Read more in the paper here.
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.
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.
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.
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.
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.
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.
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.
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.
Our findings provide the first evidence that down-regulation of MFN2 preserves cardiac function in chronic HH by promoting cardiac metabolic reprogramming.
Read more in the publication here.
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.
Read more in the publication here.
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.
Read more in the publication here.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Read more in the publication here.
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.
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.
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.
Read more in the publication here.
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.
Read more in the publication here.
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.
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.
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.
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.
Read more in the publication here.
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.
Read more in the publication here.
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
Read more in the publication here.
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.
Read more in the publication here.
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.
Read more in the publication here.
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.
Read more in the publication here.
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.
Read more in the publication here.
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.
Read more in the publication here.
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.
Read more in the publication here.
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.
Read more in the publication here.
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.
Read more in the publication here.
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.
Read more in the publication here.
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.
Read more about the article here.
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.
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.
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.
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.
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.
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.
Read more in the paper here.
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.
Read more in the paper here.
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.
Pathogenic variants in neuronal voltage-gated sodium (NaV) channel genes including SCN2A, which encodes NaV1.2, are frequently discovered in neurodevelopmental disorders with and 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 (GoF) variants cause epilepsy whereas loss-of-function (LoF) variants are associated with ASD and ID. However, this framework is based on a limited number of functional studies conducted under heterogenous experimental conditions whereas most disease-associated SCN2A variants have not been functionally annotated. We determined the functional properties of more than 30 SCN2A variants using automated patch clamp recording to assess the analytical validity of this approach 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 population variants using two distinct alternatively spliced forms of NaV1.2 that were heterologously expressed in HEK293T cells. Multiple biophysical parameters were assessed on 5,858 individual cells. We found that automated patch clamp recording provided a valid high throughput method to ascertain detailed functional properties of NaV1.2 variants with concordant findings for a subset of variants that were previously studied using manual patch clamp. Additionally, many epilepsy-associated variants in our study exhibited complex patterns of gain- and loss-of-function properties that are difficult to classify overall by a simple binary scheme. The higher throughput achievable with automated patch clamp enables study of a larger number of variants, greater standardization of recording conditions, freedom from operator bias, and enhanced experimental rigor valuable for accurate assessment of NaV channel variant dysfunction. Together, this approach will enhance our ability to discern relationships between variant channel dysfunction and neurodevelopmental disorders.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Read more and find the publication here.
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.
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.
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.
Bacterial and mammalian NaV channels provide insights into the molecular basis of channel gating and will facilitate organism-specific drug discovery.
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
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
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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).
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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).
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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).
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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 has focussed on interaction with the hERG channel and QT prolongation which can lead to potentially fatal torsades de pointes (TdP). Although this approach has been largely successful in preventing new drugs reaching the market with unexpected potential to cause TdP, it is also possible that potentially valuable therapeutics have failed due to this early screening. A new paradigm, the Comprehensive In-vitro Proarrhythmia Assay (CiPA), was introduced in 2013 to provide a more complete assessment of proarrythmic risk. An assessment of a multitude of cardiac ion channels, in addition to hERG, should provide a more accurate prediction of the proarrythmic risk of a compound compared with testing on hERG alone. Here we show recordings from HEK or CHO cells expressing the CiPA stipulated ion channels; NaV1.5, CaV1.2, hERG, KV7.1, Kir2.1 or KV4.3, activated within one single experiment on the SyncroPatch 384PE.
The NaV1.5 channel, encoded by the SCN5A gene, is a voltage-gated sodium (NaV) channel found in skeletal muscle and heart. It is TTX insensitive with an IC50 in the micromolar range. NaV1.5 is responsible for the upstroke of the cardiac action potential in both ventricular and atrial myocytes and is therefore critical for generation and propagation of the cardiac action potential in human heart. Block of this channel can lead to prolongation of the QRS interval of the electrocardiogram (ECG) and can have profound effects on the rate of cardiac deploarization and conduction velocity, thus causing potentially dangerous cardiac arrythmias. Furthermore, effects of NaV1.5 inactivation can modify cardiac repolarization. Given the importance of this channel in maintaining cardiac function, it has become an important target in compound safety screening. Local anaesthetics, such as lidocaine, have been shown to exhibit state- and use-dependence when acting on the cardiac sodium channel. The IC50 was shown to be approximately 30 times lower at depolarized holding potentials where inactivation was almost complete. For this reason, it is important to test potency of compounds at different holding potentials. Here we present high quality data with reliable pharmacology on hNaV1.5 expressing HEK293 cells at a high throughput collected on the SyncroPatch 384PE. Current-voltage plots and concentration response curves for four NaV channel blockers are shown, including lidocaine at different holding potentials.
Abstract: Mutations in the KCNH2 gene are a well-established cause of sudden cardiac death, resulting from disturbed electrical signalling, in otherwise healthy young people. Yet, the majority of missense variants identified in KCNH2 are likely to be benign. To differentiate between benign and pathogenic variants in KCNH2 we have developed a high throughput functional assay using the syncropatch 384PE automated patch clamp system. The Victor Chang Cardiac Research Institute is an independent, not-for-profit, medical research facility that is dedicated to finding cures for cardiovascular disease. Renowned for the quality of its breakthroughs, the Victor Chang Cardiac Research Institute is one of the most respected heart research facilities in the world.
Whether ion channels experience ligand-dependent dynamic ion selectivity remains of critical importance since this could support ion channel functional bias. Tracking selective ion permeability through ion channels, however, remains challenging even with patch-clamp electrophysiology. In this study, we have developed highly sensitive bioluminescence resonance energy transfer (BRET) probes providing dynamic measurements of Ca2+ and K+ concentrations and ionic strength in the nanoenvironment of Transient Receptor Potential Vanilloid-1 Channel (TRPV1) and P2X channel pores in real time and in live cells during drug challenges. Our results indicate that AMG517, BCTC, and AMG21629, three well-known TRPV1 inhibitors, more potently inhibit the capsaicin (CAPS)-induced Ca2+ influx than the CAPS-induced K+ efflux through TRPV1. Even more strikingly, we found that AMG517, when injected alone, is a partial agonist of the K+ efflux through TRPV1 and triggers TRPV1-dependent cell membrane hyperpolarization. In a further effort to exemplify ligand bias in other families of cationic channels, using the same BRET-based strategy, we also detected concentration- and time-dependent ligand biases in P2X7 and P2X5 cationic selectivity when activated by benzoyl-adenosine triphosphate (Bz-ATP). These custom-engineered BRET-based probes now open up avenues for adding value to ion-channel drug discovery platforms by taking ligand bias into account.
This publication details the discovery of a series of selective transient receptor potential cation channel subfamily M member 5 (TRPM5) agonists culminating with the identification of the lead compound (1R, 3R)-1-(3-chloro-5-fluorophenyl)-3-(hydroxymethyl)-1,2,3,4-tetrahydroisoquinoline-6-carbonitrile (39). We describe herein our biological rationale for agonism of the target, the examination of the then current literature tool molecules, and finally the process of our discovery starting with a high throughput screening hit through lead development. We also detail the selectivity of the lead compound 39 versus related family members TRPA1, TRPV1, TRPV4, TRPM4 and TRPM8, the drug metabolism and pharmacokinetics (DMPK) profile and in vivo efficacy in a mouse model of gastrointestinal motility.
Gamma-aminobutyric acid type A receptors (GABAARs) are ligand gated channels mediating inhibition in the central nervous system. Here, we identify a so far undescribed function of β-subunit homomers as proton-gated anion channels. Mutation of a single H267A in β3 subunits completely abolishes channel activation by protons. In molecular dynamic simulations of the β3 crystal structure protonation of H267 increased the formation of hydrogen bonds between H267 and E270 of the adjacent subunit leading to a pore stabilising ring formation and accumulation of Cl- within the transmembrane pore. Conversion of these residues in proton insensitive ρ1 subunits transfers proton-dependent gating, thus highlighting the role of this interaction in proton sensitivity. Activation of chloride and bicarbonate currents at physiological pH changes (pH50 is in the range 6- 6.3) and kinetic studies suggest a physiological role in neuronal and non-neuronal tissues that express beta subunits, and thus as potential novel drug target.
SMN protein deficiency causes motoneuron disease spinal muscular atrophy (SMA). SMN-based therapies improve patient motor symptoms to variable degrees. An early hallmark of SMA is the perturbation of the neuromuscular junction (NMJ), a synapse between a motoneuron and muscle cell. NMJ formation depends on acetylcholine receptor (AChR) clustering triggered by agrin and its co-receptors lipoprotein receptor-related protein 4 (LRP4) and transmembrane muscle-specific kinase (MuSK) signalling pathway. We have previously shown that flunarizine improves NMJs in SMA model mice, but the mechanisms remain elusive. We show here that flunarizine promotes AChR clustering in cell-autonomous, dose- and agrin-dependent manners in C2C12 myotubes. This is associated with an increase in protein levels of LRP4, integrin-beta-1 and alpha-dystroglycan, three agrin co-receptors. Furthermore, flunarizine enhances MuSK interaction with integrin-beta-1 and phosphotyrosines. Moreover, the drug acts on the expression and splicing of Agrn and Cacna1h genes in a muscle-specific manner. We reveal that the Cacna1h encoded protein CaV3.2 closely associates in vitro with the agrin co-receptor LRP4. In vivo, it is enriched nearby NMJs during neonatal development and the drug increases this immunolabelling in SMA muscles. Thus, flunarizine modulates key players of the NMJ and identifies CaV3.2 as a new protein involved in the NMJ biology.
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. To enable the application of this approach at scale this 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 automated 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%. 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 appropriate consideration of protocol design, including sweep length, number of repetitions, and leak correction, this protocol can be applied on APC to acquire data comparable to manual patch clamp.
The cardiac sodium ion channel (NaV1.5) is a protein with four domains (DI-DIV), each with six transmembrane segments. Its opening and subsequent inactivation results in the brief rapid influx of Na+ ions resulting in the depolarization of cardiomyocytes. The neurotoxin veratridine (VTD) inhibits NaV1.5 inactivation resulting in longer channel opening times, and potentially fatal action potential prolongation. VTD is predicted to bind at the channel pore, but alternative binding sites have not been ruled out. To determine the binding site of VTD on NaV1.5, we performed docking calculations and high-throughput electrophysiology experiments. The docking calculations identified two distinct binding regions. The first site was in the pore, close to the binding site of NaV1.4 and NaV1.5 blocking drugs in experimental structures. The second site was at the “mouth” of the pore at the cytosolic side, partly solvent-exposed. Mutations at this site (L409, E417, and I1466) had large effects on VTD binding, while residues deeper in the pore had no effect, consistent with VTD binding at the mouth site. Overall, our results suggest a VTD binding site close to the cytoplasmic mouth of the channel pore. Binding at this alternative site might indicate an allosteric inactivation mechanism for VTD at NaV1.5.
Fluoride has been used in the internal recording solution for manual and automated patch clamp experiments for decades because it helps to improve the seal resistance and promotes longer lasting recordings. In manual patch clamp, fluoride has been used to record voltage-gated Na (NaV) channels where seal resistance and access resistance are critical for good voltage control. In automated patch clamp, suction is applied from underneath the patch clamp chip to attract a cell to the hole and obtain a good seal. Since the patch clamp aperture cannot be moved to improve the seal like the patch clamp pipette in manual patch clamp, automated patch clamp manufacturers use internal fluoride to improve the success rate for obtaining GΩ seals. However, internal fluoride can affect voltage-dependence of activation and inactivation, as well as affecting internal second messenger systems and therefore, it is desirable to have the option to perform experiments using physiological, fluoride-free internal solution. We have developed an approach for high throughput fluoride-free recordings on a 384-well based automated patch clamp system with success rates >40% for GΩ seals. We demonstrate this method using hERG expressed in HEK cells, as well as NaV1.5, NaV1.7, and KCa3.1 expressed in CHO cells. We describe the advantages and disadvantages of using fluoride and provide examples of where fluoride can be used, where caution should be exerted and where fluoride-free solutions provide an advantage over fluoride-containing solutions.
The presence of the Transient Receptor Potential channel Vanilloid type 2 (TRPV2) in red blood cells (RBCs) was recently discovered. TRPV2 is a non-selective cation channel that is reported to be mechanosensitive having numerous properties in common with Piezo1. TRPV2 channels can be activated by Δ9-tetrahydrocannabinol (Δ9-THC) and the changes induced in RBC by application of Δ9-THC can be attributed to TRPV2 channel activity. A previous study shown that addition of 30 µM Δ9-THC led to a large fraction of super hydrated RBCs in a healthy donor consuming marijuana in contrast to significantly milder response from no smoker healthy donors. For further investigations three marijuana consumers with very similar smoking habits were scouted, and blood was collected. Whether this heightened sensitivity of the smokers (MS) vs. no-smokers (NS) RBCs is caused by hypersensitizing of TRPV2 was tackled by comparing MS and NS RBCs in functional patch-clamp measurements using the high - throughput automated patch clamp platform SyncroPatch 384. Whole-cell currents elicited by THC application did not show a significant different thus indicates a similar cellular conductance for MS and NS RBCs and therefore rather a lack of difference in TRPV2 expression level. We demonstrate that APC technology is a suitable tool permits for studying TRPV2 channels in RBCs.
Thanks to the crosstalk between Na+ and Ca2+ channels, Na+ and Ca2+ homeostasis interplay in so-called excitable cells enables the generation of action potential in response to electrical stimulation. Here, we investigated the impact of persistent activation of voltage-gated Na+ (NaV) channels by neurotoxins, such as veratridine (VTD), on intracellular Ca2+ concentration ([Ca2+]i) in a model of excitable cells, the rat pituitary GH3b6 cells, in order to identify the molecular actors involved in Na+-Ca2+ homeostasis crosstalk. By combining RT-qPCR, immunoblotting, immunocytochemistry, and patch-clamp techniques, we showed that GH3b6 cells predominantly express the NaV1.3 channel subtype, which likely endorses their voltage-activated Na+ currents. Notably, these Na+ currents were blocked by ICA-121431 and activated by the β-scorpion toxin Tf2, two selective NaV1.3 channel ligands. Using Fura-2, we showed that VTD induced a [Ca2+]i increase. This effect was suppressed by the selective NaV channel blocker tetrodotoxin, as well by the selective L-type CaV channel (LTCC) blocker nifedipine. We also evidenced that crobenetine, a NaV channel blocker, abolished VTD-induced [Ca2+]i elevation, while it had no effects on LTCC. Altogether, our findings highlight a crosstalk between NaV and LTCC in GH3b6 cells, providing a new insight into the mode of action of neurotoxins.
Slack channels are sodium-activated potassium channels that are encoded by the KCNT1 gene. Several KCNT1 gain of function mutations have been linked to malignant migrating partial seizures of infancy. Quinidine is an anti-arrhythmic drug that functions as a moderately potent inhibitor of Slack channels; however, quinidine use is limited by its poor selectivity, safety and pharmacokinetic profile. Slack channels represent an interesting target for developing novel therapeutics for the treatment of malignant migrating partial seizures of infancy and other childhood epilepsies; thus, ongoing efforts are directed toward the discovery of small-molecules that inhibit Slack currents. This review summarizes patent applications published in 2020–2021 that describe the discovery of novel small-molecule Slack inhibitors.
