, and the region comprising the transmembrane domain and the mitochondrial‐targeting sequence was sufficient for this process. Membrane permeabilization was inhibited by nonselective channel blockers or by N‐(n‐nonyl)deoxynojirimycin (NN‐DNJ), a viroporin inhibitor. Moreover, NN‐DNJ inhibited HBx‐induced mitochondrial depolarization in Huh‐7 cells. Based on the results of this study, we can postulate that the HBx protein itself is sufficient to induce mitochondrial membrane permeabilization. Our finding provides important information for a strategy of HBx targeting during HBV treatment

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2017 – Dipole Modifiers Regulate Lipid Lateral Heterogeneity in Model Membranes
Vesicle Prep Pro Publication in Acta Naturae (2017) Authors: Efimova S.S., Ostroumova O.S.

In this study we report on experimental observations of giant unilamellar liposomes composed of ternary mixtures of cholesterol (Chol), phospholipids with relatively low Tmelt (DOPC, POPC, or DPoPC) and high Tmelt (sphingomyelin (SM), or tetramyristoyl cardiolipin (TMCL)) and their phase behaviors in the presence and absence of dipole modifiers. It was shown that the ratios of liposomes exhibiting noticeable phase separation decrease in the series POPC, DOPC, DPoPC regardless of any high-Tmelt lipid. Substitution of SM for TMCL led to increased lipid phase segregation. Taking into account the fact that the first and second cases corresponded to a reduction in the thickness of the lipid domains enriched in low- and high-Tmelt lipids, respectively, our findings indicate that the phase behavior depends on thickness mismatch between the ordered and disordered domains. The dipole modifiers, flavonoids and styrylpyridinium dyes, reduced the phase segregation of membranes composed of SM, Chol, and POPC (or DOPC). The other ternary lipid mixtures tested were not affected by the addition of dipole modifiers. It is suggested that dipole modifiers address the hydrophobic mismatch through fluidization of the ordered and disordered domains. The ability of a modifier to partition into the membrane and fluidize the domains was dictated by the hydrophobicity of modifier molecules, their geometric shape, and the packing density of domain-forming lipids. Phloretin, RH 421, and RH 237 proved the most potent among all the modifiers examined.

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2017 – Ion- and water-binding sites inside an occluded hourglass pore of a trimeric intracellular cation (TRIC) channel
Vesicle Prep Pro Publication in BioMed Central Biology (2017) Authors: Ou X., Guo J., Wang L., Yang H., Liu X., Sun J., Liu Z.

Background:Trimeric intracellular cation (TRIC) channels are crucial for Ca2+ handling in eukaryotes and are involved in K+ uptake in prokaryotes. Recent studies on the representative members of eukaryotic and prokaryotic TRIC channels demonstrated that they form homotrimeric units with the ion-conducting pores contained within each individual monomer.Results:Here we report detailed insights into the ion- and water-binding sites inside the pore of a TRIC channel from Sulfolobus solfataricus (SsTRIC). Like the mammalian TRIC channels, SsTRIC is permeable to both K+ and Na+ with a slight preference for K+, and is nearly impermeable to Ca2+, Mg2+, or Cl–. In the 2.2-Å resolution K+-bound structure of SsTRIC, ion/water densities have been well resolved inside the pore. At the central region, a filter-like structure is shaped by the kinks on the second and fifth transmembrane helices and two nearby phenylalanine residues. Below the filter, the cytoplasmic vestibule is occluded by a plug-like motif attached to an array of pore-lining charged residues.Conclusions:The asymmetric filter-like structure at the pore center of SsTRIC might serve as the basis for the channel to bind and select monovalent cations. A Velcro-like plug-pore interacting model has been proposed and suggests a unified framework accounting for the gating mechanisms of prokaryotic and eukaryotic TRIC channels.

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2013 – Lipid-Bilayer-Spanning DNA Nanopores with a Bifunctional Porphyrin Anchor
Vesicle Prep Pro Publication in Angewandte Chemie International Edition (2013) Authors: Burns J.R., Göpfrich K., Wood J.W.,Thacker V.V.,Stulz E., Keyser U.F., Howorka S.

An artificial membrane nanopore assembled from DNA oligonucleotides carries porphyrin tags, which anchor the nanostructure into the lipid bilayer. The porphyrin moieties also act as fluorescent dyes to aid the microscopic visualization of the DNA nanopore.

Publication link
2014 – Nanoscale phase behavior on flat and curved membranes
Vesicle Prep Pro Publication in Nanotechnology (2014) Authors: Andersen T., Bahadori A., Ott D., Kyrsting A., Reihani S.N.S., Bendix P.M.

The diverse physical properties of membranes play a critical role in many membrane associated biological processes. Proteins responsible for membrane transport can be affected by the lateral membrane order and lateral segregation of proteins is often controlled by the preference of certain membrane anchors for membrane phases having a physically ordered state. The dynamic properties of coexisting membrane phases are often studied by investigating their thermal behavior. Optical trapping of gold nanoparticles is a useful tool to generate local phase transitions in membranes. The high local temperatures surrounding an irradiated gold nanoparticle can be used to melt a part of a giant unilamellar lipid vesicle (GUV) which is then imaged using phase sensitive fluorophores embedded within the bilayer. By local melting of GUVs we reveal how a protein-free, one component lipid bilayer can mediate passive transport of fluorescent molecules by localized and transient pore formation. Also, we show how tubular membrane curvatures can be generated by optical pulling from the melted region on the GUV. This will allow us to measure the effect of membrane curvature on the phase transition temperature.

Publication link
2012 – Hydrophobic gating of mechanosensitive channel of large conductance evidenced by single-subunit resolution
Vesicle Prep Pro Publication in Proceedings of the National Academy of Sciences of the United States of America (2012) Authors: Birkner JP, Poolman B, Koçer A.

Mechanosensitive (MS) ion channels are membrane proteins that detect and respond to membrane tension in all branches of life. In bacteria, MS channels prevent cells from lysing upon sudden hypoosmotic shock by opening and releasing solutes and water. Despite the importance of MS channels and ongoing efforts to explain their functioning, the molecular mechanism of MS channel gating remains elusive and controversial. Here we report a method that allows single-subunit resolution for manipulating and monitoring “mechanosensitive channel of large conductance” from Escherichia coli. We gradually changed the hydrophobicity of the pore constriction in this homopentameric protein by modifying a critical pore residue one subunit at a time. Our experimental results suggest that both channel opening and closing are initiated by the transmembrane 1 helix of a single subunit and that the participation of each of the five identical subunits in the structural transitions between the closed and open states is asymmetrical. Such a minimal change in the pore environment seems ideal for a fast and energy-efficient response to changes in the membrane tension.

Publication link
2013 – Effect of flavonoids on the phase separation in giant unilamellar vesicles formed from binary lipid mixtures
Vesicle Prep Pro Publication in Chemistry and Physics of Lipids (2013) Authors: Ostroumova O.S., Chulkov E.G., Stepanenko O.V., Schagina L.V.

Confocal fluorescence microscopy have been employed to investigate phase separation in giant unilamellar vesicles prepared from binary mixtures of unsaturated dioleoylphosphocholine with saturated phosphocholines or brain sphingomyelin in the absence and presence of the flavonoids, biochanin A, phloretin, and myricetin. It has been demonstrated that biochanin A and phloretin make uncolored domains more circular or eliminate visible phase separation in liposomes while myricetin remains the irregular shape of fluorescence probe-excluding domains. Influence of the flavonoids on the endotherms of liposome suspension composed of dioleoylphosphocholine and dimyristoylphosphocholine was investigated by the differential scanning calorimetry. Calorimetry data do not contradict to confocal imaging results.