The DNA-PK complex is activated by double-strand DNA breaks and regulates the non-homologous end-joining repair pathway; thus, targeting DNA-PK by inhibiting the DNA-PK catalytic subunit (DNA-PKcs) is potentially a useful therapeutic approach for oncology. A previously reported series of neutral DNA-PKcs inhibitors were modified to incorporate a basic group, with the rationale that increasing the volume of distribution while maintaining good metabolic stability should increase the half-life. However, adding a basic group introduced hERG activity, and basic compounds with modest hERG activity (IC50 = 10–15 μM) prolonged QTc (time from the start of the Q wave to the end of the T wave, corrected by heart rate) in an anaesthetized guinea pig cardiovascular model. Further optimization was necessary, including modulation of pKa, to identify compound 18, which combines low hERG activity (IC50 = 75 μM) with excellent kinome selectivity and favorable pharmacokinetic properties.
Cyclic α-aryl β-dicarbonyl derivatives are important scaffolds in medicinal chemistry. Palladium-catalyzed coupling reactions of haloarenes were conducted with diverse five- to seven-membered cyclic β-dicarbonyl derivatives including barbiturate, pyrazolidine-3,5-dione, and 1,4-diazepane-5,7-dione. The coupling reactions of various para- or meta-substituted aryl halides occurred efficiently when Pd(t-Bu3P)2, Xphos, and Cs2CO3 were used under 1,4-dioxane reflux conditions. Although the couplings of ortho-substituted aryl halides with pyrazolidine-3,5-dione and 1,4-diazepane-5,7-dione were moderate, the coupling with barbiturate was limited. Using the optimized reaction conditions, we synthesized several 5-aryl barbiturates as new scaffolds of CaV1.3 Ca2+ channel inhibitors. Among the synthesized molecules, 14e was the most potent CaV1.3 inhibitor with an IC50 of 1.42 μM.
Despite the identification of the high incidence red cell antigen Era nearly 40 years ago, the molecular background of this antigen, together with the other two members of the Er blood group collection, has yet to be elucidated. Whole exome and Sanger sequencing of individuals with serologically defined Er alloantibodies identified several missense mutations within the PIEZO1 gene, encoding amino acid substitutions within the extracellular domain of the Piezo1 mechanosensor ion channel. Confirmation of Piezo1 as the carrier molecule for the Er blood group antigens was demonstrated using immunoprecipitation, CRISPR/Cas9-mediated gene knockout and expression studies in an erythroblast cell line. We report the molecular bases of five Er blood group antigens: the recognised Era, Erb and Er3 antigens; and two novel high incidence Er antigens, described here as Er4 and Er5, establishing a new blood group system. Anti-Er4 and anti-Er5 are implicated in severe hemolytic disease of the fetus and newborn (HDFN). Demonstration of Piezo1, present at just a few hundred copies on the surface of the red blood cell, as the site of a new blood group system highlights the potential antigenicity of even low abundance membrane proteins and contributes to our understanding of the in vivo characteristics of this important and widely studied protein in transfusion biology and beyond.
T-type voltage-gated Ca2+ channels have been implicated in many human disorders, and there has been increasing interest in developing highly selective and potent T-type Ca2+ channel modulators for potential clinical use. However, the unique biophysical properties of T-type Ca2+ channels are not conducive for developing high-throughput screening (HTS) assays to identify modulators, particularly potentiators. To illustrate, T-type Ca2+ channels are largely inactivated and unable to open to allow Ca2+ influx at −25 mV, the typical resting membrane potential of the cell lines commonly used in cellular screening assays. To address this issue, we developed cell lines that express Kir2.3 channels to hyperpolarize the membrane potential to −70 mV, thus allowing T-type channels to return to their resting state where they can be subsequently activated by membrane depolarization in the presence of extracellular KCl. Furthermore, to simplify the HTS assay and to reduce reagent cost, we stably expressed a membrane-tethered genetic calcium sensor, GCaMP6s-CAAX, that displays superior signal to the background compared to the untethered GCaMP6s or the synthetic Ca2+ sensor Fluo-4AM. Here, we describe a novel GCaMP6s-CAAX-based calcium assay utilizing a high-throughput fluorometric imaging plate reader (Molecular Devices, Sunnyvale, CA) format that can identify both activators and inhibitors of T-type Ca2+ channels. Lastly, we demonstrate the utility of this novel fluorescence-based assay to evaluate the activities of two distinct G-protein-coupled receptors, thus expanding the use of GCaMP6s-CAAX to a wide range of applications relevant for developing cellular assays in drug discovery.
Glioblastoma is a lethal brain cancer that commonly recurs after tumor resection and chemotherapy treatment. Depolarized resting membrane potentials and an acidic intertumoral extracellular pH have been associated with a proliferative state and drug resistance, suggesting that forced hyperpolarization and disruption of proton pumps in the plasma membrane could be a successful strategy for targeting glioblastoma overgrowth. We screened 47 compounds and compound combinations, most of which were ion-modulating, at different concentrations in the NG108-15 rodent neuroblastoma/glioma cell line. A subset of these were tested in the U87 human glioblastoma cell line. A FUCCI cell cycle reporter was stably integrated into both cell lines to monitor proliferation and cell cycle response. Immunocytochemistry, electrophysiology, and a panel of physiological dyes reporting voltage, calcium, and pH were used to characterize responses. The most effective treatments on proliferation in U87 cells were combinations of NS1643 and pantoprazole; retigabine and pantoprazole; and pantoprazole or NS1643 with temozolomide. Marker analysis and physiological dye signatures suggest that exposure to bioelectric drugs significantly reduces proliferation, makes the cells senescent, and promotes differentiation. These results, along with the observed low toxicity in human neurons, show the high efficacy of electroceuticals utilizing combinations of repurposed FDA approved drugs.
Authors David J.A. Wyllie and Derek Bowie give a brief history of ionotropic glutamate receptor research and an overview of 5 papers in the 15th January 2022 issue of the Journal of Physiology. This includes the Techniques in Physiology paper by Obergrussberger et al, 2022 highlighting the advances and applications of high throughput methods for automated electrophysiological studies and specifically patch-clamp recording. Automated patch clamp devices such as the SyncroPatch 384 are increasingly becoming workhorses in the pharmaceutical industry as well as small research labs/centres.
Transient Receptor Potential Melastatin 5 (TRPM5) is an intracellular calcium-activated cation-selective ion channel expressed in a variety of cell types. Dysfunction of this channel has recently been implied in a range of disease states including diabetes, enteric infections, inflammatory responses, parasitic infection and other pathologies. However, to date, agonists and positive modulators of this channel with sufficient selectivity to enable target validation studies have not been described, limiting the evaluation of TRPM5 biology and its potential as a drug target. We developed a high-throughput assay using a fluorescent membrane potential dye and a medium- and high-throughput electrophysiology assay using QPatch HTX and SyncroPatch 384PE. By employing these assays, we conducted a primary screening campaign and identified hit compounds as TRPM5 channel positive modulators. An initial selectivity profile confirmed hit selectivity to TRPM5 and is presented here. These small molecule TRPM5 compounds have a high potential both as early tool compounds to enable pharmacological studies of TRPM5 and as starting points for the development of potent, selective TRPM5 openers or positive modulators as novel drugs targeting several pathological states.
Photoactivatable drugs targeting ligand-gated ion channels open up new opportunities for light-guided therapeutic interventions. Photoactivable toxins targeting ion channels have the potential to control excitable cell activities with low invasiveness and high spatiotemporal precision. As proof-of-concept, we develop HwTxIV-Nvoc, a UV light-cleavable and photoactivatable peptide that targets voltage-gated sodium (NaV) channels and validate its activity in vitro in HEK293 cells, ex vivo in brain slices and in vivo on mice neuromuscular junctions. We find that HwTxIV-Nvoc enables precise spatiotemporal control of neuronal NaV channel function under all conditions tested. By creating multiple photoactivatable toxins, we demonstrate the broad applicability of this toxin-photoactivation technology.
Airway epithelial damage is a common feature in respiratory diseases such as COPD and has been suggested to drive inflammation and progression of disease. These features manifest as remodeling and destruction of lung epithelial characteristics including loss of small airways which contributes to chronic airway inflammation. Histone deacetylase 6 (HDAC6) has been shown to play a role in epithelial function and dysregulation, such as in cilia disassembly, epithelial to mesenchymal transition (EMT) and oxidative stress responses, and has been implicated in several diseases. We thus used ACY-1083, an inhibitor with high selectivity for HDAC6, and characterized its effects on epithelial function including epithelial disruption, cytokine production, remodeling, mucociliary clearance and cell characteristics. Primary lung epithelial air-liquid interface cultures from COPD patients were used and the impacts of TNF, TGF-β, cigarette smoke and bacterial challenges on epithelial function in the presence and absence of ACY-1083 were tested. Each challenge increased the permeability of the epithelial barrier whilst ACY-1083 blocked this effect and even decreased permeability in the absence of challenge. TNF was also shown to increase production of cytokines and mucins, with ACY-1083 reducing the effect. We observed that COPD-relevant stimulations created damage to the epithelium as seen on immunohistochemistry sections and that treatment with ACY-1083 maintained an intact cell layer and preserved mucociliary function. Interestingly, there was no direct effect on ciliary beat frequency or tight junction proteins indicating other mechanisms for the protected epithelium. In summary, ACY-1083 shows protection of the respiratory epithelium during COPD-relevant challenges which indicates a future potential to restore epithelial structure and function to halt disease progression in clinical practice.
Hundreds of KCNQ2 variants have been identified by genetic testing of children with early onset epilepsy and/or developmental disability. Voltage-clamp recording from heterologous cells has proved useful for establishing deleterious functional effects of KCNQ2 variants, but procedures adapting these assays for standardized, higher throughput data collection and reporting are lacking. In this study, we employed automated patch clamp recording to assess in parallel the functional and pharmacological properties of 79 missense and 2 in-frame deletion variants of KCNQ2. Among the variants we studied were a training set of 18 pathogenic variants previously studied by voltage-clamp recording, 24 mostly rare population variants, and 39 disease-associated variants with unclear functional effects. Variant KCNQ2 subunits were transiently expressed in a cell line stably expressing KCNQ3 to reconstitute the physiologically relevant channel complex. Variants with severe loss-of-function were also co-expressed 1:1 with WT KCNQ2 in the KCNQ3 cell line to mimic the heterozygous genotype and assess dominant-negative behavior. In total, we analyzed electrophysiological data recorded from 9,480 cells. The functional properties of WT KCNQ2/KCNQ3 channels and pharmacological responses to known blockers and activators determined by automated patch clamp recording were highly concordant with previous findings. Similarly, functional properties of 18 known pathogenic variants largely matched previously published results and the validated automated patch clamp assay. Many of the 39 previously unstudied disease-associated KCNQ2 variants exhibited prominent loss-of-function and dominant-negative effects, providing strong evidence in support of pathogenicity. All variants, exhibit response to retigabine (10 µM), although there were differences in maximal responses. Variants within the ion selectivity filter exhibited the weakest responses whereas retigabine had the strongest effect on gain-of-function variants in the voltage-sensor domain. Our study established a high throughput method to detect deleterious functional consequences of KCNQ2 variants. We demonstrated that dominant-negative loss-of-function is a common mechanism associated with missense KCNQ2 variants but this does not occur with rare population variation in this gene. Importantly, we observed genotype-dependent differences in the response of KCNQ2 variants to retigabine.
The transient receptor potential cation channel 5 (TRPC5) plays an important role in numerous cellular processes. Due to this, it has gained considerable attention over the past few years as a potential therapeutic target. Recently, TRPC5 has been shown to be involved in the regulation of podocyte survival, indicating a potential treatment option for chronic kidney disease. In addition, a recent study has shown TRPC5 to be expressed in human sensory neurons and suggests that TRPC5 inhibition could be an effective treatment for spontaneous and tactile pain. To understand these processes more fully, potent and selective tool compounds are needed. Herein we report further exploration of the 2-aminobenzimidazole scaffold as a potent TRPC5 inhibitor, culminating in the discovery of 16 f as a potent and selective TRPC5 inhibitor.
Brugada syndrome (BrS) is a cardiac arrhythmia disorder associated with sudden death in young adults. With the exception of SCN5A, encoding the cardiac sodium channel NaV1.5, susceptibility genes remain largely unknown. Here we performed a genome-wide association meta-analysis comprising 2,820 unrelated cases with BrS and 10,001 controls, and identified 21 association signals at 12 loci (10 new). Single nucleotide polymorphism (SNP)-heritability estimates indicate a strong polygenic influence. Polygenic risk score analyses based on the 21 susceptibility variants demonstrate varying cumulative contribution of common risk alleles among different patient subgroups, as well as genetic associations with cardiac electrical traits and disorders in the general population. The predominance of cardiac transcription factor loci indicates that transcriptional regulation is a key feature of BrS pathogenesis. Furthermore, functional studies conducted on MAPRE2, encoding the microtubule plus-end binding protein EB2, point to microtubule-related trafficking effects on NaV1.5 expression as a new underlying molecular mechanism. Taken together, these findings broaden our understanding of the genetic architecture of BrS and provide new insights into its molecular underpinnings.
Impaired cerebellar Purkinje neuron firing resulting from reduced expression of large-conductance calcium-activated potassium (BK) channels is a consistent feature in models of inherited neurodegenerative Spinocerebellar Ataxia (SCA). Restoring BK channel expression improves motor function and delays cerebellar degeneration, indicating that BK channels are an attractive therapeutic target. Current BK channel activators lack specificity and potency and therefore are poor templates for future drug development. We implemented an automated patch-clamp platform for high throughput drug discovery of BK channel activators using the Nanion SyncroPatch 384PE system. We screened over 15,000 compounds for their ability to increase BK channel current amplitude under conditions of lower intracellular calcium that is present in disease. We identified several novel BK channels activators that were then re-tested on the SyncroPatch 384PE to generate concentration-response curves (CRCs). Compounds with favorable CRCs were subsequently tested for their ability to improve irregular cerebellar Purkinje neuron spiking, characteristic of BK channel dysfunction in SCA1 mice. We identified a novel BK channel activator, 4-chloro-N-(5-chloro-2-cyanophenyl)-3-(trifluoromethyl)benzene-1-sulfonamide (herein renamed BK-20), that activated BK currents more potently (pAC50 = 4.64) than NS-1619 (pAC50 = 3.7) at a free internal calcium concentration of 270 nM in a heterologous expression system and improved spiking regularity in SCA1 Purkinje neurons. BK-20 had no activity on SK1-3 channels but interestingly was a potent blocker of CaV3.1 (IC50 = 1.05 mM). Our work describes both a novel compound for further drug development in disorders with irregular Purkinje spiking and a unique platform for drug discovery in degenerative ataxias. Significance Statement Motor impairment associated with altered Purkinje cell spiking due to dysregulation of BK expression and function is a shared feature of disease in many degenerative ataxias. BK channel activators represent an outstanding therapeutic agent for ataxia. We have developed a high-throughput platform to screen for BK channel activators and identified a novel compound that can serve as a template for future drug-development for the treatment of these disabling disorders.
Purpose Up to 30% of patients with Brugada syndrome (BrS) carry loss-of-function (LoF) variants in the cardiac sodium channel gene SCN5A encoding for the protein NaV1.5. Recent studies suggested that NaV1.5 can dimerize, and some variants exert dominant negative effects. In this study, we sought to explore the generality of missense variant NaV1.5 dominant negative effects and their clinical severity. Results In heterozygous expression with WT, 32 of 35 LoF and 6 of 15 partial LoF variants showed reduction to 75% of WT-alone peak current, showing a dominant negative effect. Individuals with dominant negative LoF variants had an elevated disease burden compared with the individuals with putative haploinsufficient variants (2.7-fold enrichment in BrS cases, P = .019). Conclusion Most SCN5A missense LoF variants exert a dominant negative effect. This class of variant confers an especially high burden of BrS.
A major advancement has recently occurred in the ability to predict protein secondary structure from sequence using artificial neural networks. This new accessibility to high-quality predicted structures provides a big opportunity for the protein design community. It is particularly welcome for membrane protein design, where the scarcity of solved structures has been a major limitation of the field for decades. Here, we review the work done to date on the membrane protein design and set out established and emerging tools that can be used to most effectively exploit this new access to structures.
Cryptococcus neoformans and Cryptococcus gattii can cause fatal invasive infections, especially in immunocompromised patients. However, few antifungal drugs are available to help treat cryptococcosis. In this study, by compound library screening, we presented the first report of hit compound P163-0892, which had potent in vitro and in vivo antifungal activity against Cryptococcus spp. In vitro tests showed that P163-0892 was not cytotoxic and had highly selective and strong antifungal activities against Cryptococcus spp. with MIC values less than 1 μg/mL. Synergism of P163-0892 and fluconazole was also observed in vitro. The in vivo antifungal efficacy of P163-0892 was assessed in a wax moth larval fungal infection model, and treatment with 10 mg/kg P163-0892 caused a significant reduction in fungal burden and significant extension of the survival time. Taken together, our data indicate that the hit compound P163-0892 warrants further investigation as a novel anti-Cryptococcus agent.
α-bungarotoxin is a large, 74 amino acid toxin containing five disulphide bridges, initially identified in the venom of Bungarus multicinctus snake. Like most large toxins, chemical synthesis of α-bungarotoxin is challenging, explaining why all previous reports use purified or recombinant α-bungarotoxin. However, only chemical synthesis allows easy insertion of non-natural amino acids or new chemical functionalities. Herein, we describe a procedure for the chemical synthesis of a fluorescent-tagged α-bungarotoxin. The full-length peptide was designed to include an alkyne function at the amino-terminus through the addition of a pentynoic acid linker. Chemical synthesis of α-bungarotoxin requires hydrazide-based coupling of three peptide fragments in successive steps. After completion of the oxidative folding, an azide-modified Cy5 fluorophore was coupled by click chemistry onto the toxin. Next, we determined the efficacy of the fluorescent-tagged α-bungarotoxin to block acetylcholine (ACh)-mediated currents in response to muscle nicotinic receptor activation in TE671 cells. Using automated patch-clamp recordings, we demonstrate that fluorescent synthetic α-bungarotoxin has the expected nanomolar affinity for the nicotinic receptor. The blocking effect of fluorescent α-bungarotoxin could be displaced by incubation with a 20-mer peptide mimicking the α-bungarotoxin binding site. In addition, TE671 cells could be labelled with fluorescent toxin, as witnessed by confocal microscopy, and this labelling was partially displaced by the 20-mer competitive peptide. We thus demonstrate that synthetic fluorescent-tagged α-bungarotoxin preserves excellent properties for binding onto muscle nicotinic receptors.
Cholesterol is a major regulator of multiple types of ion channels. While there is increasing information about cholesterol binding sites, the molecular mechanisms through which cholesterol binding alters channel function are virtually unknown. In this study, we used a combination of Martini coarse-grained simulations, a network theory-based analysis, and electrophysiology to determine the effect of cholesterol on the dynamic structure of the Kir2.2 channel. We found that increasing membrane cholesterol reduced the likelihood of contact between specific regions of the cytoplasmic and transmembrane domains of the channel, most prominently at the subunit-subunit interfaces of the cytosolic domains. This decrease in contact was mediated by pairwise interactions of specific residues and correlated to the stoichiometry of cholesterol binding events. The predictions of the model were tested by site-directed mutagenesis of two identified residues, V265 and H222, and high throughput electrophysiology.
Sequencing Mendelian arrhythmia genes in individuals without an indication for arrhythmia genetic testing can identify carriers of pathogenic or likely pathogenic (P/LP) variants. However, the extent to which these variants are associated with clinically meaningful phenotypes before or after return of variant results is unclear. In addition, the majority of discovered variants are currently classified as variants of uncertain significance, limiting clinical actionability.
Channelrhodopsins (ChRs) are proteins that guide phototaxis in protists and exhibit light-gated channel conductance when their genes are heterologously expressed in mammalian cells. ChRs are widely used as molecular tools to control neurons and cardiomyocytes with light (optogenetics). Cation- and anion-selective ChRs (CCRs and ACRs, respectively) enable stimulation and inhibition of neuronal activity by depolarization and hyperpolarization of the membrane, respectively. More than 400 natural ChR variants have been identified so far, and high-throughput polynucleotide sequencing projects add many more each year. However, electrophysiological characterization of new ChRs lags behind because it is mostly done by time-consuming manual patch clamp (MPC). Here we report using a high-throughput automated patch clamp (APC) platform, SyncroPatch 384i from Nanion Technologies, for ChR research. We find that this instrument can be used for determination of the light intensity dependence and current-voltage relationships in ChRs and discuss its advantages and limitations.
As a branch of quantitative systems toxicology, in silico simulations are of growing attractiveness to guide preclinical cardiosafety risk assessments. Traditionally, a cascade of in vitro/in vivo assays has been applied in pharmaceutical research to screen out molecules at risk for cardiac side effects and prevent subsequent risk for patients. Drug cardiosafety assessments typically employ early mechanistic, hazard-oriented in silico/in vitro assays for compound inhibition of cardiac ion channels, followed by induced pluripotent stem cells (iPSCs) or tissue-based models such as the rabbit Purkinje fiber assay, which includes the major mechanisms contributing to action potential (AP) genesis. Additionally, multiscale simulation techniques based on mathematical models have become available, which are performed in silico ‘at the heart’ of compound triage to substitute Purkinje tests and increase translatability through mechanistic interpretability. To adhere to the 3R principle and reduce animal experiments, we performed a comparative benchmark and investigated a variety of mathematical cardiac AP models, including a newly developed minimalistic model specifically tailored to the AP of rabbit Purkinje cells, for their ability to substitute experiments. The simulated changes in AP duration (dAPD90) at increasing drug concentrations were compared to experimental results from 588 internal Purkinje fiber studies covering 555 different drugs with diverse modes of action. Using our minimalistic model, 80% of the Purkinje experiments could be quantitatively reproduced. This result allows for significant saving of experimental effort in early research and justifies the embedding of electrophysiological simulations into the DMTA (Design, Make, Test, Analyze) cycle in pharmaceutical compound optimization.
Over 95% of Polycythemia Vera (PV) patients carry the V617F mutation in the tyrosine kinase Janus kinase 2 (JAK2), resulting in uncontrolled erythroid proliferation and a high risk of thrombosis. Using mass spectrometry, we analyzed the RBC membrane proteome and showed elevated levels of multiple Ca2+ binding proteins as well as endoplasmic-reticulum-residing proteins in PV RBC membranes compared with RBC membranes from healthy individuals. In this study, we investigated the impact of JAK2V617F on (1) calcium homeostasis and RBC ion channel activity and (2) protein expression and sorting during terminal erythroid differentiation. Our data from automated patch-clamp show modified calcium homeostasis in PV RBCs and cell lines expressing JAK2V617F, with a functional impact on the activity of the Gárdos channel that could contribute to cellular dehydration. We show that JAK2V617F could play a role in organelle retention during the enucleation step of erythroid differentiation, resulting in modified whole cell proteome in reticulocytes and RBCs in PV patients. Given the central role that calcium plays in the regulation of signaling pathways, our study opens new perspectives to exploring the relationship between JAK2V617F, calcium homeostasis, and cellular abnormalities in myeloproliferative neoplasms, including cellular interactions in the bloodstream in relation to thrombotic events.
High throughput genomics has greatly facilitated identification of genetic variants. However, determining which variants contribute to disease causation is challenging with more than half of all missense variants now classified as variants of uncertain significance (VUS). A VUS leaves patients and their clinicians unable to utilize the variant information in clinical decision-making. In long QT syndrome type 2, KCNH2 channel function is directly associated with disease presentation. Therefore, functional phenotyping of KCNH2 variants can provide direct evidence to aid variant classification. Here, we investigated the expression of all codon variants in exon 2 of KCNH2 using a massively parallel trafficking assay and for a subset of 458 single nucleotide variants compared the results with peak tail current density and gating using automated patch clamp electrophysiology. Trafficking could correctly classify loss of peak tail current density variants with an AUC reaching 0.94 compared to AUCs of 0.75 to 0.8 for in silico variant classifiers. We suggest massively parallel trafficking assays can provide prospective and accurate functional assessment for all missense variants in KCNH2 and most likely many other ion channels and membrane proteins.
Crucial conventional patch-clamp approaches to investigate cellular electrophysiology suffer from low-throughput and require considerable experimenter expertise. Automated patch-clamp APC) approaches are more experimenter independent and offer high-throughput, but by design are predominantly limited to assays containing small, homogenous cells. In order to enable high-throughput APC assays on larger cells such as native cardiomyocytes isolated from mammalian hearts, we employed a fixed-well APC plate format. A broad range of We identified 35 LoF variants (10% of wild type [WT] peak current) and 15 partial LoF variants (10%-50% of WT peak current) that we assessed for dominant negative effects. SCN5A variants were studied in HEK293T cells, alone or in heterozygous coexpression with WT SCN5A using automated patch clamp. To assess the clinical risk, we compared the prevalence of dominant negative vs putative haploinsufficient (frameshift, splice, or nonsense) variants in a BrS consortium and the Genome Aggregation Database population database. detailed electrophysiological parameters including action potential, L-type calcium current and basal inward rectifier current were reliably acquired from isolated swine atrial and ventricular cardiomyocytes using APC. Effective pharmacological modulation also indicated that this technique is applicable for drug screening using native cardiomyocyte material. Furthermore, sequential acquisition of multiple parameters from a single cell was successful in a high throughput format, substantially increasing data richness and quantity per experimental run. When appropriately expanded, these protocols will provide a foundation for effective mechanistic and phenotyping studies of human cardiac electrophysiology. Utilizing scarce biopsy samples, regular high throughput characterization of primary cardiomyocytes using APC will facilitate drug development initiatives and personalized treatment strategies for a multitude of cardiac diseases.
Loss-of-function (LOF) variants in the KV11.1 potassium channel cause long QT syndrome (LQTS). Most variants disrupt intracellular channel transport (trafficking) to the cell membrane. Since some channel inhibitors improve trafficking of KV11.1 variants, a high-throughput screening (HTS) assay to detect trafficking enhancement would be valuable to the identification of drug candidates. The thallium (Tl+) flux assay technique, widely used for drug screening, was optimized using human embryonic kidney (HEK-293) cells expressing a trafficking-deficient KV11.1 variant in 384-well plates. Assay quality was assessed using Z prime (Z’) scores comparing vehicle to E-4031, a drug that increases KV11.1 membrane trafficking. The optimized assay was validated by immunoblot, electrophysiology experiments, and a pilot drug screen. The combination of: 1) truncating the trafficking-deficient variant KV11.1-G601S (KV11.1-G601S-G965*X) with the addition of 2) KV11.1 channel activator (VU0405601) and 3) cesium (Cs+) to the Tl+ flux assay buffer resulted in an outstanding Z’ of 0.83. To validate the optimized trafficking assay, we carried out a pilot screen that identified three drugs (ibutilide, azaperone, and azelastine) that increase KV11.1 trafficking. The new assay exhibited 100% sensitivity and specificity. Immunoblot and voltage-clamp experiments confirmed that all three drugs identified by the new assay improved membrane trafficking of two additional LQTS KV11.1 variants. We report two new ways to increase target-specific activity in trafficking assays—genetic modification and channel activation—that yielded a novel HTS assay for identifying drugs that improve membrane expression of pathogenic KV11.1 variants.
Many genes, including KCNH2, contain “hotspot” domains associated with a high density of variants associated with disease. This has led to the suggestion that variant location can be used as evidence supporting classification of clinical variants. However, it is not known what proportion of all potential variants in hotspot domains cause loss of function. Here, we have used a massively parallel trafficking assay to characterize all single-nucleotide variants in exon 2 of KCNH2, a known hotspot for variants that cause long QT syndrome type 2 and an increased risk of sudden cardiac death. Forty-two percent of KCNH2 exon 2 variants caused at least 50% reduction in protein trafficking, and 65% of these trafficking-defective variants exerted a dominant-negative effect when co-expressed with a WT KCNH2 allele as assessed using a calibrated patch-clamp electrophysiology assay. The massively parallel trafficking assay was more accurate (AUC of 0.94) than bioinformatic prediction tools (REVEL and CardioBoost, AUC of 0.81) in discriminating between functionally normal and abnormal variants. Interestingly, over half of variants in exon 2 were found to be functionally normal, suggesting a nuanced interpretation of variants in this “hotspot” domain is necessary. Our massively parallel trafficking assay can provide this information prospectively.
Neuropsychiatric disorders such as schizophrenia are challenging to treat due to the biological complexity of the disease and the lack of knowledge of the underlying pathophysiology. Whole exome and genome sequencing studies have identified disease-linked rare variants in patients with large effect size. Here, we functionally characterize the schizophrenia linked variant V1282F in SCN2A, encoding the sodium channel NaV1.2. This variant was introduced into isogenic lines of hiPSCs using CRISPR/CAS9 genome editing tools. hiPSCs were then differentiated into cortical neurons to understand how the variant and gene may be contributing to disease. We observed a significant (~25%) decrease in sodium current in the V1282F neurons compared to control neurons, suggesting the mutation is causing a loss-of-channel function. These results were supported by recordings in recombinant cells overexpressing either the mutant or wildtype NaV1.2, with the mutant channel having significantly (~75%) lower current amplitude than wildtype. We hypothesize that this phenotype may contribute to disease either through the direct loss of neuronal activity or through subsequent abnormal neurodevelopment.
Modern sequencing technologies have revolutionized our detection of gene variants. However, in most genes, including KCNH2, the majority of missense variants are currently classified as variants of uncertain significance (VUSs). The aim of this study was to investigate the utility of an automated patch-clamp assay for aiding clinical variant classification in KCNH2. The assay was designed according to recommendations proposed by the Clinical Genome Sequence Variant Interpretation Working Group. Thirty-one variants (17 pathogenic/likely pathogenic, 14 benign/likely benign) were classified internally as variant controls. They were heterozygously expressed in Flp-In HEK293 cells for assessing the effects of variants on current density and channel gating in order to determine the sensitivity and specificity of the assay. All 17 pathogenic variant controls had reduced current density, and 13 of 14 benign variant controls had normal current density, which enabled determination of normal and abnormal ranges for applying evidence of moderate or supporting strength for VUS reclassification. Inclusion of functional assay evidence enabled us to reclassify 6 out of 44 KCNH2 VUSs as likely pathogenic. The high-throughput patch-clamp assay can provide moderate-strength evidence for clinical interpretation of clinical KCNH2 variants and demonstrates the value of developing automated patch-clamp assays for functional characterization of ion channel gene variants.
The insecticide deltamethrin of the pyrethroid class mainly targets voltage-gated sodium channels (NaVs). Deltamethrin prolongs the opening of NaVs by slowing down fast inactivation and deactivation. Pyrethroids are supposedly safe for humans, however, they have also been linked to the gulf-war syndrome, a neuropathic pain condition that can develop following exposure to certain chemicals. Inherited neuropathic pain conditions have been linked to mutations in the NaV subtypes NaV1.7, NaV1.8, and NaV1.9. Here, we examined the effect of deltamethrin on the human isoforms NaV1.7, NaV1.8, and NaV1.9_C4 (chimera containing the C-terminus of rat NaV1.4) heterologously expressed in HEK293T and ND7/23 cells using whole-cell patch-clamp electrophysiology. For all three NaV subtypes, we observed increased persistent and tail currents that are typical for NaV channels modified by deltamethrin. The most surprising finding was an enhanced slow inactivation induced by deltamethrin in all three NaV subtypes. An enhanced slow inactivation is contrary to the prolonged opening caused by pyrethroids and has not been described for deltamethrin or any other pyrethroid before. Furthermore, we found that the fraction of deltamethrin-modified channels increased use-dependently. However, for NaV1.8, the use-dependent potentiation occurred only when the holding potential was increased to −90 mV, a potential at which the tail currents decay more slowly. This indicates that use-dependent modification is due to an accumulation of tail currents. In summary, our findings support a novel mechanism whereby deltamethrin enhances slow inactivation of voltage-gated sodium channels, which may, depending on the cellular resting membrane potential, reduce neuronal excitability and counteract the well-described pyrethroid effects on channel activation.