Poster PDF
2015 – Organellar Transporters and Ion Channels – How to access their electrophysiology by using the SURFE2R technology and Planar Patch Clamp
SURFE2R N1, SyncroPatch 96 (a predecessor model of the SyncroPatch 384) and Port-a-Patch poster, GRC - Organellar Channels and Transporters 2015
Poster PDF
2018 – Expression and pharmacology of GluA2-containing AMPA receptors in cell lines and stem cell-derived neurons
Port-a-Patch, Patchliner and SyncroPatch 384PE (a predecessor model of the SyncroPatch 384) poster, Europhysiology Meeting 2018
Poster PDF
2015 – The backstage pass to study your favorite TRP channel
Port-a-Patch, Patchliner and SyncroPatch 384PE (a predecessor model of the SyncroPatch 384) poster, TRP Meeting 2015
Poster PDF
2021 – Activation and inhibition of assay-ready TRPA1 and TRPV cells: an automated patch clamp study
Port-a-Patch, Patchliner and SyncroPatch 384i (a predecessor model of SyncroPatch 384), Biophysical Society Meeting 2021
Webinar
12.09.2018 | Webinar: CiPA study: Bridging ion channel and myocyte data
Authors: Dr. Sonja Stölzle-Feix
CiPA myocyte phase II validation study results: cross-site comparison using the CardioExcyte 96;
HTS Phase I study: an update on progress of the CiPA Ion Channel Work Stream using the SyncroPatch 384PE and Patchliner

Get up-to-date with the CiPA progress of the Myocyte and Ion Channel Work Goups: Since 2005 the S7B and E14 guidances from ICH and FDA have been in place to assess a potential drug candidate's ability to cause long QT syndrome. To refine these guidelines, the FDA proposed the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative, where the assessment of drug effects on cardiac repolarization was one subject of investigation. Within the myocyte validation study, effects of pharmaceutical compounds on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were assessed and this article will focus on the evaluation of the proarrhythmic potential of 23 blinded drugs in four hiPSC-CM cell lines.

Experiments were performed on the CardioExcyte 96 at different sites. A combined readout of contractility (via impedance) and electrophysiology endpoints (field potentials) was performed.Our data demonstrates that hERG blockers such as dofetilide and further high risk categorized compounds prolong the field potential duration. Arrhythmia were detected in both impedance as well as field potential recordings. Intermediate risk compounds induced arrhythmia in almost all cases at the highest dose. In the case of low risk compounds, either a decrease in FPDmax was observed, or not a significant change from pre-addition control values.

With exceptions, hiPSC-CMs are sensitive and exhibit at least 10% delayed or shortened repolarization from pre-addition values and arrhythmia after drug application and thus can provide predictive cardiac electrophysiology data. The baseline electrophysiological parameters vary between iPS cells from different sources, therefore positive and negative control recordings are recommended.

Webinar
12.09.2018 | Webinar: CiPA study: Bridging ion channel and myocyte data
Authors: Tim Strassmaier

Get up-to-date with the CiPA progress of the Myocyte and Ion Channel Work Goups: Since 2005 the S7B and E14 guidances from ICH and FDA have been in place to assess a potential drug candidate's ability to cause long QT syndrome. To refine these guidelines, the FDA proposed the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative, where the assessment of drug effects on cardiac repolarization was one subject of investigation. Within the myocyte validation study, effects of pharmaceutical compounds on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were assessed and this article will focus on the evaluation of the proarrhythmic potential of 23 blinded drugs in four hiPSC-CM cell lines.

Experiments were performed on the CardioExcyte 96 at different sites. A combined readout of contractility (via impedance) and electrophysiology endpoints (field potentials) was performed.Our data demonstrates that hERG blockers such as dofetilide and further high risk categorized compounds prolong the field potential duration. Arrhythmia were detected in both impedance as well as field potential recordings. Intermediate risk compounds induced arrhythmia in almost all cases at the highest dose. In the case of low risk compounds, either a decrease in FPDmax was observed, or not a significant change from pre-addition control values.

With exceptions, hiPSC-CMs are sensitive and exhibit at least 10% delayed or shortened repolarization from pre-addition values and arrhythmia after drug application and thus can provide predictive cardiac electrophysiology data. The baseline electrophysiological parameters vary between iPS cells from different sources, therefore positive and negative control recordings are recommended.

Poster PDF
2015 – Complementary automated patch clamp, extracellular field potential and impedance recordings of iPSCs: safety screening tool box for the future
Webinar
2018 – HTS Phase I study: an update on progress of the CiPA Ion Channel Work Stream using the SyncroPatch 384PE and Patchliner
Presenter: Tim Strassmaier, Nanion Technologies Inc. USA, Source: Webinar: "CiPA study: Bridging ion channel and myocyte data", September 12, 2018
Title: CiPA myocyte phase II validation study results: cross-site comparison using the CardioExcyte 96

The CiPA HTS Ion Channel Working Group finalized its phase I study in 2017. Amongst other external sites, Nanion Technologies in Germany, USA and Japan participated with the Patchliner and the SyncroPatch 384PE in this study. A comparative view of the ion channel targets and a cross-platform and cross-site comparison will be presented. Furthermore, results from the myocyte Work Stream using arrhythmogenic compounds will be compared and confirmed with patch clamp data derived from the HTS Work Stream.

Please note: The original webinar presentation contained 8 slides with data of an upcoming publication. Due to confidentiality reasons, the relevant slides were cut out of the movie.

Poster PDF
2018 – Combining electrophysiology and contractility recordings for more complete assessment of hiPSC-CMs
SyncroPatch 384PE (a predecessor model of the SyncroPatch 384), Patchliner and CardioExcyte 96 poster, Europhysiology Meeting 2018
Webinar
28.07.2015 | Webinar: High Throughput and High Fidelity: Automated Patch Clamp in Screening and Research
Authors: Alfred L. George; Dr. Carlos Vanoye,
User meeting video
14.10.2020 | Webinar: Development and validation of ASIC1a ligand-gated ion channel drug discovery assays on automated patch clamp platforms.
Authors: Marc Rogers, This is an on-demand webinar from Nan]i[on and Friends 2020.

Whilst voltage-gated ion channels formed the bulk of academic and industrial effort in developing and utilising APC assays for ion channel drug discovery, recent years have seen increasing interest in ligand-gated receptors. These targets offer specific challenges for APC systems in terms of lower channel expression, rapid application and wash-off of ligands, and loss of responsiveness due to short- and long-term desensitisation. In this presentation I will outline successful development of pipette- and tip-based APC assay formats for the rapidly-activating ASIC1A channel on the Patchliner and SyncroPatch384i.

User meeting video
15.10.2020 | Webinar: Benchmarking best practices and calibration standards for HTS hERG recordings for improved proarrhythmic assessment
Dr. Alison Obergrussberger (Nanion Technologies), This is an on-demand webinar from Nan]i[on and Friends 2020.

The use of automated patch clamp (APC) electrophysiology in cardiac safety screening has increased over the years, and APC is now an established and accepted technique in most, if not all, safety testing laboratories. Since the introduction of the ICH S7B non-clinical guidance in November 2005 which requires all new drugs to be tested for activity on the IKr current carried by hERG expressed in recombinant cell lines using the patch-clamp technique, very few drugs have been withdrawn from the market due to pro-arrhythmic complications. APC has become the major workhorse in safety testing laboratories and is now considered to be the gold standard. Furthermore, with the introduction of the comprehensive in vitro pro-arrhythmia assay (CiPA) which recommends expanding electrophysiological recordings to include other cardiac ion channels, APC will continue to play a major role in cardiac safety testing. Recently, a large study comparing the results of a set of standard compounds tested on different instruments at different sites has been published[1] which highlights the need for standardized protocols for reliable results, for example, for hERG recordings.

We have undertaken a study to identify key parameters that can affect IC50 values of compounds acting on hERG using the medium and high throughput APC systems, Patchliner, SyncroPatch 384PE and SyncroPatch 384i. Effects of experimental parameters such as voltage protocol, incubation time, labware, compound storage time and replicate number on IC50 values of a set of CiPA compounds will be presented and recommendations for best practices for hERG measurements using APC is provided. Furthermore, as outlined in the 2020 Best Practice Consideration for In vitro Studies, ‘The concentration of compound to which the cells were exposed should be verified by applying a validated analytical method to the solution collected from the cell chamber’[2] in patch clamp studies. Nanion has implemented a new procedure that enables sample collection from used wells from the NPC-384 chips and this will be described.