The ability to discriminate between different ionic species, termed ion selectivity, is a key feature of ion channels and forms the basis for their physiological function. Members of the degenerin/epithelial sodium channel (DEG/ENaC) superfamily of trimeric ion channels are typically sodium selective, but to a surprisingly variable degree. While acid-sensing ion channels (ASICs) are weakly sodium selective (sodium:potassium ratio ∼10:1), ENaCs show a remarkably high preference for sodium over potassium (>500:1). This discrepancy may be expected to originate from differences in the pore-lining second transmembrane segment (M2). However, these show a relatively high degree of sequence conservation between ASICs and ENaCs, and previous functional and structural studies could not unequivocally establish that differences in M2 alone can account for the disparate degrees of ion selectivity. By contrast, surprisingly little is known about the contributions of the first transmembrane segment (M1) and the preceding pre-M1 region. In this study, we used conventional and noncanonical amino acid–based mutagenesis in combination with a variety of electrophysiological approaches to show that the pre-M1 and M1 regions of mASIC1a channels are major determinants of ion selectivity. Mutational investigations of the corresponding regions in hENaC show that these regions contribute less to ion selectivity, despite affecting ion conductance. In conclusion, our work suggests that the remarkably different degrees of sodium selectivity in ASICs and ENaCs are achieved through different mechanisms. These results further highlight how M1 and pre-M1 are likely to differentially affect pore structure in these related channels.
Neurogranin (Ng), encoded by the schizophrenia risk gene NRGN, is a calmodulin-binding protein enriched in the postsynaptic compartments, and its expression is reduced in the postmortem brains of patients with schizophrenia. Experience-dependent translation of Ng is critical for encoding contextual memory, and Ng regulates developmental plasticity in the primary visual cortex during the critical period. However, the overall impact of Ng on the neuronal signaling that regulates synaptic plasticity is unknown.
Slowpoke (Slo) potassium channels display extraordinarily high conductance, are synergistically activated by a positive transmembrane potential and high intracellular Ca2+ concentrations and are important targets for insecticides and antiparasitic drugs. However, it is unknown how these compounds modulate ion translocation and whether there are insect-specific binding pockets. Here, we report structures of Drosophila Slo in the Ca2+-bound and Ca2+-free form and in complex with the fungal neurotoxin verruculogen and the anthelmintic drug emodepside. Whereas the architecture and gating mechanism of Slo channels are conserved, potential insect-specific binding pockets exist. Verruculogen inhibits K+ transport by blocking the Ca2+-induced activation signal and precludes K+ from entering the selectivity filter. Emodepside decreases the conductance by suboptimal K+ coordination and uncouples ion gating from Ca2+ and voltage sensing. Our results expand the mechanistic understanding of Slo regulation and lay the foundation for the rational design of regulators of Slo and other voltage-gated ion channels.
Human ether-à-go-go-related gene potassium channel 1 (hERG) is a voltage-gated potassium channel, the voltage-sensing domain (VSD) of which is targeted by a gating-modifier toxin, APETx1. APETx1 is a 42-residue peptide toxin of sea anemone Anthopleura elegantissima and inhibits hERG by stabilizing the resting state. A previous study that conducted cysteine-scanning analysis of hERG identified two residues in the S3-S4 region of the VSD that play important roles in hERG inhibition by APETx1. However, mutational analysis of APETx1 could not be conducted as only natural resources have been available until now. Therefore, it remains unclear where and how APETx1 interacts with the VSD in the resting state.
In a program to identify pain treatments with low addiction potential, we isolated five steroids, conosteroids A–E (1–5), from the hypobranchial gland of the mollusk Conus geographus. Compounds 1–5 were active in a mouse dorsal root ganglion (DRG) assay that suggested that they might be analgesic. A synthetic analogue 6 was used for a detailed pharmacological study. Compound 6 significantly increased the pain threshold in mice in the hot-plate test at 2 and 50 mg/kg. Compound 6 at 500 nM antagonizes type-A γ-aminobutyric acid receptors (GABAARs). In a patch-clamp experiment, out of the six subunit combinations tested, 6 exhibited subtype selectivity, most strongly antagonizing α1β1γ2 and α4β3γ2 receptors (IC50 1.5 and 1.0 μM, respectively). Although the structures of 1–6 differ from those of known neuroactive steroids, they are cell-type-selective modulators of GABAARs, expanding the known chemical space of neuroactive steroids.
The SCN5A R1623Q mutation is one of the most common genetic variants associated with severe congenital long QT syndrome 3 (LQT3) in fetal and neonatal patients. To investigate the properties of the R1623Q mutation, we established an induced pluripotent stem cell (iPSC) cardiomyocyte (CM) model from a patient with LQTS harboring a heterozygous R1623Q mutation. The properties and pharmacological responses of iPSC-CMs were characterized using a multi-electrode array system. The biophysical characteristic analysis revealed that R1623Q increased open probability and persistent currents of sodium channel, indicating a gain-of-function mutation. In the pharmacological study, mexiletine shortened FPDcF in R1623Q-iPSC-CMs, which exhibited prolonged field potential duration corrected by Fridericia’s formula (FPDcF, analogous to QTcF). Meanwhile, E4031, a specific inhibitor of human ether-a-go-go-related gene (hERG) channel, significantly increased the frequency of arrhythmia-like early after depolarization (EAD) events. These characteristics partly reflect the patient phenotypes. To further analyze the effect of neonatal isoform, which is predominantly expressed in the fetal period, on the R1623Q mutant properties, we transfected adult form and neonatal isoform SCN5A of control and R1623Q mutant SCN5A genes to 293T cells. Whole-cell automated patch-clamp recordings revealed that R1623Q increased persistent Na+ currents, indicating a gain-of-function mutation. Our findings demonstrate the utility of LQT3-associated R1623Q mutation-harboring iPSC-CMs for assessing pharmacological responses to therapeutic drugs and improving treatment efficacy. Furthermore, developmental switching of neonatal/adult NaV1.5 isoforms may be involved in the pathological mechanisms underlying severe long QT syndrome in fetuses and neonates.
Machine learning is widely used in drug development to predict activity in biological assays based on chemical structure. However, the process of transitioning from one experimental setup to another for the same biological endpoint has not been extensively studied. In a retrospective study, we here explore different modeling strategies of how to combine data from the old and new assays when training conformal prediction models using data from hERG and NaV assays. We suggest to continuously monitor the validity and efficiency of models as more data is accumulated from the new assay and select a modeling strategy based on these metrics. In order to maximize the utility of data from the old assay, we propose a strategy that augments the proper training set of an inductive conformal predictor by adding data from the old assay but only having data from the new assay in the calibration set, which results in valid (well-calibrated) models with improved efficiency compared to other strategies. We study the results for varying sizes of new and old assays, allowing for discussion of different practical scenarios. We also conclude that our proposed assay transition strategy is more beneficial, and the value of data from the new assay is higher, for the harder case of regression compared to classification problems.
Incorporation of noncanonical amino acids (ncAAs) can endow proteins with novel functionalities, such as crosslinking or fluorescence. In ion channels, the function of these variants can be studied with great precision using standard electrophysiology, but this approach is typically labor intensive and low throughput. Here, we establish a high-throughput protocol to conduct functional and pharmacological investigations of ncAA-containing human acid-sensing ion channel 1a (hASIC1a) variants in transiently transfected mammalian cells. We introduce 3 different photocrosslinking ncAAs into 103 positions and assess the function of the resulting 309 variants with automated patch clamp (APC). We demonstrate that the approach is efficient and versatile, as it is amenable to assessing even complex pharmacological modulation by peptides. The data show that the acidic pocket is a major determinant for current decay, and live-cell crosslinking provides insight into the hASIC1a–psalmotoxin 1 (PcTx1) interaction. Further, we provide evidence that the protocol can be applied to other ion channels, such as P2X2 and GluA2 receptors. We therefore anticipate the approach to enable future APC-based studies of ncAA-containing ion channels in mammalian cells.
Ion channels are attractive drug targets for many therapeutic applications. However, high-throughput screening (HTS) of drug candidates is difficult and remains very expensive. We thus assessed the suitability of the bioluminescence resonance energy transfer (BRET) technique as a new HTS method for ion-channel studies by taking advantage of our recently characterized intra- and intermolecular BRET probes targeting the transient receptor potential vanilloid type 1 (TRPV1) ion channel. These BRET probes monitor conformational changes during TRPV1 gating and subsequent coupling with calmodulin, two molecular events that are intractable using reference techniques such as automated calcium assay (ACA) and automated patch-clamp (APC). We screened the small-sized Prestwick chemical library, encompassing 1200 compounds with high structural diversity, using either intra- and intermolecular BRET probes or ACA. Secondary screening of the detected hits was done using APC. Multiparametric analysis of our results shed light on the capability of calmodulin inhibitors included in the Prestwick library to inhibit TRPV1 activation by capsaicin. BRET was the lead technique for this identification process. Finally, we present data exemplifying the use of intramolecular BRET probes to study other transient receptor potential (TRP) channels and non-TRPs ion channels. Knowing the ease of use of BRET biosensors and the low cost of the BRET technique, these assays may advantageously be included for extending ion-channel drug screening.
Transient receptor potential cation channel subfamily M member 5 (TRPM5) is a nonselective monovalent cation channel activated by intracellular Ca2+ increase. Within the gastrointestinal system, TRPM5 is expressed in the stoma, small intestine, and colon. In the search for a selective agonist of TRPM5 possessing in vivo gastrointestinal prokinetic activity, a high-throughput screening was performed and compound 1 was identified as a promising hit. Hit validation and hit to lead activities led to the discovery of a series of benzo[d]isothiazole derivatives. Among these, compounds 61 and 64 showed nanomolar activity and excellent selectivity (>100-fold) versus related cation channels. The in vivo drug metabolism and pharmacokinetic profile of compound 64 was found to be ideal for a compound acting locally at the intestinal level, with minimal absorption into systemic circulation. Compound 64 was tested in vivo in a mouse motility assay at 100 mg/kg, and demonstrated increased prokinetic activity.
KCNH2 is one of the 59 medically actionable genes recommended by the American College of Medical Genetics for reporting of incidental findings from clinical genomic sequencing. However, half of the reported KCNH2 variants in the ClinVar database are classified as variants of uncertain significance. In the absence of strong clinical phenotypes, there is a need for functional phenotyping to help decipher the significance of variants identified incidentally. Here, we report detailed methods for assessing the molecular phenotype of any KCNH2 missense variant. The key components of the assay include quick and cost-effective generation of a bicistronic vector to co-express WT and any KCNH2 variant allele, generation of stable Flp-In HEK293 cell lines and high-throughput automated patch-clamp electrophysiology analysis of channel function. Stable cell lines take 3-4 weeks to produce and can be generated in bulk, which will then allow up to 30 variants to be phenotyped per week after 48 hours of channel expression. This high throughput functional genomics assay will enable a much more rapid assessment of the extent of loss of function of any KCNH2 variant.
Protoxin II (ProTx II) is a high affinity gating modifier that is thought to selectively block the NaV1.7 voltage-dependent Na+ channel, a major therapeutic target for the control of pain. We aimed at producing ProTx II analogues entitled with novel functionalities for cell distribution studies and biochemical characterization of its NaV channel targets.
MAT2a is a methionine adenosyltransferase that synthesizes the essential metabolite S-adenosylmethionine (SAM) from methionine and ATP. Tumors bearing the co-deletion of p16 and MTAP genes have been shown to be sensitive to MAT2a inhibition, making it an attractive target for treatment of MTAP-deleted cancers. A fragment-based lead generation campaign identified weak but efficient hits binding in a known allosteric site. By use of structure-guided design and systematic SAR exploration, the hits were elaborated through a merging and growing strategy into an arylquinazolinone series of potent MAT2a inhibitors. The selected in vivo tool compound 28 reduced SAM-dependent methylation events in cells and inhibited proliferation of MTAP-null cells in vitro. In vivo studies showed that 28 was able to induce antitumor response in an MTAP knockout HCT116 xenograft model.
Gain-of-function (GOF) mutations in the KCNQ1 voltage-gated potassium channel can induce cardiac arrhythmia. Here it was tested whether any of the known human GOF disease mutations in KCNQ1 act by increasing the amount of KCNQ1 that reaches the cell surface—“super-trafficking”. Seven out of the 15 GOF mutants tested were seen to surface-traffic more efficiently than the wild type (WT) channel. Among these we found that levels of R231C KCNQ1 in the plasma membrane were 5-fold higher than the WT channel. This was shown to arise from the combined effects of enhanced efficiency of translocon-mediated membrane integration of the S4 voltage-sensor helix and from enhanced post-translational folding/trafficking that is related to energetic linkage of C231 with the V129 and F166 side chains. Whole-cell electrophysiology recordings confirmed that R231C KCNQ1 in complex with KCNE1 exhibits constitutive conductance, but also revealed that the single channel activity of this mutant is only 20% that of WT. The GOF phenotype associated with R231C therefore reflects the effects of super-trafficking and constitutive channel activation, which together offset reduced channel activity. These investigations show that membrane protein super-trafficking can contribute to human disease.
Mutations in KCNQ2, which encodes a pore-forming K+ channel subunit responsible for neuronal M-current, cause neonatal epileptic encephalopathy, a complex disorder presenting with severe early-onset seizures and impaired neurodevelopment. The condition is exceptionally difficult to treat, partially because the effects of KCNQ2 mutations on the development and function of human neurons are unknown. Here, we used induced pluripotent stem cells (iPSCs) and gene editing to establish a disease model and measured the functional properties of differentiated excitatory neurons. We find that patient iPSC-derived neurons exhibit faster action potential repolarization, larger post-burst afterhyperpolarization and a functional enhancement of Ca2+-activated K+ channels. These properties, which can be recapitulated by chronic inhibition of M-current in control neurons, facilitate a burst-suppression firing pattern that is reminiscent of the interictal electroencephalography pattern in patients. Our findings suggest that dyshomeostatic mechanisms compound KCNQ2 loss-of-function leading to alterations in the neurodevelopmental trajectory of patient iPSC-derived neurons.
The patch-clamp technique and more recently the high throughput patch-clamp technique have contributed to major advances in the characterization of ion channels. However, the whole-cell voltage-clamp technique presents certain limits that need to be considered for robust data generation. One major CaVeat is that increasing current amplitude profoundly impacts the accuracy of the biophysical analyses of macroscopic ion currents under study. Using mathematical kinetic models of a cardiac voltage-gated sodium channel and a cardiac voltage-gated potassium channel, we demonstrated how large current amplitude and series resistance artefacts induce an undetected alteration in the actual membrane potential and affect the characterization of voltage-dependent activation and inactivation processes. We also computed how dose–response curves are hindered by high current amplitudes. This is of high interest since stable cell lines frequently demonstrating high current amplitudes are used for safety pharmacology using the high throughput patch-clamp technique. It is therefore critical to set experimental limits for current amplitude recordings to prevent inaccuracy in the characterization of channel properties or drug activity, such limits being different from one channel type to another. Based on the predictions generated by the kinetic models, we draw simple guidelines for good practice of whole-cell voltage-clamp recordings.
The gene KCNT1 encodes the sodium-activated potassium channel KNa1.1 (Slack, Slo2.2). Variants in the KCNT1 gene induce a gain-of-function (GoF) phenotype in ionic currents and cause a spectrum of intractable neurological disorders in infants and children, including epilepsy of infancy with migrating focal seizures (EIMFS) and autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). Effective treatment options for KCNT1-related disease are absent, and novel therapies are urgently required. We describe the development of a novel class of oxadiazole KNa1.1 inhibitors, leading to the discovery of compound 31 that reduced seizures and interictal spikes in a mouse model of KCNT1 GoF.
The rising interest in KV7 modulators originates from their ability to evoke fundamental electrophysiological perturbations in a tissue-specific manner. A large number of therapeutic applications are, in part, based on the clinical experience with two broad-spectrum KV7 agonists, flupirtine and retigabine. Since precise molecular structures of human KV7 channel subtypes in closed and open states have only very recently started to emerge, computational studies have traditionally been used to analyze binding modes and direct the development of more potent and selective KV7 modulators with improved safety profiles. Herein, the synthetic and medicinal chemistry of small molecule modulators and the representative biological properties are summarized. Furthermore, new therapeutic applications supported by in vitro and in vivo assay data are suggested.
Cation and anion channelrhodopsins (CCRs and ACRs, respectively) primarily from two algal species, Chlamydomonas reinhardtii and Guillardia theta, have become widely used as optogenetic tools to control cell membrane potential with light. We mined algal and other protist polynucleotide sequencing projects and metagenomic samples to identify 75 channelrhodopsin homologs from three channelrhodopsin families, including one revealed in dinoflagellates in this study. We carried out electrophysiological analysis of 33 natural channelrhodopsin variants from different phylogenetic lineages and 10 metagenomic homologs in search of sequence determinants of ion selectivity, photocurrent desensitization, and spectral tuning in channelrhodopsins. Our results show that association of a reduced number of glutamates near the conductance path with anion selectivity depends on a wider protein context, because prasinophyte homologs with the identical glutamate pattern as in cryptophyte ACRs are cation-selective. Desensitization is also broadly context-dependent, as in one branch of stramenopile ACRs and their metagenomic homologs its extent roughly correlates with phylogenetic relationship of their sequences. Regarding spectral tuning, two prasinophyte CCRs exhibit red-shifted spectra to 585 nm, although their retinal-binding pockets do not match those of previously known similarly red-shifted channelrhodopsins. In cryptophyte ACRs we identified three specific residue positions in the retinal-binding pocket that define the wavelength of their spectral maxima. Lastly, we found that dinoflagellate rhodopsins with a TCP motif in the third transmembrane helix and a metagenomic homolog exhibit channel activity.
Voltage-gated sodium channels (NaVs) are promising targets for analgesic and antiepileptic therapies. Although specificity between NaV subtypes may be desirable to target specific neural types, such as nociceptors in pain, many broadly acting NaV inhibitors are clinically beneficial in neuropathic pain and epilepsy. Here, we present the first systematic characterization of vixotrigine, a NaV blocker. Using recombinant systems, we find that vixotrigine potency is enhanced in a voltage- and use-dependent manner, consistent with a state-dependent block of NaVs. Furthermore, we find that vixotrigine potently inhibits sodium currents produced by both peripheral and central nervous system NaV subtypes, with use-dependent IC50 values between 1.76 and 5.12 μM. Compared with carbamazepine, vixotrigine shows higher potency and more profound state-dependent inhibition but a similar broad spectrum of action distinct from NaV1.7- and NaV1.8-specific blockers. We find that vixotrigine rapidly inhibits NaVs and prolongs recovery from the fast-inactivated state. In native rodent dorsal root ganglion sodium channels, we find that vixotrigine shifts steady-state inactivation curves. Based on these results, we conclude that vixotrigine is a broad-spectrum, state-dependent NaV blocker.
We applied a set of in silico and in vitro assays, compliant with the CiPA (Comprehensive In Vitro Proarrhythmia Assay) paradigm, to assess the risk of chloroquine or hydroxychloroquine‐mediated QT prolongation and Torsades de Pointes (TdP), alone and combined with erythromycin and azithromycin, drugs repurposed during the first wave of COVID‐19. Each drug or drug combination was tested in patch clamp assays on 7 cardiac ion channels, in in silico models of human ventricular electrophysiology (Virtual Assay®) using control (healthy) or high‐risk cell populations, and in human induced pluripotent stem cell (hiPSC)‐derived cardiomyocytes. In each assay, concentration‐response curves encompassing and exceeding therapeutic free plasma levels were generated. Both chloroquine and hydroxychloroquine showed blocking activity against some potassium, sodium and calcium currents. Chloroquine and hydroxychloroquine inhibited IKr (IC50: 1µM and 3‐7 µM, respectively) and IK1 currents (IC50: 5 and 44 µM, respectively). When combining hydroxychloroquine with azithromycin, no synergistic effects were observed. The two macrolides had no or very weak effects on the ion currents (IC50 > 300 ‐ 1000µM). Using Virtual Assay®, both antimalarials affected several TdP indicators, chloroquine being more potent than hydroxychloroquine. Effects were more pronounced in the high‐risk cell population. In hiPSC‐derived cardiomyocytes, all drugs showed early‐after‐depolarizations, except azithromycin. Combining chloroquine or hydroxychloroquine with a macrolide did not aggravate their effects. In conclusion, our integrated nonclinical CiPA dataset confirmed that, at therapeutic plasma concentrations relevant for malaria or off‐label use in COVID‐19, chloroquine and hydroxychloroquine use is associated with a proarrhythmia risk, which is higher in populations carrying predisposing factors but not worsened with macrolide combination.
Protamine sulfate (PS) is the only available option to reverse the anticoagulant activity of unfractionated heparin (UFH), however it can cause cardiovascular and respiratory complications. We explored the toxicity of PS and its complexes with UFH in zebrafish, rats, and mice. The involvement of nitric oxide (NO) in the above effects was investigated. Concentration–dependent lethality, morphological defects, and decrease in heart rate (HR) were observed in zebrafish larvae. PS affected HR, blood pressure, respiratory rate, peak exhaled CO2, and blood oxygen saturation in rats. We observed hypotension, increase of HR, perfusion of paw vessels, and enhanced respiratory disturbances with increases doses of PS. We found no effects of PS on human hERG channels or signs of heart damage in mice. The hypotension in rats and bradycardia in zebrafish were partially attenuated by the inhibitor of endothelial NO synthase. The disturbances in cardiovascular and respiratory parameters were reduced or delayed when PS was administered together with UFH. The cardiorespiratory toxicity of PS seems to be charge–dependent and involves enhanced release of NO. PS administered at appropriate doses and ratios with UFH should not cause permanent damage of heart tissue, although careful monitoring of cardiorespiratory parameters is necessary.
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. Historically, the effects of compounds onion channel biophysical properties and voltage-dependent activation/inactivation could only be assessed using low-throughput, manual patch clamp recording techniques. In recent years, automated patch clamp (APC) platforms have drastically increased throughput. In contrast to their broad utilization in compound screening, APC platforms have rarely been used for mechanism of action studies, in large part due to the lack of sophisticated, scalable analysis methods to process the large amount of data generated by APC platforms. In the current study, we developed a highly efficient and scalable software workflow to overcome this challenge. This method, to our knowledge the first of its kind, enables automated curve fitting and complex analysis of compound effects. Using voltage-gated sodium channels as an example, we were able to immediately assess the effects of test compounds on a spectrum of biophysical properties, including peak current, voltage-dependent steady state activation/inactivation, and time constants of activation and fast inactivation. Overall, this automated data analysis method provides a novel solution for in-depth analysis of large-scale APC data, and thus will significantly impact ion channel research and drug discovery.
CACNA1I is implicated in the susceptibility to schizophrenia by large-scale genetic association studies of single nucleotide polymorphisms. However, the channelopathy of CACNA1I in schizophrenia is unknown. CACNA1I encodes CaV3.3, a neuronal voltage-gated calcium channel that underlies a subtype of T-type current that is important for neuronal excitability in the thalamic reticular nucleus (TRN) and other regions of the brain. Here, we present an extensive functional characterization of 57 naturally occurring rare and common missense variants of CACNA1I derived from a Swedish schizophrenia cohort of more than 10,000 individuals. Our analysis of this allelic series of coding CACNA1I variants revealed that reduced CaV3.3 channel current density was the dominant phenotype associated with rare CACNA1I coding alleles derived from control subjects, whereas rare CACNA1I alleles from schizophrenia patients encoded CaV3.3 channels with altered responses to voltages. CACNA1I variants associated with altered current density primarily impact the ionic channel pore, and those associated with altered responses to voltage impact the voltage-sensing domain. CaV3.3 variants associated with altered voltage dependence of the CaV3.3 channel and those associated with peak current density deficits were significantly segregated across affected and unaffected groups (Fisher’s exact test, P = 0.034). Our results, together with recent data from the SCHEMA (Schizophrenia Exome Sequencing Meta-analysis) cohort, suggest that reduced CaV3.3 function may protect against schizophrenia risk in rare cases. We subsequently modeled the effect of the biophysical properties of CaV3.3 channel variants on TRN excitability and found that compared with common variants, ultrarare CaV3.3 coding variants derived from control subjects significantly decreased TRN excitability (P = 0.011). When all rare variants were analyzed, there was a nonsignificant trend between variants that reduced TRN excitability and variants that either had no effect or increased TRN excitability across disease status. Taken together, the results of our functional analysis of an allelic series of >50 CACNA1I variants in a schizophrenia cohort reveal that loss of function of CaV3.3 is a molecular phenotype associated with reduced disease risk burden, and our approach may serve as a template strategy for channelopathies in polygenic disorders.
Malignant migrating partial seizures of infancy is a rare, devastating form of epilepsy most commonly associated with gain-of-function mutations in the potassium channel, Slack. Not only is this condition almost completely pharmacoresistant, there are not even selective drug-like tools available to evaluate whether inhibition of these overactivated, mutant Slack channels may represent a viable path forward toward new antiepileptic therapies. Therefore, we used a highthroughput thallium flux assay to screen a drug-like, 100 000-compound library in search of inhibitors of both wild-type and a disease-associated mutant Slack channel. Using this approach, we discovered VU0606170, a selective Slack channel inhibitor with low micromolar potency. Critically, VU0606170 also proved effective at significantly decreasing the firing rate in overexcited, spontaneously firing cortical neuron cultures. Taken together, our data provide compelling evidence that selective inhibition of Slack channel activity can be achieved with small molecules and that inhibition of Slack channel activity in neurons produces efficacy consistent with an antiepileptic effect. Thus, the identification of VU0606170 provides a much-needed tool for advancing our understanding of the role of the Slack channel in normal physiology and disease as well as its potential as a target for therapeutic intervention.
The novel coroNaVirus, SARS-CoV-2, has been identified as the causative agent for the current coroNaVirus disease (COVID-19) pandemic. 3CL protease (3CLpro) plays a pivotal role in the processing of viral polyproteins. We report peptidomimetic compounds with a unique benzothiazolyl ketone as a warhead group, which display potent activity against SARS-CoV-2 3CLpro. The most potent inhibitor YH-53 can strongly block the SARS-CoV-2 replication. X-ray structural analysis revealed that YH-53 establishes multiple hydrogen bond interactions with backbone amino acids and a covalent bond with the active site of 3CLpro. Further results from computational and experimental studies, including an in vitro absorption, distribution, metabolism, and excretion profile, in vivo pharmacokinetics, and metabolic analysis of YH-53 suggest that it has a high potential as a lead candidate to compete with COVID-19.
In the recent years, the progress in genetic analysis and next-generation sequencing technologies have opened up exciting landscapes for diagnosis and study of molecular mechanisms, allowing the determination of a particular mutation for individual patients suffering from hereditary red blood cell-related diseases or anemia. However, the huge amount of data obtained makes the interpretation of the results and the identification of the pathogenetic variant responsible for the diseases sometime difficult. Moreover, there is increasing evidence that the same mutation can result in varying cellular properties and different symptoms of the disease. Even for the same patient, the phenotypic expression of the disorder can change over time. Therefore, on top of genetic analysis, there is a further request for functional tests that allow to confirm the pathogenicity of a molecular variant, possibly to predict prognosis and complications (e.g., vaso-occlusive pain crises or other thrombotic events) and, in the best case, to enable personalized theranostics (drug and/or dose) according to the disease state and progression. The mini-review will reflect recent and future directions in the development of diagnostic tools for red blood cell-related diseases and anemias. This includes point of care devices, new incarnations of well-known principles addressing physico-chemical properties, and interactions of red blood cells as well as high-tech screening equipment and mobile laboratories.
There is an increasing expectation that computational approaches may supplement existing human decision-making. Frontloading of models for cardiac safety prediction is no exception to this trend, and ongoing regulatory initiatives propose use of high-throughput in vitro data combined with computational models for calculating proarrhythmic risk. Evaluation of these models requires robust assessment of the outcomes. Using FDA Adverse Event Reporting System reports and electronic healthcare claims data from the Truven-MarketScan US claims database, we quantify the incidence rate of arrhythmia in patients and how this changes depending on patient characteristics. First, we propose that such datasets are a complementary resource for determining relative drug risk and assessing the performance of cardiac safety models for regulatory use. Second, the results suggest important determinants for appropriate stratification of patients and evaluation of additional drug risk in prescribing and clinical support algorithms and for precision health.
Malfunctions of voltage-gated sodium and calcium channels (SCN and CACNA1 genes) have been associated with severe neurologic, psychiatric, cardiac and other diseases. Altered channel activity is frequently grouped into gain or loss of ion channel function (GOF or LOF, respectively) which is not only corresponding to clinical disease manifestations, but also to differences in drug response. Experimental studies of channel function are therefore important, but laborious and usually focus only on a few variants at a time. Based on known gene-disease-mechanisms, we here infer LOF (518 variants) and GOF (309 variants) of likely pathogenic variants from disease phenotypes of variant carriers. We show regional clustering of inferred GOF and LOF variants, respectively, across the alignment of the entire gene family, suggesting shared pathomechanisms in the SCN/CACNA1 genes. By training a machine learning model on sequence- and structure-based features we predict LOF- or GOF- associated disease phenotypes (ROC = 0.85) of likely pathogenic missense variants. We then successfully validate the GOF versus LOF prediction on 87 functionally tested variants in SCN1/2/8A and CACNA1I (ROC = 0.73) and in exome-wide data from > 100.000 cases and controls. Ultimately, functional prediction of missense variants in clinically relevant genes will facilitate precision medicine in clinical practice.
Levodopa-induced dyskinesia (LID) poses a significant health care challenge for Parkinson's disease (PD) patients. Amantadine is currently the only drug proven to alleviate LID. Although its efficacy in treating LID is widely assumed to be mediated by blockade of N-methyl-D-aspartate (NMDA) glutamate receptors, our experiments demonstrate that at therapeutically relevant concentrations, amantadine preferentially blocks inward-rectifying K+ channel type 2 (Kir2) channels in striatal spiny projection neurons (SPNs) - not NMDA receptors. In so doing, amantadine enhances dendritic integration of excitatory synaptic potentials in SPNs and enhances - not antagonizes - the induction of long-term potentiation (LTP) at excitatory, axospinous synapses. Taken together, our studies suggest that the alleviation of LID in PD patients is mediated by diminishing the disparity in the excitability of direct- and indirect-pathway SPNs in the on state, rather than by disrupting LTP induction. This insight points to a pharmacological approach that could be used to effectively ameliorate LID and improve the quality of life for PD patients.