Poster PDF
2022 – Characterization of ion channel currents endogenously expressed in Neuro2A cells using automated patch clamp
Poster PDF
2022 – The role of LRRC8 in the hypotonic stress response of human keratinocytes
Poster PDF
2020 – Kinetic and pharmacological properties of P2X3 and P2X2/3 receptors
SyncroPatch 384PE (a predecessor model of the SyncroPatch 384 instrument) and Patchliner poster, 64th Annual Meeting of the Biophysical Society
Poster PDF
2021 – Development and validation of ASIC1a ligand-gated ion channel drug discovery assays on automated patch clamp platforms
Poster PDF
2020 – Reliable Identification of hERG Liability in Drug Discovery by Automated Patch Clamp
SyncroPatch 384i (a predecessor model of SyncroPatch 384) and Patchliner poster, 64th Annual Meeting of the Biophysical Society
Poster PDF
2018 – Investigating pain pathways by inhibition of voltage-gated sodium channels
SyncroPatch 384PE (a predecessor model of the SyncroPatch 384) and Patchliner poster, FENS Meeting 2018
Poster PDF
2017 – Cardiomyocytes in Voltage Clamp and Current Clamp by Automated Patch Clamp
SyncroPatch 384PE (a predecessor model of the SyncroPatch 384) and Patchliner poster, BPS Meeting 2017
Poster PDF
2021 – Reliable identification of cardiac liability in drug discovery using automated patch clamp: Considerations and best practices for high throughput recordings of NaV 1.5
Patchliner and SyncroPatch 384i (a predecessor model of SyncroPatch 384), Physiology 202
Application Note PDF
VRAC – “The role of LRRC8 in the hypotonic stress response of human keratinocytes”
SyncroPatch 384 and Patchliner application note:    

The human skin is constantly exposed to various stress factors such as temperature changes, mechanical stress, different humidity levels, air pollution or radiation. These factors can have a tremendous impact on the skin and can contribute to barrier disruption and inflammation, dry and fragile skin as well as premature ageing. Recent advances in different research areas point to an important role of LRRC8 volume regulated anion channels (VRACs) in a plethora of different physiological processes. The function of LRRC8 has been characterized in human keratinocytes and in the native human epidermis and the LRRC8 ion channel has been proposed to be a novel molecular target to modulate keratinocyte differentiation in a recent patent.LRRC8A (also named SWELL1) has been identified as the first essential component of VRACs in various cell types. LRRC8A is composed of four transmembrane domains and a C-terminal domain containing up to 17 leucine-rich repeats. Together with four additional LRRC8 family members (LRRC8B-E) it assembles into hetero-hexameric complexes. Interestingly, the LRRC8 subunit composition differs between cell types and influences VRAC properties such as inactivation kinetics, voltage-dependence and selectivity of the transported osmolyte. The generation of LRRC8A-/- knockout HaCaT keratinocytes have provided evidence for the essential function of LRRC8A in hypotonic stress response of human keratinocytes.In this Application Note we show electrophysiological data from WT and LRRC8A-/- knockout HaCaT keratinocytes which corroborate the essential function of LRRC8A in keratinocytes.

Poster PDF
2016 – Next level toxicity screening: From single channel to overall cell behavior
Orbit mini, CardioExcyte 96 and SyncroPatch 384PE (a predecessor model of the SyncroPatch 384) poster, Meeting of the French Society of Toxinology (SFET) 2015 
Poster PDF
2018 – Optogenetic technologies enable high throughput ion channel drug discovery and toxicity screening
SyncroPatch 384PE (a predecessor model of the SyncroPatch 384) and CardioExcyte 96 poster, Biophysics Annual Meeting 2018
Presentation PDF
2018 – Innovations for cell monitoring in safety and toxicity assays
Presenter: Dr. Elena Dragicevic, Senior Scientist / Sales Manager, Nanion Technologies; Source: SOT, Ncardia & Nanion hosted session, March 12. 2018
Application Note PDF
TRPC5 – “Internal perfusion of Ca2+ to activate hTRPC5 on Nanion’s SyncroPatch 384PE”
SyncroPatch 384PE (a predecessor model of the SyncroPatch 384) application note   Cells were kindly provided by Charles River.

Transient Receptor Potential Canonical (TRPC) channels are a subfamily of TRP channels. The TRPC family contains at least 7 subunits and are predominantly expressed in neuronal cells where they may play an important role in Ca2+ flux. TRPC5 channels are non-selective cation channels expressed in many areas of the brain particularly the hippocampus, amygdala and cerebellum, amongst others. Although the physiological and pathophysiological role of TRPC5 is not fully known, it does appear to be important in neuronal function, in particular during development where it is involved in hippocampal neurite outgrowth and growth cone morphology. Knockout mouse studies have also revealed that TRPC5 plays an essential role in innate fear. TRPC5 is expressed in some regions outside of the CNS including the heart where it contributes to cardiac hypertrophy in heart failure. TRPC5 is activated by intracellular calcium. Using the SyncroPatch 384PE, TRPC5 expressed in HEK cells could be activated by perfusion of the intracellular solution to contain free-Ca2+. This current was potentiated by the lanthanide, gadolinium (Gd3+), as expected and blocked by 2-APB with an IC50 consistent with that reported in the literature.

Poster PDF
2018 – High throughput automatic patch clamp: Applications for Safety Pharmacology
SyncroPatch 384PE (a predecessor model of the SyncroPatch 384) poster, JSPS Meeting 2018
Application Note PDF
TREK-1 – “Activation and inhibition of TREK-1 on Nanion’s SyncroPatch 384PE”
SyncroPatch 384PE (a predecessor model of the SyncroPatch 384) application note   Cells were kindly provided by SB Drug Discovery.

The TWIK-related K+ channel (TREK-1) is a two pore domain (K2P) K+ channel encoded by the KCNK2 gene. The protein is a homodimer, each subunit comprised of transmembrane (TM) helical regions (M1-M4), two pore domains (P1 & P2), an extracellular region with 2 helices, and intracellular N and C termini. The pore helices and pore loops form the K+ selectivity filter. First discovered in 1996, it plays an important physiological role in background K+ conductance and thus plays a major role in regulating resting membrane potential. TREK-1 widely is expressed throughout the CNS and spinal cord, particularly in the cortex, hippocampus, thalamus, hypothalamus, cerebellum and basal ganglia. Additionally, TREK-1 is expressed in high levels in small and medium sized dorsal root ganglion (DRG) neurons. It is also expressed in other regions such as lung, heart, kidney, skeletal muscle and human myometrium where it is up-regulated during pregnancy5 and may play a role in maintaining a negative membrane potential prior to labor. TREK-1 is modulated by a variety of different physical and chemical stimuli including mechanical (stretch), temperature, intracellular acidosis, poly-unsaturated fatty acids and phospoholipids. It has also been shown to be opened by volatile anaesthetics and is likely to be an important target for these agents. TREK-1 has been proposed to play a pivotal role in cerebral ischemia, epilepsy, depression, pain perception and temperature sensing and is an interesting therapeutic target. Human TREK-1 expressed in HEK cells were recorded on the SyncroPatch 384PE with good success rates. hTREK-1 was activated by BL-1249 and blocked by THA.

Application Note PDF
TRPA1 – “High Throughput Activation and Block of hTRPA1 on Nanion’s SyncroPatch 384PE”
SyncroPatch 384PE (a predecessor model of the SyncroPatch 384) application note: Cells were kindly provided by Millipore.

Transient receptor potential (TRP) channels have become important potential targets in drug discovery for the treatment of, for example, pain, respiratory diseases such as asthma, cancer and immune disorders, multiple kidney diseases and skeletal disorders1 . The transient receptor potential ankyrin 1 (TRPA1), a member of the TRP family of cation channels, plays a predominant role in the sensation of noxious cold2 and inflammatory pain3 . The channel is activated by a range of environmental irritants causing pain, pungent compounds found in foods such as garlic, mustard and cinnamon, as well as metabolites produced during oxidative stress4 . Consistent with its proposed function in nociception, TRPA1 has been shown to be expressed in sensory neurons of the dorsal root ganglion (DRG) and trigeminal ganglion, both of which transmit painful responses2. Thus, within drug development, much attention is paid to the TRPA1 channel. Preclinical data and data from a recent human genetic study5 highlight TRPA1 antagonists as a promising new approach for the treatment of acute and chronic pain. Indeed, a TRPA1 antagonist has shown positive results in a proof-ofconcept study for diabetic neuropathic pain6 . The most challenging aspects involved in the screening of the TRPA1 channel are the channel’s mechanosensitivity, fast desensitization and activity dependence on intracellular calcium. Here, we present high quality data with reliable pharmacology on hTRPA1 expressing HEK cells collected on the SyncroPatch 384PE. Data is presented showing activation of the TRPA1 channel by SCMA and inhibition by A-967079.

Application Note PDF
TASK-1 – “Activation and Inhibition of TASK-1 on Nanion’s SyncroPatch 384PE”
SyncroPatch 384PE (a predecessor model of SyncroPatch 384) application note:   Cells were kindly provided by SB Drug Discovery.

The resting membrane potential of excitable cells is determined by leak conductances predominantly mediated by KCNK and two-pore-domain potassium channels (K2P). K2P channels are characterized by the presence of two pore forming regions and four trans-membrane spanning (4TMS) regions in each channel subunit and form functional dimers. These channels are essential for the production of background leak type potassium currents that act to regulate resting membrane potential and levels of cellular excitability. The TWIKrelated acid-sensitive K+ channel 1 (K2P3.1 or TASK-1) is a member of the K2P channel family and is encoded by the KCNK3 gene. TASK-1 is ubiquitously expressed throughout the CNS but also in other tissues such as in the heart, adrenal gland, lung, pancreas,kidney, intestine and prostate. TASK-1 has been implicated in atrial fibrillation (AF) pathophysiology and was suggested as an atrial-selective antiarrhythmic drug target. TASK-1 is activated by extracellular acidosis and inhibited by anandamide and by local anesthetics including bupivicaine. Volatile general anestethics such as halothan and xenon stimulate TASK-1.

Application Note PDF
TMEM16A (ANO1) – “Internal perfusion of Ca2+ to activate TMEM16A/ANO1 on the SyncroPatch 384PE”
SyncroPatch 384PE (a predecessor model of SyncroPatch 384) application note   Cells were kindly provided by SB Drug Discovery.

TMEM16A/Anoctamin1 is a Ca2+-activated Cl− channel (CACC) which has a broad functional spectrum in processes including trans-epithelial ion transport, olfaction, photo-transduction, smooth muscle contraction, nociception, cell proliferation and control of neuronal excitability. TMEM16A has been implicated to play a role in a number of health disorders and may be an important therapeutic target in cystic fibrosis, asthma, pain and some human cancers. TMEM16A is activated by elevated cytosolic calcium concentrations. In conventional patch clamp experiments, exchange of the intracellular solution to include calcium in order to initiate channel activity is challenging and typically performed using inside-out patches or by comparing the effect of internal calcium between different cells in the whole cell configuration. Furthermore, current run-down or desensitization are common problems associated with recording this ion channel. Here, we present data from HEK293 cells expressing hTMEM16A in whole cell and perforated patch mode using fluoride-free internal solution on the SyncroPatch 384PE. The data show that intracellular solution can be exchanged in a very robust manner to investigate calcium sensitivity, voltage dependence and pharmacology of the channel.

Application Note PDF
P2X3 – “Activation and inhibition of P2X3 channels recorded on the SyncroPatch 384PE”
SyncroPatch 384PE (a predecessor model of SyncroPatch 384) application note:   Cells were engineered and kindly provided by Axxam S.p.A., Milan.  

P2X receptors are ligand-gated ion channels that open in response to extracellular ATP. They are permeable to small monovalent cations, some having significant divalent or anion permeability. P2X receptors are found on many cell types including smooth muscle cells, sensory neurones, epithelia, bone and leukocytes. A role for P2X receptors has been suggested in transmission of thermal stimuli, chemosensory signalling, taste and pain. To date, 7 P2X receptor genes have been cloned and studied in heterologous expression systems. Functional receptors are trimeric, which can be homomeric or heteromeric. The P2X2 and P2X3 receptors can function either as homomers or as P2X2/3 heteromers. When expressed together, a mixture of P2X2 and P2X3 homomers as well as P2X2/3 heteromers are likely to exist, which may be distinguished through their biophysical and pharmacological properties. Both P2X3 homomers and P2X2/3 heteromeric receptors have been implicated in nociception and pain signalling and may be important therapeutic targets for analgesic drugs. Additionally, the P2X3 and P2X2/3 receptor antagonist MK- 7264 (gefapixant), has recently progressed to Phase III trials for refractory or unexplained chronic cough.Here, we present data collected on the SyncroPatch 384PE showing activation and inhibition of P2X3 currents expressed in CHO cells with rapid and brief application of ligand (‘Ligand Puff’). ATP or αβ-methylene ATP (αβ-MeATP) activated P2X3 receptors with an EC50 value similar to values found in the literature. P2X3 receptors could be repetitively activated by ATP and blocked by A-317491 with an IC50 value in good agreement with the literature.

Application Note PDF
Qualification of Patch Ready Cells on a SyncroPatch 384PE
SyncroPatch 384PE (a predecessor model of SyncroPatch 384) application note:   Patch Ready Cells provided by acCELLerate

Recombinant cell lines that functionally express human cardiac ion channels are a valuable tool for testing new drugs for potential side effects that induce proarrhythmia. It can be difficult to maintain a constant quality of these cell lines in a continually passaged culture making this process incompatible with routine screening in high-throughput mode. Here we demonstrate the preparation of Patch Ready Cells prepared from five cell lines expressing recombinant ion chan-nels (B’SYS, Switzerland) which are recommended by the CiPA initiative for drug safety testing. The Patch Ready Cells have been tested by automated patch-clamp on a SyncroPatch 384PE (Nanion, Germany) to demonstrate their applicability in high-throughput cardiotoxicity testing.

Application Note PDF
NMDA Receptors (NR1/NR2B) – “Activation and Inhibition of human NMDA Channels on Nanion`s SyncroPatch 384PE”
SyncroPatch 384PE (a predecessor model of SyncroPatch 384) application note   Cells were kindly provided by Charles River.

N-Methyl-D-aspartate (NMDA) receptors are a member of the ionotropic glutamate receptor family, ligandgated ion channels that mediate the majority of excitatory neurotransmission in the mammalian CNS. They are expressed primarily in the CNS but also in peripheral locations such as pancreatic islet cells, sensory nerve terminals in skin and cardiac ganglia. Seven subunits of the NMDA receptor have been identified, NR1, NR2A-D and NR3A-B2 , they assemble as a tetramer consisting of two NR1 subunits and either two NR2 subunits or a combination of NR2 and NR3 subunits. Activation of NMDA receptors requires the simultaneous binding of glutamate and glycine. Calcium entry through NMDA receptors plays an important role in development and synaptic plasticity and is proposed to underlie higher processes such as learning and memory. It is also proposed to play a role in a number of neurological diseases such as epilepsy and Alzheimer’s. Indeed, memantine is an NMDA antagonist which has been approved for the treatment of moderate to severe Alzheimer’s. NMDA antagonists may also be targets for the treatment of neuropathic pain, major depression and Parkinson’s disease. Here we present high quality data at a high throughput collected on the SyncroPatch 384PE showing activation and inhibition of NMDA NR1/ NR2B expressed in HEK cells. Stable recordings of NMDA receptor were achieved and modulation of the response by spermine and ketamine is shown.

Application Note PDF
P2X2 / P2X3 – “Pharmacology of P2X2/3 channels recorded on the SyncroPatch 384PE”
SyncroPatch 384PE (a predecessor model of SyncroPatch 384) application note:   Cells were engineered and kindly provided by Axxam S.p.A., Milan.  

P2X receptors are ligand-gated ion channels that open in response to extracellular ATP. They are permeable to small monovalent cations, some having significant divalent or anion permeability. P2X receptors are found on many cell types including smooth muscle cells, sensory neurones, epithelia, bone and leukocytes. A role for P2X receptors has been suggested in transmission of thermal stimuli, chemosensory signalling, taste and pain. To date, 7 P2X receptor genes have been cloned and studied in heterologous expression systems. Functional receptors are trimeric, which can be homomeric or heteromeric. The P2X2 and P2X3 receptors can function either as homomers or as P2X2/3 heteromers. When expressed together, a mixture of P2X2 and P2X3 homomers as well as P2X2/3 heteromers are likely to exist, which may be distinguished through their biophysical and pharmacological properties. P2X2/3 receptors have been implicated in nociception and pain signalling and may be important therapeutic targets for analgesic drugs.Here we present data collected on the SyncroPatch 384PE showing activation and inhibition of P2X2/3 currents expressed in CHO cells with rapid and brief application of ligand (‘Ligand Puff’). ATP activated P2X2/3 receptors in a concentration-dependent manner with an EC50 similar to those reported in the literature for a mixture of homomeric and heteromeric P2X2/3 receptors. P2X2/3 receptors could be repetitively activated by ATPand blocked by suramin with an IC50 in good agreement with the literature.