Acid-sensing ion channels (ASICs) are proton-gated cation channels that contribute to synaptic plasticity, as well as initiation of pain and neuronal death following ischemic stroke. As such, there is a great interest in understanding the in vivo regulation of ASICs, especially by endogenous neuropeptides that potently modulate ASICs. The most potent endogenous ASIC modulator known to date is the opioid neuropeptide big dynorphin (BigDyn). BigDyn is upregulated in chronic pain and increases ASIC-mediated neuronal death during acidosis. Understanding the mechanism and site of action of BigDyn on ASICs could thus enable the rational design of compounds potentially useful in the treatment of pain and ischemic stroke. To this end, we employ a combination of electrophysiology, voltage-clamp fluorometry, synthetic BigDyn analogs and non-canonical amino acid-mediated photocrosslinking. We demonstrate that BigDyn binding induces ASIC1a conformational changes that are different from those induced by protonation and likely represent a distinct closed state. Using alanine-substituted BigDyn analogs, we find that the BigDyn modulation of ASIC1a is mediated through electrostatic interactions of basic amino acids in the BigDyn N-terminus. Furthermore, neutralizing acidic amino acids in the ASIC1a extracellular domain reduces BigDyn effects, suggesting a binding site at the acidic pocket. This is confirmed by photocrosslinking using the non-canonical amino acid azido-phenylalanine. Overall, our data define the mechanism of how BigDyn modulates ASIC1a, identify the acidic pocket as the binding site for BigDyn and thus highlight this CaVity as an important site for the development of ASIC-targeting therapeutics. Significance: Neuropeptides such as big dynorphin (BigDyn) play important roles in the slow modulation of fast neurotransmission, which is mediated by membrane-embedded receptors. In fact, BigDyn is the most potent known endogenous modulator of one such receptor, the acid-sensing ion channel (ASIC), but the mode of action remains unknown. In this work, we employ a broad array of technologies to unravel the details of where big dynorphin binds to ASIC and how it modulates its activity. As both BigDyn and ASIC are implicated in pain pathways, this work might pave the way toward future analgesics.
The clinical administration of GABAergic medications leads to hypotension which has classically been attributed to the modulation of neuronal activity in the central and peripheral nervous systems. However, certain types of peripheral smooth muscle cells have been shown to express GABAA receptors, which modulate smooth muscle tone, by the activation of these chloride channels on smooth muscle cell plasma membranes. Limited prior studies demonstrate that non-human large-caliber capacitance blood vessels mounted on a wire myograph are responsive to GABAA ligands. We questioned whether GABAA receptors are expressed in human resistance arteries and whether they modulate myogenic tone. We demonstrate the novel expression of GABAA subunits on vascular smooth muscle from small-caliber human omental and mouse tail resistance arteries. We show that GABAA receptors modulate both plasma membrane potential and calcium responses in primary cultured cells from human resistance arteries. Lastly, we demonstrate functional physiologic modulation of myogenic tone via GABAA receptor activation in human and mouse arteries. Together, these studies demonstrate a previously unrecognized role for GABAA receptors in the modulation of myogenic tone in mouse and human resistance arteries.
In this issue of SLAS Discovery, we present a special collection of manuscripts, including three original research papers and one review, that reflect recent advances and continuing challenges in the development and application of assay technologies to drug discovery for ion channel targets. First, though, we provide our perspectives on the specific challenges and opportunities in this field.
Rationale: Partial or complete loss of function variants in SCN5A are the most common genetic cause of the arrhythmia disorder Brugada Syndrome (BrS1). However, the pathogenicity of SCN5A variants is often unknown or disputed; 80% of the 1,390 SCN5A missense variants observed in at least one individual to date are variants of uncertain significance (VUS). The designation of VUS is a barrier to the use of sequence data in clinical care. Objective: We selected 83 variants for study: 10 previously studied control variants, 10 suspected benign variants, and 63 suspected Brugada Syndrome-associated variants, selected on the basis of their frequency in the general population and in patients with Brugada Syndrome. We used high-throughput automated patch clamping to study the function of the 83 variants, with the goal of reclassifying variants with functional data. Methods and Results: Ten previously studied variants had functional properties concordant with published manual patch clamp data. All 10 suspected benign variants had wildtype-like function. 22 suspected BrS variants had loss of channel function (10% normalized peak current) and 23 variants had partial loss of function (10-50% normalized peak current). The 73 previously unstudied variants were initially classified as likely benign (n=2), likely pathogenic (n=11), or VUS (n=60). After the patch clamp studies, 16 variants were benign/likely benign, 47 were pathogenic/likely pathogenic, and only 10 were still VUS. 8/22 loss of function variants were partially rescuable by incubation at lower temperature or pretreatment with a sodium channel blocker. Structural modeling identified likely mechanisms for loss of function including altered thermostability, and disruptions to alpha helices, disulfide bonds, or the permeation pore. Conclusions: High-throughput automated patch clamping enabled the reclassification of the majority of tested VUS’s in SCN5A.
GS-967 and eleclazine (GS-6615) are novel sodium channel inhibitors exhibiting antiarrhythmic effects in various in vitro and in vivo models. The antiarrhythmic mechanism has been attributed to preferential suppression of late sodium current (INaL). Here, we took advantage of a throughput automated electrophysiology platform (SyncroPatch 768PE) to investigate the molecular pharmacology of GS-967 and eleclazine on peak sodium current (INaP) recorded from human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. We compared GS-967 and eleclazine to the antiarrhythmic drug lidocaine, the prototype INaL inhibitor ranolazine, and the slow inactivation enhancing drug lacosamide. In human induced pluripotent stem cell-derived cardiomyocytes, GS-967 and eleclazine caused a reduction of INaP in a frequency-dependent manner consistent with use-dependent block (UDB). GS-967 and eleclazine had similar efficacy but evoked more potent UDB of INaP (IC50=0.07 and 0.6 μM, respectively) than ranolazine (7.8 μM), lidocaine (133.5 μM) and lacosamide (158.5 μM). In addition, GS-967 and eleclazine exerted more potent effects on slow inactivation and recovery from inactivation compared to the other sodium channel blocking drugs we tested. The greater UDB potency of GS-967 and eleclazine was attributed to the significantly higher association rates (KON) and moderate unbinding rate (KOFF) of these two compounds with sodium channels. We propose that substantial UDB contributes to the observed antiarrhythmic efficacy of GS-967 and eleclazine. Significance statement:We investigated the molecular pharmacology of GS-967 and eleclazine on sodium channels in human induced pluripotent stem cell derived cardiomyocytes using a high throughput automated electrophysiology platform. Sodium channel inhibition by GS-967 and eleclazine has unique features including accelerating the onset of slow inactivation and impairing recovery from inactivation. These effects combined with rapid binding and moderate unbinding kinetics explain potent use-dependent block, which we propose contributes to their observed antiarrhythmic efficacy.
Background: KCHN2 encodes the KV11.1 potassium channel responsible for IKr, a major repolarization current during the cardiomyocyte action potential. Variants in KCNH2 that decrease IKr can cause Type 2 Long QT syndrome, usually due to mistrafficking to the cell surface. Accurately discriminating between variants with normal and abnormal trafficking would help clinicians identify and treat individuals at risk of a major cardiac event. The volume of reported non-synonymous KCNH2 variants preclude the use of conventional electrophysiologic methods for functional study. Objective: To report a high-throughput, multiplexed screening method for KCNH2 genetic variants capable of measuring the cell surface abundance of hundreds of missense variants in KCNH2. Methods: We develop a method to quantitate KCNH2 variant trafficking on a pilot region of 11 residues in the S5 helix, and generate trafficking scores for 220/231 missense variants in this region. Results: For 5/5 variants, high-throughput trafficking scores validated when tested in single variant flow cytometry and confocal microscopy experiments. We additionally compare our results with planar patch electrophysiology and find that loss-of-trafficking variants do not produce IKr, but that some variants which traffic normally may still be functionally compromised. Conclusions: Here, we describe a new method for detecting trafficking-deficient variants in KCNH2 in a multiplexed assay. This new method accurately generates trafficking data for variants in KCNH2 and can be readily extended to all residues in KV11.1 and to other cell surface proteins. Clinical Implications: Hundreds of KCNH2 variants have been observed to date, and thousands more will be found as clinical and population sequencing efforts become increasingly widespread. The major mechanism of KV11.1 loss of function is misfolding and failure to traffic to the cell surface. Deep mutational scanning of KCNH2 trafficking is a scalable, high-throughput method that can help identify new loss of function variants and decipher the large number of KCNH2 variants being found in the population.
Voltage-gated Na+ (NaV) channels regulate homeostasis in bacteria and control membrane electrical excitability in mammals. Compared to their mammalian counterparts, bacterial NaV channels possess a simpler, fourfold symmetric structure and have facilitated studies of the structural basis of channel gating. However, the pharmacology of bacterial NaV remains largely unexplored. Here we systematically screened 39 NaV modulators on a bacterial channel (NaChBac) and characterized a selection of compounds on NaChBac and a mammalian channel (human NaV1.7). We found that while many compounds interact with both channels, they exhibit distinct functional effects. For example, the local anesthetics ambroxol and lidocaine block both NaV1.7 and NaChBac but affect activation and inactivation of the two channels to different extents. The voltage-sensing domain targeting toxin BDS-I increases NaV1.7 but decreases NaChBac peak currents. The pore binding toxins aconitine and veratridine block peak currents of NaV1.7 and shift activation (aconitine) and inactivation (veratridine) respectively. In NaChBac, they block the peak current by binding to the pore residue F224. Nonetheless, aconitine has no effect on activation or inactivation, while veratridine only modulates activation of NaChBac. The conservation and divergence in the pharmacology of bacterial and mammalian NaV channels provide insights into the molecular basis of channel gating and will facilitate organism-specific drug discovery.
Herein we report the optimization of a series of tricyclic indazoles as selective estrogen receptor degraders (SERD) and antagonists for the treatment of ER+ breast cancer. Structure based design together with systematic investigation of each region of the molecular architecture led to the identification of N-[1-(3-fluoropropyl)azetidin-3-yl]-6-[(6S,8R)-8-methyl-7-(2,2,2-trifluoroethyl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinolin-6-yl]pyridin-3-amine (28). This compound was demonstrated to be a highly potent SERD that showed a pharmacological profile comparable to fulvestrant in its ability to degrade ERα in both MCF-7 and CAMA-1 cell lines. A stringent control of lipophilicity ensured that 28 had favorable physicochemical and preclinical pharmacokinetic properties for oral administration. This, combined with demonstration of potent in vivo activity in mouse xenograft models, resulted in progression of this compound, also known as AZD9833, into clinical trials.
The function of the voltage-gated KCNQ1 potassium channel is regulated by co-assembly with KCNE auxiliary subunits. KCNQ1-KCNE1 channels generate the slow delayed rectifier current, IKs, which contributes to the repolarization phase of the cardiac action potential. A three amino acid motif (F57-T58-L59, FTL) in KCNE1 is essential for slow activation of KCNQ1-KCNE1 channels. However, how this motif interacts with KCNQ1 to control its function is unknown. Combining computational modeling with electrophysiological studies, we developed structural models of the KCNQ1-KCNE1 complex that suggest how KCNE1 controls KCNQ1 activation. The FTL motif binds at a cleft between the voltage-sensing and pore domains and appears to affect the channel gate by an allosteric mechanism. Comparison with the KCNQ1-KCNE3 channel structure suggests a common transmembrane-binding mode for different KCNEs and illuminates how specific differences in the interaction of their triplet motifs determine the profound differences in KCNQ1 functional modulation by KCNE1 versus KCNE3.
Conventionally, manual patch-clamp electrophysiological approaches are the gold standard for studying ion channel function in neurons. However, these approaches are labor-intensive, yielding low-throughput results, and are therefore not amenable for compound profiling efforts during the early stages of drug discovery. The SyncroPatch 384PE has been successfully implemented for pharmacological experiments in heterologous overexpression systems that may not reproduce the function of voltage-gated ion channels in a native, heterogeneous environment. Here, we describe a protocol allowing the characterization of endogenous voltage-gated potassium (KV) and sodium (NaV) channel function in developing primary rat cortical cultures, allowing investigations at a significantly improved throughput compared with manual approaches. Key neuronal marker expression and microelectrode array recordings of electrophysiological activity over time correlated well with neuronal maturation. Gene expression data revealed high molecular diversity in KV and NaV subunit composition throughout development. Voltage-clamp experiments elicited three major current components composed of inward and outward conductances. Further pharmacological experiments confirmed the endogenous expression of functional KV and NaV channels in primary cortical neurons. The major advantages of this approach compared with conventional manual patch-clamp systems include unprecedented improvements in experimental ease and throughput for ion channel research in primary neurons. These efforts demonstrated feasibility for primary neuronal ion channel investigation with the SyncroPatch, providing the foundation for future studies characterizing biophysical changes in endogenous ion channels in primary systems associated with disease or development.
In response to the ongoing shift of the regulatory cardiac safety paradigm, a recent White Paper proposed general principles for developing and implementing proarrhythmia risk prediction models. These principles included development strategies to validate models, and implementation strategies to ensure a model developed by one lab can be used by other labs in a consistent manner in the presence of lab-to-lab experimental variability. While the development strategies were illustrated through the validation of the model under the Comprehensive In vitro Proarrhythmia Assay (CiPA), the implementation strategies have not been adopted yet.
Mathematical models of ion channels, which constitute indispensable components of action potential models, are commonly constructed by fitting to whole-cell patch-clamp data. In a previous study, we fitted cell-specific models to hERG1a (KV11.1) recordings simultaneously measured using an automated high-throughput system, and studied cell-cell variability by inspecting the resulting model parameters. However, the origin of the observed variability was not identified. Here, we study the source of variability by constructing a model that describes not just ion current dynamics, but the entire voltage-clamp experiment. The experimental artefact components of the model include: series resistance, membrane and pipette capacitance, voltage offsets, imperfect compensations made by the amplifier for these phenomena, and leak current. In this model, variability in the observations can be explained by either cell properties, measurement artefacts, or both. Remarkably, by assuming that variability arises exclusively from measurement artefacts, it is possible to explain a larger amount of the observed variability than when assuming cell-specific ion current kinetics. This assumption also leads to a smaller number of model parameters. This result suggests that most of the observed variability in patch-clamp data measured under the same conditions is caused by experimental artefacts, and hence can be compensated for in post-processing by using our model for the patch-clamp experiment. This study has implications for the question of the extent to which cell-cell variability in ion channel kinetics exists, and opens up routes for better correction of artefacts in patch-clamp data.
Using structure- and ligand-based design principles, a novel series of piperidyl chromane arylsulfonamide NaV1.7 inhibitors was discovered. Early optimization focused on improvement of potency through refinement of the low energy ligand conformation and mitigation of high in vivo clearance. An in vitro hepatotoxicity hazard was identified and resolved through optimization of lipophilicity and lipophilic ligand efficiency to arrive at GNE-616 (24), a highly potent, metabolically stable, subtype selective inhibitor of NaV1.7. Compound 24 showed a robust PK/PD response in a NaV1.7-dependent mouse model, and site-directed mutagenesis was used to identify residues critical for the isoform selectivity profile of 24.
The scorpion toxin AmmTx3 is a specific blocker of KV4 channels. It was shown to have interesting potential for neurological disorders. In this study, we report the first chemical synthesis of AmmTx3 by using the native chemical ligation strategy and validate its biological activity. We determined its 3D structure by nuclear magnetic resonance spectroscopy, and pointed out that AmmTx3 possesses the well-known CSαβ structural motif, which is found in a large number of scorpion toxins. Overall, this study establishes an easy synthetic access to biologically active AmmTx3 toxin.