Application Note PDF
NaV1.8 – “Stability and reproducibility of hNaV1.8 recordings on Nanion’s SyncroPatch 384PE”
SyncroPatch 384PE (a predecessor model of SyncroPatch 384) application note   Cells were kindly provided by Charles River.

The NaV1.8 gene (originally named PN3 or SNS; gene symbol SCN10A) encodes a voltage-gated sodium (NaV) channel, selectively expressed in dorsal root ganglion (DRG) neurons. In contrast to the fast and rapidly inactivating TTX-sensitive channels, NaV1.8 is TTX resistant and exhibits slower kinetics with a depolarized voltage-dependence of activation and inactivation. hNaV1.8 is an interesting drug target for inflammatory and neuropathic pain because modulation of this ion channel by inflammatory mediators appears to be a key mechanism of DRG nociceptor sensitization and activation. Interestingly, the development of potent and selective NaV1.8 inhibitors has shown promising results in reducing neuropathic pain in animal models and this has fueled interest in the search for selective NaV1.8 inhibitors. The bottleneck for drug discovery involving ion channels is often the electrophysiological assays. Nanion’s SyncroPatch 384PE offers a high throughput gigaseal platform which records up to 384/768 experiments simultaneously which helps to address this problem. It enables the recording of high quality data with reliable pharmacology, and biophysical characterizations of the protein. Our results show current-voltage relationships consistent with published results and very stable recordings using multi-hole chips. Furthermore, we show activation of hNaV1.8 from different states results in altered compound affinity. We demonstrate the suitability of Nanion’s SyncroPatch 384PE for high throughput screening of hNaV1.8.

Application Note PDF
Neurons – “Electrophysiological recordings of LGIC and AA transporters in iCell® GlutaNeurons”
Patchliner, SyncroPatch 384PE (a predecessor model of the SyncroPatch 384) and SURFE2R N1 application note:  Cells were kindly provided by FUJIFILM Cellular Dynamics, Inc.  

Human neurons derived from induced pluripotent stem cells (hiPSCs) are becoming increasingly important for studying basic neuronal physiology and can provide good models for studying neurological disorders. hiPSC derived neurons provide a viable alternative to primary cells and animal models in the drug discovery industry for finding novel therapeutics to treat seizure-related and neurodegenerative disorders. iCell® GlutaNeurons are glutamatergic-enriched cortical neurons derived from hiPSCs. Single cell gene transcription analysis has shown the presence of glutamate receptors: AMPA, kainate and NMDA, as well as glutamate and GABA transporters. Ionotropic glutamate receptors mediate the majority of excitatory neurotransmission in the mammalian CNS and removal of glutamate from the synaptic cleft by reuptake via glutamate transporters plays a role in regulating neuronal excitability. GABA is the major inhibitory neurotransmitter in the brain and is important in controlling excitability. After release, GABA is removed from the extracellular space by GABA transporters(GATs), thus terminating inhibitory synaptic transmission. Both GABA and glutamate transporters may provide novel therapeutic targets for, e.g. Parkinson’s disease, Alzheimer’s disease, and epilepsy.We recorded ligand-gated ion channel currents mediated by GABAA and AMPA receptors from iCell® GlutaNeurons on the Patchliner and SyncroPatch 384PE. Furthermore, we could measure GABA and glutamate transporters in these neurons using the SURFE2R N1 device.

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NaV1.5 and hERG – “There is no F in APC: Reliable fluoride-free recordings on the SyncroPatch 384”
SyncroPatch 384 application note:   Cells were kindly provided by SB Drug Discovery and Charles River.

Automated patch clamp (APC) instruments are used for a wide variety of applications ranging from basic research into channelopathies and biophysical characteristics of ion channels, through to routine cardiac safety testing. Their use in cardiac safety screening has increased over the years and APC is now an established and accepted technique in most, if not all, safety testing laboratories. It is well known that fluoride is often used in the internal solution in APC experiments to improve the seal resistance. The presence of external calcium (or other divalent cation) further improves the seal by a mechanism thought to be due to the formation of CaF2 crystals at the interface between the pipette or micro-pore and the cell as described in a recent patent application.Even in manual patch clamp experiments, fluoride has been used to record voltage gated Na+ channels for over 20 years, despite known effects on voltage dependence of conductance, and steady-state fast inactivation and its inhibition of protein phosphatase. Fluoride is used because it improves the seal and allows stable measurements to be performed over long periods of time. However, because there are some experiments where it is advantageous to use physiological, fluoride-free internal solutions and external solution that does not use divalent ‘seal enhancer’ solutions, we have developed a method that allows fluoride-free, physiological solutions to be used with good success rates. We demonstrate this using the cardiac ion channels hERG expressed in HEK293 cells (SB Drug Discovery) and NaV1.5 expressed in CHO cells (Charles River).

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NaV1.7 – “Characterization of hNaV1.7 on Nanion’s SyncroPatch 384PE”
SyncroPatch 384PE (a predecessor model of SyncroPatch 384) application note   Cells were kindly provided by Anaxon.

The NaV1.7 gene (SCN9A) encodes a voltage-gated sodium (NaV) channel, primarily expressed in the peripheral nervous system. It has been isolated from rat dorsal root ganglion (DRG) neurons, human medullary thyroid cancer cells (hNE-Na) and PC12 cells. Different NaV channels play a key role in modulation of action potentials in the central and peripheral nervous systems. In particular, the fast upstroke of the action potential is mediated by NaV channels. NaV channels are in part characterized by their TTX-sensitivity (TTX-resistant [TTXr], TTX-sensitive [TTXs]). NaV1.7 is a TTXs channel and is sensitive to TTX in the nanomolar range. The role of hNaV1.7 has yet to be fully elucidated but is proposed to play an important part in nociception and pain sensing. NaV1.7 has been implicated to play a role in disease pain states, in particular inflammatory pain and hypersensitivity to heat following burn injury. Common to many of the voltage-gated ion channels, a number of compounds exhibit both state- and use-dependence. For this reason, it is important to be able to reliably measure the effects of compounds using different voltage protocols to investigate state and use-dependency. In this Application Note we present data using the SyncroPatch 384PE characterizing CHO cells stably expressing hNaV1.7. The current-voltage relationship and the state- and use-dependence effects of the sodium channel blocker, tetracaine, are shown.

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nAChRα4β2 – “Pharmacology of human α4β2 nAChR recorded on the SyncroPatch 384PE”
SyncroPatch 384PE (a predecessor model of the SyncroPatch 384) application note:   Cells were engineered and kindly provided by SB Drug Discovery.  

Nicotinic Acetylcholine Receptors (nAChR) are cationpermeable ion channels, which mediate fast synaptic transmission when activated by the endogenous neurotransmitter acetylcholine (ACh) and the exogenous natural alkaloid, nicotine. Neuronal nAChR form pentameric channels which are composed of two α (α2 to α10) and three β subunits (β2 to β4). Mutations of nAChR are associated with some forms of epilepsy and many other neurological disorders such as Alzheimer’s Disease, Parkinson’s, Tourette’s Syndrome, Schizophrenia and depression. The most abundantly expressed nAChR in the mammalian brain are the α7 homomeric and α4β2 heteromeric receptors. In contrast to the α7, a4β2 nAChR has a high affinity for nicotine. This property, the up-regulation during chronic exposure to nicotine, and the receptor expression location in addiction sensitive regions of the brain like the ventral tegmental area, strongly indicate that the a4β2 nAChR is a potential target for addiction to nicotine. Here we present data collected on the SyncroPatch 384PE showing activation and block of α4β2 nAChR currents expressed in HEK cells with rapid application of ligand (‘Ligand Puff’). ACh activates α4β2 nAChR in a concentration dependent manner with an EC50 value similar to those reported in the literature. Reproducible currents were achieved when cells were preincubated with acetylcholinesterase (AChE). Finally, α4β2 nAChR were blocked by dihydro-b-erythroidine hydrobromide (DHßE), a well known competitive antagonist of the α4 subunit3 with an IC50 in good agreement with the literature.

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NaV1.5 – “Increase throughput by recording in unattended mode on the SyncroPatch 384”
SyncroPatch 384PE and SyncroPatch 384i (a predecessor model of the SyncroPatch 384) application note:   Cells were kindly provided by Charles River.  

High throughput screening (HTS) is used in the pharmaceutical industry to aid drug discovery. Large numbers of chemical compounds can be tested for biological activity using a range of techniques. The patch clamp technique remains the gold standard to test activity of compounds on ion channels and automated patch clamp (APC) is increasingly adopted in HTS labs as an alternative to conventional patch clamp given its increased ease-of-use and higher throughput. APC is employed in all aspects of drug discovery from hit finding and lead optimization through to target validation and safety testing. This is only possible due to the increase in throughput toward HTS capabilities, the compatibility with HTS workflows, and a lower cost per data point which can compete with other techniques such as fluorescence imaging (using, for example, the FLIPRTM instrument) and calcium imaging with the added benefit of real-time kinetics of drug effects. Indeed, all the major contract research organizations worldwide use APC for ion channel screening and cardiac safety testing. Increased automation, including unattended operation, is also an important factor for increasing throughput, and instruments can reliably work beyond an 8-h day provided they are serviced with enough cells, solutions, and compounds. For this to work effectively, data must be reliable with high success rates, low false positive and negative rates along with reproducible IC50 values.

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hNaV1.9 – “High Throughput Pharmacology of NaV1.9 Channels on Nanion’s SyncroPatch 384”
SyncroPatch 384 application note:   Cells kindly provided by Icagen, Inc., USA.

The SCN11A gene encodes the voltage-gated sodium channel NaV1.9 which is predominantly expressed in small-diameter sensory neurons of dorsal root ganglia (DRG) and trigeminal ganglia. NaV1.9 is characterized by slow activation with little depolarization near the resting membrane potential generating a persistent, tetrodotoxin (TTX)-insensitive current which inactivates only slowly. These properties suggest that the conductance mediated by NaV1.9 mainly contributes to amplification of depolarizing responses to subthreshold stimuli leading to lower action potential (AP) firing thresholds and increase in AP firing frequency. The role of hNaV1.9 has yet to be fully elucidated but is proposed to be involved in nociception of inflammatory and neuropathic pain. Several gain-of function mutations in SCN11A have been identified which result in either painful neuropathy or an insensitivity to pain. Given its proposed role in pain perception, NaV1.9 has gained some attention as a potential target for the development of novel pain therapeutics. Here we present high quality data with reliable pharmacology on hNaV1.9 expressing HEK293 cells at a high throughput collected on the SyncroPatch 384. Biophysical properties of NaV1.9 expressed in HEK cells (cells kindly provided by Icagen, Inc., USA) and concentration response curves for three NaV channel blockers are shown, including use-dependence of tetracaine.

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hTRPA1 – “Reproducible activation and pharmacology of hTRPA1 on the SyncroPatch 384”
SyncroPatch 384PE (a predecessor model of SyncroPatch 384) application note:   Cells were kindly provided by Charles River

The transient receptor potential ankyrin 1 (TRPA1) is a calcium permeable non-selective cation channel that belongs to the transient receptor potential (TRP) superfamily. The TRPA1 channel is expressed in the sensory neurons of the nodose ganglia, dorsal root ganglia, and trigeminal ganglia, and also non-neuronal cells such as cardiomyocytes, lung fibroblasts and pancreatic β cells. TRPA1 is activated by a range of natural pungent compounds including allyl isothiocyanate (AITC),cinnamaldehyde, and allicin. TRPA1 can also be activated by cold temperature and has been proposed to act as a mechanosensor. Not only has TRPA1 been proposed to play a role in nociception and certain pain conditions, but has also in cardiovascular conditions such as atherosclerosis, heart failure, arrhythmia, vasodilation, and hypertension. Thus, within drug development, much attention is paid to the TRPA1 channel. For example, TRPA1 has been identified as a potential target for persistent chronic painful states including inflammation, neuropathic pain, diabetes, fibromyalgia, bronchitis, and emphysema. Indeed, the TRPA1 antagonist GR 17536 from Glenmark showed efficacy in a Phase IIa proof-of-concept clinical trial for peripheral diabetic neuropathy.

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HCN2 receptor – “Biophysical modulation of hHCN2 by bPAC recorded on the SyncroPatch 384PE”
SyncroPatch 384PE (a predecessor model of the SyncroPatch 384) application note:   Cells were engineered and kindly provided by Axxam S.p.A.

Neuronal and cardiac rhythmicity is predominantly controlled by hyperpolarization activated cyclic nucleotide gated (HCN) channels. The HCN family comprises four members (HCN1-4) which are ubiquitously expressed in the central and peripheral nervous system. Activated by hyperpolarization, HCN channels open slowly with no inactivation. Cyclic AMP (and other second messenger proteins) affects the activation properties independent of phosphorylation, modulating the voltage dependence of current activation and accelerating the kinetics of channel opening. HCN mediates a Na+/K+ conductance (Ih) which contributes to the establishment of the resting membrane potential. It is therefore not surprising that HCN channels play an important role in the regulation of neuronal firing and excitability as well as pacemaking. Disruption of HCN function slows down the heart rate and provides a potential target for the treatment of neuronal disorders such as epilepsy and neuropathic pain.Here we present data collected on the SyncroPatch 384PE showing pharmacology and modulation of hHCN2 expressed in HEK cells. In addition, these cells heterologously express a light-sensitive bacterial phospho-adelynate cyclase (bPAC). We demonstrate two ways of triggering the cAMP pathway in order to modulate the HCN2 channel opening kinetics. First, we used the internal perfusion system of the SyncroPatch 384PE for direct application of cAMP to the intracellular environment. Second, we triggered the cAMP pathway by optical stimulation of bPAC. Further, we showed voltage dependent block of Ih with Cs+ and ZD7288. Ivabradine, a drug used for symptomatic management of stable heart related chest pain and heart failure blocked the channel with an IC50 of 0.1 mM in good agreement the literature.

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hERG – “High Throughput Pharmacology of hERG Channels on Nanion’s SyncroPatch 384PE”
SyncroPatch 384PE (a predecessor model of the SyncroPatch 384) application note   

The hERG gene encodes a potassium channel responsible for the repolarization of the IKr current in cardiac cells. This channel is important in the repolarization of the cardiac action potential. Abnormalities in this channel can lead to long or short QT syndrome, leading to potentially fatal cardiac arrhythmia. Given the importance of this channel in maintaining cardiac function, and disturbances of channel activity by certain compounds such as antiarrhythmias and anti-psychotics, it has become an important target in compound safety screening. A large range of therapeutic agents with diverse chemical structures have been reported to induce long QT syndrome by inhibiting the hERG channel. These include antihistamines (e.g. Terfenadine), gastrointestinal prokinetic agents (e.g. Cisapride), amongst others. Therefore, it is important to test new therapeutics for actions on the hERG channel early on in the drug discovery process. Here we present high quality data with reliable pharmacology on hERG expressing CHO cells at a high throughput collected on the SyncroPatch 384PE. Current-voltage plots, and concentration response curves for the compounds pimozide, astemizole, cisapride and terfenadine are shown. The IC50 values for these compounds are within the expected range and success rates of 80% for completed experiments were recorded.

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hASIC1a – “Pharmacology of human ASIC1a channels on Nanion’s SyncroPatch 384i”
SyncroPatch 384i (a predecessor model of the SyncroPatch 384) application note:   Cells were kindly provided by Charles River.

Acid-sensing ion channels (ASICs) are protongated ion channels which are highly sensitive to extracellular acidosis and are permeable to cations, predominantly Na+. They are members of the sodium-selective cation channels belonging to the epithelial sodium channel/degenerin (ENaC/DEG)family.