Objective Pathogenic variants in KCNB1, encoding the voltage‐gated potassium channel KV2.1, are associated with developmental and epileptic encephalopathy (DEE). Previous functional studies on a limited number of KCNB1 variants indicated a range of molecular mechanisms by which variants affect channel function, including loss of voltage sensitivity, loss of ion selectivity, and reduced cell‐surface expression.Methods We evaluated a series of 17 KCNB1 variants associated with DEE or other neurodevelopmental disorders (NDDs) to rapidly ascertain channel dysfunction using high‐throughput functional assays. Specifically, we investigated the biophysical properties and cell‐surface expression of variant KV2.1 channels expressed in heterologous cells using high‐throughput automated electrophysiology and immunocytochemistry–flow cytometry.Results Pathogenic variants exhibited diverse functional defects, including altered current density and shifts in the voltage dependence of activation and/or inactivation, as homotetramers or when coexpressed with wild‐type KV2.1. Quantification of protein expression also identified variants with reduced total KV2.1 expression or deficient cell‐surface expression. Interpretation Our study establishes a platform for rapid screening of KV2.1 functional defects caused by KCNB1 variants associated with DEE and other NDDs. This will aid in establishing KCNB1 variant pathogenicity and the mechanism of dysfunction, which will enable targeted strategies for therapeutic intervention based on molecular phenotype.
Voltage-gated sodium (NaV) channels are targets of disease mutations, toxins, and therapeutic drugs. Despite recent advances, the structural basis of voltage sensing, electromechanical coupling, and toxin modulation remains ill-defined. Protoxin-II (ProTx2) from the Peruvian green velvet tarantula is an inhibitor cystine-knot peptide and selective antagonist of the human NaV1.7 channel. Here, we visualize ProTx2 in complex with voltage-sensor domain II (VSD2) from NaV1.7 using X-ray crystallography and cryoelectron microscopy. Membrane partitioning orients ProTx2 for unfettered access to VSD2, where ProTx2 interrogates distinct features of the NaV1.7 receptor site. ProTx2 positions two basic residues into the extracellular vestibule to antagonize S4 gating-charge movement through an electrostatic mechanism. ProTx2 has trapped activated and deactivated states of VSD2, revealing a remarkable ∼10 Å translation of the S4 helix, providing a structural framework for activation gating in voltage-gated ion channels. Finally, our results deliver key templates to design selective NaV channel antagonists.
Predicting how pharmaceuticals may affect heart rhythm is a crucial step in drug-development, and requires a deep understanding of a compound’s action on ion channels. In vitro hERG-channel current recordings are an important step in evaluating the pro-arrhythmic potential of small molecules, and are now routinely performed using automated high-throughput patch clamp platforms. These machines can execute traditional voltage clamp protocols aimed at specific gating processes, but the array of protocols needed to fully characterise a current is typically too long to be applied in a single cell. Shorter high-information protocols have recently been introduced which have this capability, but they are not typically compatible with high-throughput platforms. We present a new high-information 15 s protocol to characterise hERG (KV11.1) kinetics, suitable for both manual and high-throughput systems. We demonstrate its use on the Nanion SyncroPatch 384PE, a 384 well automated patch clamp platform, by applying it to CHO cells stably expressing hERG1a. From these recordings we construct 124 cell-specific variants/parameterisations of a hERG model at 25 °C. A further 8 independent protocols are run in each cell, and are used to validate the model predictions. We then combine the experimental recordings using a hierarchical Bayesian model, which we use to quantify the uncertainty in the model parameters, and their variability from cell to cell, which we use to suggest reasons for the variability. This study demonstrates a robust method to measure and quantify uncertainty, and shows that it is possible and practical to use high-throughput systems to capture full hERG channel kinetics quantitatively and rapidly. Statement of Significance We present a method for high-throughput characterisation of hERG potassium channel kinetics, via fitting a mathematical model to results of over one hundred single cell patch clamp measurements collected simultaneously on an automated voltage clamp platform. The automated patch clamp data are used to parameterise a mathematical ion channel model fully, opening a new era of automated and rapid development of mathematical models from quick and cheap experiments. The method also allows ample data for independent validation of the models and enables us to study experimental variability and propose its origins. In future the method can be applied to characterise changes to hERG currents in different conditions, for instance at different temperatures (see Part II of the study) or under mutations or the action of pharmaceuticals; and should be easily adapted to study many other currents.
Ion channel behaviour can depend strongly on temperature, with faster kinetics at physiological temperatures leading to considerable changes in currents relative to room temperature. These temperature-dependent changes in voltage-dependent ion channel kinetics (rates of opening, closing and inactivating) are commonly represented with Q10 coefficients or an Eyring relationship. In this paper we assess the validity of these representations by characterising channel kinetics at multiple temperatures. We focus on the hERG channel, which is important in drug safety assessment and commonly screened at room temperature, so that results require extrapolation to physiological temperature. In Part I of this study we established a reliable method for high-throughput characterisation of hERG1a (KV11.1) kinetics, using a 15 second information-rich optimised protocol. In this Part II, we use this protocol to study the temperature dependence of hERG kinetics using CHO cells over-expressing hERG1a on the Nanion SyncroPatch 384PE, a 384-well automated patch clamp platform, with temperature control. We characterise the temperature dependence of hERG gating by fitting the parameters of a mathematical model of hERG kinetics to data obtained at five distinct temperatures between 25 and 37 °C, and validate the models using different protocols. Our models reveal that activation is far more temperature sensitive than inactivation, and we observe that the temperature dependency of the kinetic parameters is not represented well by Q10 coefficients: it broadly follows a generalised, but not the standardly-used, Eyring relationship. We also demonstrate that experimental estimations of Q10 coefficients are protocol-dependent. Our results show that a direct fit using our 15 second protocol best represents hERG kinetics at any given temperature, and suggests that predictions from the Generalised Eyring theory may be preferentially used if no experimentally-derived data are available. Statement of Significance Ion channel currents are highly sensitive to temperature changes. Yet because many experiments are performed more easily at room temperature, it is common to extrapolate findings to physiological temperatures through the use of Q10 coefficients or Eyring rate theory. By applying short, information-rich protocols that we developed in Part I of this study we identify how kinetic parameters change over temperature. We find that the commonly-used Q10 and Eyring formulations are incapable of describing the parameters’ temperature dependence, a more Generalised Eyring relationship works well, but remeasuring kinetics and refitting a model is optimal. The findings have implications for the accuracy of the many applications of Q10 coefficients in electrophysiology, and suggest that care is needed to avoid misleading extrapolations in their many scientific and industrial pharmaceutical applications.
Background: Voltage-gated potassium channel KV4.2 (encoded by KCND2 gene) contributes to the cardiac transient outward potassium current (Ito1). This current is the main contributor to the repolarisation phase 1 of the cardiac action potential. The toxin AmmTx3, identified from the venom of the scorpion Androctonus mauretanicus, is a blocker of KV4.x channels, and have interesting therapeutic potential for neurological disorders due to its effect in cerebellar granule neurons. Its effects on cardiac KV4.2 channels remains unclear. Conclusion :AmmTx3 toxin can be chemically synthesized and used as a KV4.2 channel inhibitor to contributed to the better understanding of the exact role of Ito1 in cardiac electrophysiology. Those first results seem to be a promising evidence that AmmTx3 could a potential inhibitor of Ito current in early repolarisation syndrome.
AimsCurrent treatment for congenital long QT syndrome Type 2 (cLQTS2), an electrical disorder that increases the risk of life-threatening cardiac arrhythmias, is aimed at reducing the incidence of arrhythmia triggers (beta-blockers) or terminating the arrhythmia after onset (implantable cardioverter-defibrillator). An alternative strategy is to target the underlying disease mechanism, which is reduced rapid delayed rectifier current (IKr) passed by KV11.1 channels. Small molecule activators of KV11.1 have been identified but the extent to which these can restore normal cardiac signalling in cLQTS2 backgrounds remains unclear. Here, we examined the ability of ICA-105574, an activator of KV11.1 that impairs transition to the inactivated state, to restore function to heterozygous KV11.1 channels containing either inactivation enhanced (T618S, N633S) or expression deficient (A422T) mutations. Methods and results ICA-105574 effectively restored KV11.1 current from heterozygous inactivation enhanced or expression defective mutant channels in heterologous expression systems. In a human-induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model of cLQTS2 containing the expression defective KV11.1 mutant A422T, cardiac repolarization, estimated from the duration of calcium transients in isolated cells and the rate corrected field potential duration (FPDc) in culture monolayers of cells, was significantly prolonged. The KV11.1 activator ICA-105574 was able to reverse the prolonged repolarization in a concentration-dependent manner. However, at higher doses, ICA-105574 produced a shortening of the FPDc compared to controls. In vitro and in silico analysis suggests that this overcorrection occurs as a result of a temporal redistribution of the peak IKr to much earlier in the plateau phase of the action potential, which results in early repolarization. Conclusions KV11.1 activators, which target the primary disease mechanism, provide a possible treatment option for cLQTS2, with the CaVeat that there may be a risk of overcorrection that could itself be pro-arrhythmic.
Pathogenic variants in KCNB1, encoding the voltage-gated potassium channel KV2.1, are associated with developmental and epileptic encephalopathies (DEE). Previous functional studies on a limited number of KCNB1 variants indicated a range of molecular mechanisms by which variants affect channel function, including loss of voltage sensitivity, loss of ion selectivity, and reduced cell-surface expression. We evaluated a series of 17 KCNB1 variants associated with DEE or neurodevelopmental disorder (NDD) to rapidly ascertain channel dysfunction using high-throughput functional assays. Specifically, we investigated the biophysical properties and cell-surface expression of variant KV2.1 channels expressed in heterologous cells using high-throughput automated electrophysiology and immunocytochemistry-flow cytometry. Pathogenic variants exhibited diverse functional defects, including altered current density and shifts in the voltage-dependence of activation and/or inactivation, as homotetramers or when co-expressed with wild-type KV2.1. Quantification of protein expression also identified variants with reduced total KV2.1 expression or deficient cell-surface expression. Our study establishes a platform for rapid screening of functional defects of KCNB1 variants associated with DEE and other NDDs, which will aid in establishing KCNB1 variant pathogenicity and may enable discovery of targeted strategies for therapeutic intervention based on molecular phenotype.
Background KCNH2 encodes the human ether-à-go-go-related gene (hERG) potassium channel, which passes the rapid delayed rectifier potassium current, IKr. Loss-of-function variants in KCNH2 cause long QT syndrome type 2 (LQTS2) which is associated with a markedly increased risk of cardiac arrhythmias. The majority of rare KCNH2 variants however are likely to be benign. Objective To develop a high-throughput assay for discriminating between pathogenic and benign KCNH2 variants. Methods Nonsynonymous homozygous KCNH2 variants stably expressed in Flp-In human embryonic kidney 293 (HEK293) cell lines were phenotyped using an automated patch-clamp platform (SyncroPatch 384PE) and a cell surface ELISA assay. Functional phenotyping of heterozygous KCNH2 variants stably expressed in Flp-In HEK293 using a bicistronic vector was performed using SyncroPatch 384PE. Results In homozygous KCNH2 variant cell lines, discrepancies between current density and cell surface expression levels measured by ELISA can be explained by changes in gating properties of the variant channels. Amongst 30 heterozygous KCNH2 variant cell lines studied, the assay correctly predicted the ClinVar ascribed classification for 17/17 pathogenic/likely pathogenic/benign variants. Of 13 pore-domain variants studied, 11 had a dominant-negative expression defect whilst the remaining two had enhanced inactivation gating resulting in a dominant-negative phenotype. Conclusions High-throughput electrophysiological phenotyping of heterozygous KCNH2 variants can accurately distinguish between dominant-negative, haploinsufficient loss-of-function, and benign variants. This assay will help with future classification of KCNH2 variants
Human ether-a-go-go-related gene (hERG) trafficking inhibition is known to be one of the mechanisms of indirect hERG inhibition, resulting in QT prolongation and lethal arrhythmia. Pentamidine, an antiprotozoal drug, causes QT prolongation/Torsades de Pointes (TdP) via hERG trafficking inhibition, but 17-AAG, a geldanamycin derivative heat shock protein 90 (Hsp90) inhibitor, has not shown torsadogenic potential clinically, despite Hsp90 inhibitors generally being hypothesized to cause TdP by hERG trafficking inhibition. In the present study, we investigated the underlying mechanisms of both drugs’ actions on hERG channels using hERG-overexpressing CHO cells (hERG-CHOs) and human embryonic stem cell-derived cardiomyocytes (hES-CMs). The effects on hERG tail current and protein levels were evaluated using population patch clamp and Western blotting in hERG-CHOs. The effects on field potential duration (FPD) were recorded by a multi-electrode array (MEA) in hES-CMs. Neither drug affected hERG tail current acutely. Chronic treatment with each drug inhibited hERG tail current and decreased the mature form of hERG protein in hERG-CHOs, whereas the immature form of hERG protein was increased by pentamidine but decreased by 17-AAG. In MEA assays using hES-CMs, pentamidine time-dependently prolonged FPD, but 17-AAG shortened it. The FPD prolongation in hES-CMs upon chronic pentamidine exposure is relevant to its clinically reported arrhythmic risk. CaV1.2 or NaV1.5 current were not reduced by chronic application of either drug at a relevant concentration to hERG trafficking inhibition in human embryonic kidney (HEK293) cells. Therefore, the reason why chronic 17-AAG shortened the FPD despite the hERG trafficking inhibition occur is still unknown.
Objective: We identified a novel de novo KCNT1 variant in a patient with early‐infantile epileptic encephalopathy (EIEE) and status dystonicus, a life‐threatening movement disorder. We determined the functional consequences of this variant on the encoded KNa1.1 channel to investigate the molecular mechanisms responsible for this disorder. Methods: A retrospective case review of the proband is presented. We performed manual and automated electrophysiologic analyses of the KCNT1‐L437F variant expressed heterologously in Chinese hamster ovary (CHO) cells in the presence of channel activators/blockers. Results: The KCNT1‐L437F variant, identified in a patient with refractory EIEE and status dystonicus, confers a gain‐of‐function channel phenotype characterized by instantaneous, voltage‐dependent activation. Channel openers do not further increase L437F channel function, suggesting maximal activation, whereas channel blockers similarly block wild‐type and variant channels. We further demonstrated that KCNT1 current can be measured on a high‐throughput automated electrophysiology platform with potential value for future screening of novel and repurposed pharmacotherapies. Interpretation: A novel pathogenic variant in KCNT1 associated with early‐onset, medication‐refractory epilepsy and dystonia causes gain‐of‐function with rapid activation kinetics. Our findings extend the genotype–phenotype relationships of KCNT1 variants to include severe dystonia.
The transient receptor potential cation channel 5 (TRPC5) has been previously shown to affect podocyte survival in the kidney. As such, inhibitors of TRPC5 are interesting candidates for the treatment of chronic kidney disease (CKD). Herein, we report the synthesis and biological characterization of a series of N-heterocyclic-1-benzyl-1H-benzo[d]imidazole-2-amines as selective TRPC5 inhibitors. Work reported here evaluates the benzimidazole scaffold and substituents resulting in the discovery of AC1903, a TRPC5 inhibitor that is active in multiple animal models of CKD.
Highlights: Development of six potent diazirine-containing photoprobes based on Huwentoxin-IV. Photoprobes specifically photolabel purified bacterial-NaV1.7 VSD2 chimeric channels. Proteomic mass spectrometry identifies binding site on S1-S2 loop and S3 helix. Proposed model of HwTx-IV binding reveals importance of K27 and R29. Summary: Voltage-gated sodium (NaV) channels respond to changes in the membrane potential of excitable cells through the concerted action of four voltage-sensor domains (VSDs). Subtype NaV1.7 plays an important role in the propagation of signals in pain-sensing neurons and is a target for the clinical development of novel analgesics. Certain inhibitory cystine knot (ICK) peptides produced by venomous animals potently modulate NaV1.7; however, the molecular mechanisms underlying their selective binding and activity remain elusive. This study reports on the design of a library of photoprobes based on the potent spider toxin Huwentoxin-IV and the determination of the toxin binding interface on VSD2 of NaV1.7 through a photocrosslinking and tandem mass spectrometry approach. Our Huwentoxin-IV probes selectively crosslink to extracellular loop S1-S2 and helix S3 of VSD2 in a chimeric channel system. Our results provide a strategy that will enable mapping of sites of interaction of other ICK peptides on NaV channels.