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hClC-1 – “Characterization of hClC-1 on the SyncroPatch 384”
SyncroPatch 384 application note:   Cells were kindly provided by Charles River

The voltage-gated chloride channel gene (ClC) family is highly conserved and their members are present in both pro- and eukaryotes. In mammals, nine different ClC subtypes have been identified, which differ according to tissue distribution and subcellular location. ClC-1 is exclusively expressed in skeletal muscles. ClCs function as homodimers, allowing chloride and other anions to be conducted through each single protopore. Gating of the single monomer is fast while the common gate to open and close the pores simultaneously is slow. The channel gating can be modulated by intracellular and extracellular chloride as well as pH. ClC proteins mediate chloride flux across cellular membranes in most cell types and participate in maintenance of resting membrane potential. Plasma membrane chloride channels play an important role in reducing muscle excitability. ClC-1 contributes to membrane repolarization and stabilizes the membrane voltage in skeletal muscle. Experimental block of the chloride conductance mediated by ClC-1 facilitated muscle hyperexcitability, manifested as myotonia. Here we present data conducted on the SyncroPatch 384 showing characteristic biophysical properties and pharmacology of hClC-1 expressed in CHO cells. We applied voltage protocols including various test potentials to study the voltage dependence of compounds. In order to investigate compound binding properties we used the internal perfusion system of the SyncroPatch 384 for direct application of anthracene-9-carboxylic acid (9-AC) to the intracellular environment. Moreover, we also investigated the effect of 9-AC and niflumic acid (NFA) when they are applied from the extracellular side.

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CaV3.2 – “High Throughput Pharmacology of CaV3.2 Channels on Nanion’s SyncroPatch 384PE”
SyncroPatch 384PE (a predecessor model of the SyncroPatch 384) application note:   Cells were kindly provided by Millipore.  

The CaV3.2 channel is one of the three low voltage activated (LVA) T-type calcium channels. The LVA currents differ from the high voltage activated (HVA) calcium currents in their activation and inactivation kinetics. LVA currents are activated at lower voltages (typically activating above -50 mV and peaking at around -20 mV), they display faster inactivation, slower deactivation and a smaller conductance of Ba2+ ions as compared with the HVA currents. The CaV3.2 channel contains the α1H subunit, encoded by the CACNA1H gene on the human chromosome 16p13.3. T-type channels are expressed in a wide variety of organs throughout the human body, including nervous tissue, heart, kidney, smooth muscle, and many endocrine organs. They have been implicated in a variety of physiological processes including neuronal firing, smooth muscle contraction and hormone secretion. More recently, CaV3.2 has been shown to play a role in nociception and pain. Here we present high quality data with reliable pharmacology on CaV3.2 expressing HEK cells at a high throughput collected on the SyncroPatch 384PE. Current-voltage plots and concentration response curves for the compounds nitrendipine, nifedipine, mibefradil and amiloride are shown. The IC50 values for these compounds are within the expected range and success rates of up to 79% for completed experiments were recorded.

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CFTR – “Different modes of activation of CFTR recorded on Nanion’s SyncroPatch 384PE”
SyncroPatch 384PE (a predecessor model of the SyncroPatch 384) application note   

Cystic Fibrosis (CF) is an autosomal recessive genetic disorder which affects a number of organs, in particular the lungs, pancreas and sweat glands. It is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator protein (CFTR). CFTR is a regulated epithelial chloride channel and mutations cause a reduction in activity of the channel via a variety of mechanisms. This results in defective electrolyte transport in airway epithelia and thereby, chronic lung infection and premature mortality. Therefore, compounds which increase activity of CFTR have therapeutic potential for treating CF. The CFTR protein is composed of 5 domains: there are 2 transmembrane (TM) domains, 2 nucleotidebinding domains (NBDs) and 1 regulatory domain (R). The TM domains form the pore of the channel, channel activity is determined by phosphorylation of the R domain and gating is controlled by hydrolysis of ATP at the NBD. CFTR is activated via a number of reagents including internal fluoride, cAMP and external forskolin. Here we show activation of CFTR expressed in CHO cells on the SyncroPatch 384PE by internal perfusion of F- or external application of forskolin. The current was blocked with the specific blocker, inh-172 with an IC50 in good agreement with the literature. In addition, CFTR activated by internal cAMP was potentiated by VX-770. Using F- -free internal solution and activation by submaximal cAMP or forskolin, potentiators of CFTR can be investigated as potential therapeutics to treat CF.

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CaV1.2 – “Stability and Pharmacology of CaV1.2 Channels on Nanion’s SyncroPatch 384PE”
SyncroPatch 384PE (a predecessor model of the SyncroPatch 384) application note   Cells were kidly provided by Charles River.

The CaV1.2 channel is a voltage-gated calcium channel (VGCC) expressed in a variety of mammalian tissues and is essential for multiple processes including CNS function, cardiac and smooth muscle contraction and Ca2+-selective pore, contains the voltage sensor and many of the binding sites for regulatory modulators and drugs and accessory subunits α2δ, β and γ which are involved in anchorage, trafficking and regulatory functions. The CaV1.2 channel contains the alpha-1C subunit, encoded by the CACNA1C gene on the human chromosome 12p133. Mutations in the L-type Ca2+ channels have been associated with inherited arrhythmic disorders such as Timothy, Brugada and early repolarization syndromes. In addition, in the light of the CiPA initiative, the L-type channel is likely to become an important target for cardiac safety testing. Here we present high quality data with reliable pharmacology on CaV1.2 expressing CHO cells at a high throughput collected on the SyncroPatch 384PE. Current-voltage plots and concentration response curves for the compounds nifedipine and verapamil are shown. The IC50 values for these compounds are within the expected range and success rates of >70% for completed experiments were recorded. Importantly, CaV1.2 recorded on the Syncro- Patch 384PE exhibited stable peak amplitudes during the course of the experiment and displayed little or no rundown.

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CaV1.2 – “Stable Recordings and High Throughput Pharmacology of Assay Ready CaV1.2 Cells on the SyncroPatch 384”
SyncroPatch 384 application note:   Cells were kindly provided by NMI TT Pharmaservices, Steinbeis-Innovationszentrum Zellkulturtechnik and acCELLerate

The CaV1.2 channel is a voltage-gated calcium channel (VGCC) expressed in a variety of mammalian tissues and is essential for multiple processes including CNS function, cardiac and smooth muscle contraction and neuroendocrine regulation. VGCC are composed of 4 subunits, which include an alpha 1 subunit which forms the Ca2+-selective pore, contains the voltage sensor and many of the binding sites for regulatory modulators and drugs and accessory subunits α2δ, β and γ which are involved in anchorage, trafficking and regulatory functions. The CaV1.2 channel contains the alpha-1C subunit, encoded by the CACNA1C gene on the human chromosome 12p13. Mutations in the L-type Ca2+ channels have been associated with inherited arrhythmic disorders such as Timothy, Brugada and early repolarization syndromes. In addition, the L-type Ca2+ channel is an important target for cardiac safety testing, especially in the light of the CiPA initiative. Here we present high quality data with reliable pharmacology on Assay Ready CaV1.2 Cells at a high throughput collected on the SyncroPatch 384. Current-voltage plots and concentration response curves for the compounds nifedipine and verapamil are shown. The IC50 values for these compounds are within the expected range and success rates of >75% for completed experiments were recorded. Importantly, CaV1.2 recorded on the Syncro- Patch 384 exhibited stable peak amplitude during the course of the experiment and displayed low rundown.

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Assay Ready TRP-Channel Expressing Cells – a Flexible Tool to Screen for New Drug Candidates
SyncroPatch 384i (a predecessor model of SyncroPatch 384) application note:   Patch Ready Cells provided by acCELLerate

Looking for new targets in pain and cancer therapy, the transient receptor potential chan-nels (TRP-channels) gained a lot of interest during the last decade. The ligand-gated calcium channels play an important role in the perception of pain and temperature and are often dysregulated in tumor tissues. They have become appealing targets for Drug Discovery. Re-combinant cell lines which stably express different TRP-channels have been successfully used for lead identification and compound profiling.Looking for new targets in pain and cancer therapy, the transient receptor potential chan-nels (TRP-channels) gained a lot of interest during the last decade. The ligand-gated calcium channels play an important role in the perception of pain and temperature and are often dysregulated in tumor tissues. They have become appealing targets for Drug Discovery. Re-combinant cell lines which stably express different TRP-channels have been successfully used for lead identification and compound profiling.Assay ready cryopreserved aliquots prepared from these cell lines can be used instantly after thawing without prior cultivation. Here, we demonstrate that Assay Ready Cells prepared from TRP-channel expressing cell lines resemble the pharmacology of cells from continuous culture in different end-point assays. The cells were successfully qualified for plate-based fluorescent calcium-flux assays and for recording of activated ion channel currents using automated patch clamping.

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CaV1.2 – “High Throughput Pharmacology of CaV1.2 Channels on Nanion’s SyncroPatch 384PE”
SyncroPatch 384PE (a predecessor model of the SyncroPatch 384) application note:   Cells were kindly provided by SB Drug Discovery.  

The CaV1.2 channel is a voltage-gated calcium channel (VGCC) expressed in a variety of mammalian tissues and is essential for multiple processes including CNS function, cardiac and smooth muscle contraction and neuroendocrine regulation. VGCC are composed of 4 subunits, which include an alpha 1 subunit which forms the Ca2+-selective pore, contains the voltage sensor and many of the binding sites for regulatory modulators and drugs and accessory subunits α2δ, β and γ which are involved in anchorage, trafficking and regulatory functions. The CaV1.2 channel contains the alpha-1C subunit, encoded by the CACNA1C gene on the human chromosome 12p13. Mutations in the L-type Ca2+ channels have been associated with inherited arrhythmic disorders such as Timothy, Brugada and early repolarization syndromes. In addition, in the light of the CiPA initiative, the L-type channel is likely to become an important target for cardiac safety testing. Here we present high quality data with reliable pharmacology on CaV1.2 expressing HEK cells at a high throughput collected on the SyncroPatch 384PE. Current-voltage plots and concentration response curves for the compounds nifedipine and verapamil are shown. The IC50 values for these compounds are within the expected range and success rates of >75% for completed experiments were recorded. Importantly, CaV1.2 recorded on the SyncroPatch 384PE exhibited stable peak amplitude during the course of the experiment and displayed little or no rundown.

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AMPA receptor (GluA2) – “Activation, potentiation and inhibition of AMPA receptors on the SyncroPatch 384PE”
SyncroPatch 384PE (a predecessor model of SyncroPatch 384) application note:   Cells were kindly provided by SB Drug Discovery.

AMPA receptors are cation-permeable ionotropic glutamate receptors of the non-NMDA receptor subfamily. To date four subunits, GluA1-4, have been identified which are of similar size (approx. 900 kDa) and share 68-73% amino acid sequence identity. The functional receptor exists as a tetramer, either as homomers or heteromers (GluA1 and GluA4). The vast majority of excitatory fast synaptic transmission in the mammalian central nervous system is mediated by AMPA receptors of differing subunit combinations. It is well known that glutamate is a neurotoxin and it is proposed that overactivation of ionotropic glutamate receptors may underlie many neurodegenerative disorders such as ischemic stroke, epilepsy, Parkinson’s and dementia, amongst others. Here we present data collected on the SyncroPatch 384PE showing recordings of GluA2-mediated currents. Glutamate activated GluA2 receptors with an EC50 similar to those reported in the literature. CNQX inhibited and LY404187 enhanced GluA2-mediated responses.

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ASIC3 – “Activation and Inhibition of human ASIC3 Channels on Nanion’s SyncroPatch 384PE”
SyncroPatch 384PE (a predecessor model of SyncroPatch 384) application note:   Cells were kindly provided by Millipore.  

Acid-sensing ion channels (ASICs) are ligand-gated ion channels activated by protons. They are members of the sodium-selective cation channels belonging to the epithelial sodium channel/degenerin (ENaC/DEG) family. ASICs are highly sensitive to extracellular acidosis and are permeable to cations, predominantly Na+. So far, 6 different ASIC subunits have been identified encoded by 4 genes. They are found expressed throughout the CNS and PNS and have a proposed role in nociception and pain, and other neurological diseases such as ischaemia and inflammation. The ASIC3 channel was first identified in the late 1990’s. It was found to be localized to primary afferent nociceptive fibers innervating the skin, muscles, joints and viscera, in agreement with a role in pain perception. Furthermore, ASIC3 is expressed in higher amounts in nociceptive neurons innervating muscle (~ 50%) compared to skin (~ 10%), which indicates that ASIC3 may play an important role in detecting muscle acidosis. Here we present high quality data at a high throughput collected on the SyncroPatch 384PE showing activation and inhibition of ASIC3 expressed in HEK cells. The pH which elicited a halfmaximal response was in good agreement with the literature. The IC50 for block of the ASIC3 current by amiloride, a known blocker of ASIC and ENaC channels, was also in good agreement with the literature. Success rates of over 80% for completed experiments were recorded.

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NaV1.5 – “NaV1.5-ΔKPQ late INa current properties and pharmacology on the SyncroPatch 384i”
SyncroPatch 384i (a predecessor model of the SyncroPatch 384) application note   Cells were kindly provided by Metrion Biosciences.

The cardiac late Na current (late INa) generates persistent currents throughout the plateau phase of the cardiac action potential. Several mutations in the SCN5A gene cause a form of hereditary long QT syndrome (LQT3)1-3. The ΔKPQ mutation deletes residues Lys 1505, Pro 1506 and Gln 1507, resulting in a sustained, non-inactivating current during long (over 50 ms) depolarizations1,2. This sustained current causes prolongation of the action potential which can result in fatal ventricular arrhythmias such as Torsade de Pointes (TdP)1.One aim of the Comprehensive In Vitro Pro-arrhythmia Assay (CiPA) initiative is to improve drug safety testing in pre-clinical development by evaluating the pro-arrhythmic risk of a compound4,5. Validation studies confirm that testing the effect of compounds on an increased number of human cardiac ion channel currents including INa (NaV1.5 peak and late current) as well as IKr (hERG) leads to improved prediction of their clinical risk. Late INa can be recorded in WT NaV1.5 channels using the toxin ATX-II or veratridine, or using a cell line with LQT3 mutations in NaV1.5 without the need for pharmacological enhancement. The latter might also reduce the risk of cross-reactions between late-current enhancers and test compounds.Here we present data collected on the Syncro- Patch 384i showing the peak and late INa current re¬corded from WT and NaV1.5-ΔKPQ cell lines. Peak current could be reliably recorded from both cell types. In WT cells, late INa was negligible in the absence of ATX-II, whereas the late INa from NaV1.5-ΔKPQ cells could be reliably recorded. Peak current from WT, and peak and late INa from NaV1.5-ΔKPQ was inhibited by ranolazine and mexiletine and IC50 values agreed well with the literature6.

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AChR α1β1γδ – “Reproducible activation and pharmacology of α1β1γδ nAChR on the SyncroPatch 384”
SyncroPatch 384 application note:   Cells were obtained from CLS

Nicotinic acetylcholine receptors (nAChR) are acetylcholine- (ACh) and nicotine-gated cation permeable ion channels, which mediate fast synaptic transmission at central synapses and neuromuscular junctions. Neuromuscular nAChR form heteromeric proteins composed of four subunits: α, β, γ (or ε) and δ. Depending on the developmental stage, the AChR subunit stoichiometry changes from α1β1γδ (embryonic) to α1β1εδ (adult). Several inherited and acquired diseases are associated with nAChR dysfunction, most of which lead to impaired neuromuscular transmission and muscle weakness. The acquired autoimmune disease myasthenia gravis (MG) is caused by autoantibodies targeting muscle nAChRs that disrupts nerve-muscle communication resulting in muscle weakness and fatigue. Inherited diseases called congenital myasthenic syndromes (CMS) are associated with several abnormalities affecting ACh-release, acetylcholinesterase activity, nAChR function and/or nAChR number. Treatment has been limited to nonselective, chronic immunosuppressive therapies which have longterm toxicities. More selective and targeted therapies are now under development. Here we present data collected on the SyncroPatch 384 showing activation and block of nAChRα1β1γδ expressed in human TE671 cells with rapid application of ligand or co-application with blockers. We found that ACh activates nAChRα1β1γδ receptors with an EC50 value similar to those reported in the literature. We recorded highly reproducible currents in response to ACh and obtained IC50 values for mecamylamine and α-conotoxin GI that were in good agreement with the literature.

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Cardiac Ion Channels – “Simultaneous Assessment of CiPA Stipulated Ion Channels on the SyncroPatch 384PE”
SyncroPatch 384PE (a predecessor model of the SyncroPatch 384) application note   Cells were kindly provided by Charles River.

The cardiac action potential is defined by multiple voltage-dependent ion channels. A drug candidate’s capacity to interact with the ion channels involved in the depolarization or repolarization phases of the cardiac action potential is important for drug safety assessment. Until now, safety testing ha