Phlotoxin-1 (PhlTx1) is a peptide previously identified in tarantula venom (Phlogius species) that belongs to the inhibitory cysteine-knot (ICK) toxin family. Like many ICK-based spider toxins, the synthesis of PhlTx1 appears particularly challenging, mostly for obtaining appropriate folding and concomitant suitable disulfide bridge formation. Herein, we describe a procedure for the chemical synthesis and the directed sequential disulfide bridge formation of PhlTx1 that allows for a straightforward production of this challenging peptide. We also performed extensive functional testing of PhlTx1 on 31 ion channel types and identified the voltage-gated sodium (NaV) channel NaV1.7 as the main target of this toxin. Moreover, we compared PhlTx1 activity to 10 other spider toxin activities on an automated patch-clamp system with Chinese Hamster Ovary (CHO) cells expressing human NaV1.7. Performing these analyses in reproducible conditions allowed for classification according to the potency of the best natural NaV1.7 peptide blockers. Finally, subsequent in vivo testing revealed that intrathecal injection of PhlTx1 reduces the response of mice to formalin in both the acute pain and inflammation phase without signs of neurotoxicity. PhlTx1 is thus an interesting toxin to investigate NaV1.7 involvement in cellular excitability and pain.
Background The voltage-gated K+-channel KV11.1 has a central role in cardiac repolarization. Blockage of KV11.1 has been linked to severe cardiovascular side effects, such as acquired long QT syndrome (aLQTS), torsade de pointes arrhythmia and sudden cardiac death (SCD). KV11.1 is susceptible to unspecific drug interactions due to the presence of two aromatic amino acids residing in the inner vestibule of the pore. These aromatic residues are also present in the equine orthologue of KV11.1. This suggests that equine KV11.1 may also be prone to high-affinity block by a range of different chemical entities, which potentially could cause severe cardiac side effects and SCD in horses. Aim To screen a series of commonly used drugs in equine medicine for interaction with KV11.1. Methods High-throughput screening of selected compounds on human KV11.1 expressed in a mammalian cell line was performed using an automated patch clamp system, the SyncroPatch 384PE (Nanion Technologies, Munich, Germany). Results were validated on equine KV11.1 expressed in CHO-K1 cells by manual patch clamp. Results Acepromazine maleat (IC50 = 0.5 μM) trimethoprim (IC50 = 100 μM), diphenhydramine hydrochloride (IC50 = 2 μM) and cyproheptadine hydrochloride (IC50 = 1.84 μM) inhibited equine KV11.1 current at clinically relevant drug concentrations. Conclusion The results suggest that drug interaction with KV11.1 can occur in horses and that some drugs potentially may induce repolarization disorders in horses.
Piezo1 is a mechanosensitive ion channel that is believed to be expressed in red blood cells (RBCs), mainly supported by the findings that mutations of PIEZO1 gene are associated with the RBC disease Hereditary Xerocytosis. So far several mutations have been reported, e.g. R2456H, T2127M and E2496ELE, to exhibit a partial gain-of-function phenotype with generation of mechanically activated currents that inactivate more slowly than wild type. However, characterisation of the mutated ion channel has almost exclusively been performed based on heterologous expression in cell lines and recordings in RBCs were rather of episodic character. Here we present a patient with a novel PIEZO1 mutation (R2110W) and a patch clamp based high-throughput screening assay for Piezo1 activity. It is the first electrophysiologic single-cell based screening ever performed on RBCs, demonstrating the Piezo1 gain-of-function mutation directly on RBCs. Thus we provide a putative routine approach for detecting functional (Piezo1) channel mutations as the molecular cause of rare anaemia that can become a standard method in specialised haematological centres.
The medical staff is often powerless to treat patients affected by drug abuse or misuse and poisoning. In the case of envenomation, the treatment of choice remains horse sera administration that poses a wealth of other medical conditions and threats. Previously, we have demonstrated that DNA-based aptamers represent powerful neutralizing tools for lethal animal toxins of venomous origin. Herein, we further pursued our investigations in order to understand whether all toxin-interacting aptamers possessed equivalent potencies to neutralize αC-conotoxin PrXA in vitro and in vivo. We confirmed the high lethality in mice produced by αC-conotoxin PrXA regardless of the mode of injection and further characterized myoclonus produced by the toxin. We used high-throughput patch-clamp technology to assess the effect of αC-conotoxin PrXA on ACh-mediated responses in TE671 cells, responses that are carried by muscle-type nicotinic receptors. We show that 2 out of 4 aptamers reduce the affinity of the toxin for its receptor, most likely by interfering with the pharmacophore. In vivo, more complex responses on myoclonus and mice lethality are observed depending on the type of aptamer and mode of administration (concomitant or differed). Concomitant administration always works better than differed administration indicating the stability of the complex in vivo. The most remarkable conclusion is that an aptamer that has no or a limited efficacy in vitro may nevertheless be functional in vivo probably owing to an impact on the biodistribution or pharmacokinetics of the toxin in vivo. Overall, the results highlight that a blind selection of aptamers against toxins leads to efficient neutralizing compounds in vivo regardless of the mode of action. This opens the door to the use of aptamer mixtures as substitutes to horse sera for the neutralization of life-threatening animal venoms, an important WHO concern in tropical areas.
Integral to the cell surface is channels, pumps, and exchanger proteins that facilitate the movement of ions across the membrane. Ion channels facilitate the passive movement of ions down an electrochemical gradient. Ion pumps actively use energy to actively translocate ions, often against concentration or voltage gradients, while ion exchangers utilize energy to couple the transport of different ion species such that one ion moves down its gradient and the released free energy is used to drive the movement of a different ion against its electrochemical gradient. Some ion pumps and exchangers may be electrogenic, i.e., the ion transport they support is not electrically neutral and generates a current. Functions of these pore-forming membrane proteins include the establishment of membrane potentials, gating of ions flows across the cell membrane to elicit action potentials and other electrical signals, as well as the regulation of cell volumes. The major forms of ion channels include voltage-, ligand-, and signal-gated channels. In this review, we describe mammalian voltage dependent Na (NaV) channels.
Sweating is a fundamental process required for human thermoregulation. In today’s modern society, however, extensive sweating is rather considered unpleasant or embarrassing, or can even cause severe psychosocial pressure. Sweat reduction by antiperspirants is therefore of huge cosmetic interest. Currently, the global use of aluminum salts as antiperspirants is controversial, but no alternatives exist so far. We developed a new concept for sweat reduction which is based on directly targeting primary fluid secretion in human sweat glands. We identified a long searched for key player in human sweat glands - the ion channel TMEM16A, also known as ANO1. We extensively characterized TMEM16A and its function in native human sweat glands and sweat gland tissue culture cells by using a wide variety of different techniques such as immunohistological staining, chloride flux assays, automated patch clamping as well as state-of-the-art CRISPR/ Cas9 genome editing technology. We generated a proprietary cell-based assay to emulate TMEM16A function in a cellular sweat gland environment. We combined this cell-based assay with our cherry-picked compound libraries and performed high-throughput screening campaigns which uncovered smallmolecule modulators of TMEM16A. In silico and in vitro toxicological assessments as well as stability and formulation tests were performed and yielded compounds that are currently being tested for their sweat reduction efficacy in vivo.
Many ion channels, including NaV1.7, CaV1.3, and KV1.3, are linked to human pathologies and are important therapeutic targets. To develop efficacious and safe drugs, subtype-selective modulation is essential, but has been extremely difficult to achieve. We postulate that this challenge is caused by the poor assay design, and investigate the NaV1.7 membrane potential assay, one of the most extensively employed screening assays in modern drug discovery. The assay uses veratridine to activate channels, and compounds are identified based on the inhibition of veratridine-evoked activities. We show that this assay is biased toward nonselective pore blockers and fails to detect the most potent, selective voltage-sensing domain 4 (VSD4) blockers, including PF-05089771 (PF-771) and GX-936. By eliminating a key binding site for pore blockers and replacing veratridine with a VSD-4 binding activator, we directed the assay toward non–pore-blocking mechanisms and discovered NaV1.7-selective chemical scaffolds. Hence, we address a major hurdle in NaV1.7 drug discovery, and this mechanistic approach to assay design is applicable to CaV3.1, KV1.3, and many other ion channels to facilitate drug discovery.
Background: SCN5A mutations can lead to different cardiac diseases. Recently, SCN5A mutations have been linked to the clinical entity multifocal ectopic Purkinje-related premature contractions (MEPPC) characterized by ventricular ectopy and dilated cardiomyopathy. Methods & Results: A family with a uniform MEPPC-like phenotype, associated with complex atrial and ventricular arrhythmias and dilated cardiomyopathy caused by a high frequency of ventricular ectopy was clinically assessed. Screening of the SCN5A gene revealed a missense mutation in the linker between segments 3 and 4 in domain 1 of the NaV1.5 protein, resulting in a glycine to aspartate substitution at position 213 (G213D). The phenotype co-segregated with the missense mutation. Electrophysiological studies of wild type (WT) hNaV1.5 and hNaV1.5_G213D expressed in CHO-K cells showed that the voltage of half-maximal activation (V½) was significantly more negative for hNaV1.5_G213D compared to WT (V½ = −38.7 ± 0.5 mV for WT and V½ = −42.4 ± 0.5 mV for G213D; P 0.001). This suggests activation of NaV1.5_G231D at more negative potentials. The V½ of steady-state inactivation was significantly shifted towards more positive values for NaV1.5_G213D (V½ = −86.7 ± 0.2 mV for WT and −82.2 ± 0.3 mV for G213D; P 0.001), also contributing to a gain-of-function phenotype. Flecainide and amiodarone markedly reduced premature atrial and ventricular contractions in four patients. Conclusion: The NaV1.5_G213D mutation is associated with a gain-of-function phenotype, multifocal atrial and ventricular ectopy and dilated cardiomyopathy. Since patients with a MEPPC-like phenotype may specifically benefit from Class-1 antiarrhythmic drugs or amiodarone, clinical identification of this disease entity is important. Note: Electrophysiological analysis of heteromers (NaV1.5 and NaV1.5_G213D mutation) were executed on the SyncroPatch 384PE. Data are not shown in the publication.
Ion channels represent nearly a quarter of all targets that currently available medications modulate, and their dysfunction underlies increasing number of human diseases. Functional analysis of ion channels have traditionally been a bottleneck in large-scale analyses. Recent technological breakthroughs in automated planar electrophysiology have democratized the technique to enable high-throughput patch clamping at scale. In this chapter, we describe the methodology to perform a phenotypic screen on voltage-gated calcium channels across many different genetic coding variations and against small-molecule modulators. We first describe the procedures to establish inducible heterologous ion channel expression in HEK293 cells, where each cell incorporates one copy of a target protein cDNA—a step that is critical for producing stable and consistent expression of ion channels. We then describe the experimental and analytical methods for analyzing the function of ion channels using high-throughput planar electrophysiology.
Background: The explosive growth in known human gene variation presents enormous challenges to current approaches for variant classification that have implications for diagnosis and treatment of many genetic diseases. For disorders caused by mutations in cardiac ion channels as in congenital arrhythmia syndromes, in vitro electrophysiological evidence has high value in discriminating pathogenic from benign variants, but these data are often lacking because assays are cost, time, and labor intensive. Methods: We implemented a strategy for performing high-throughput functional evaluations of ion channel variants that repurposed an automated electrophysiological recording platform developed previously for drug discovery. Results: We demonstrated the success of this approach by evaluating 78 variants in KCNQ1, a major gene involved in genetic disorders of cardiac arrhythmia susceptibility. We benchmarked our results with traditional electrophysiological approaches and observed a high level of concordance. This strategy also enabled studies of dominant-negative behavior of variants exhibiting severe loss-of-function. Overall, our results provided functional data useful for reclassifying >65% of the studied KCNQ1 variants. Conclusions: Our results illustrate an efficient and high-throughput paradigm linking genotype to function for a human cardiac ion channel that will enable data-driven classification of large numbers of variants and create new opportunities for precision medicine.
Ion channels regulate a variety of physiological processes and represent an important class of drug target. Among the many methods of studying ion channel function, patch clamp electrophysiology is considered the gold standard by providing the ultimate precision and flexibility. However, its utility in ion channel drug discovery is impeded by low throughput. Additionally, characterization of endogenous ion channels in primary cells remains technical challenging. In recent years, many automated patch clamp (APC) platforms have been developed to overcome these challenges, albeit with varying throughput, data quality and success rate. In this study, we utilized SyncroPatch 768PE, one of the latest generation APC platforms which conducts parallel recording from two-384 modules with giga-seal data quality, to push these 2 boundaries. By optimizing various cell patching parameters and a two-step voltage protocol, we developed a high throughput APC assay for the voltage-gated sodium channel NaV1.7. By testing a group of NaV1.7 reference compounds’ IC50, this assay was proved to be highly consistent with manual patch clamp (R > 0.9). In a pilot screening of 10,000 compounds, the success rate, defined by > 500 MΩ seal resistance and >500 pA peak current, was 79%. The assay was robust with daily throughput ~ 6,000 data points and Z’ factor 0.72. Using the same platform, we also successfully recorded endogenous voltage-gated potassium channel KV1.3 in primary T cells. Together, our data suggest that SyncroPatch 768PE provides a powerful platform for ion channel research and drug discovery.
Voltage-gated KV1.3 and Ca2+-dependent KCa3.1 are the most prevalent K+ channels expressed by human and rat T cells. Despite the preferential upregulation of KV1.3 over KCa3.1 on autoantigen-experienced effector memory T cells, whether KV1.3 is required for their induction and function is unclear. Here we show, using KV1.3-deficient rats, that KV1.3 is involved in the development of chronically activated antigen-specific T cells. Several immune responses are normal in KV1.3 knockout (KO) rats, suggesting that KCa3.1 can compensate for the absence of KV1.3 under these specific settings. However, experiments with KV1.3 KO rats and KV1.3 siRNA knockdown or channel-specific inhibition of human T cells show that maximal T-cell responses against autoantigen or repeated tetanus toxoid stimulations require both KV1.3 and KCa3.1. Finally, our data also suggest that T-cell dependency on KV1.3 or KCa3.1 might be irreversibly modulated by antigen exposure.
GS-458967, 6-(4-(Trifluoromethoxy)phenyl)-3-(trifluoromethyl)-[1,2,4]triazolo[4,3-a]pyridine (GS967) is a recently described, novel, sodium channel inhibitor exhibiting potent antiarrhythmic effects in various in vitro and in vivo models. The antiarrhythmic mechanism has been attributed to preferential suppression of late sodium current. However, there has been no reported systematic investigation of the effects of this compound on isolated sodium channels. Here, we examined the effects of GS967 on peak (INaP) and late (INaL) sodium current recorded from cells that heterologously expressed human cardiac voltage-gated sodium channel, the principle cardiac sodium channel. As previously described, we observed that GS967 exerted tonic block of INaL (63%) to a significantly greater extent than INaP (19%). However, GS967 also caused a reduction of INaP in a frequency-dependent manner, consistent with use-dependent block (UDB). GS967 evoked more potent UDB of INaP (IC50 = 0.07 µM) than ranolazine (16 µM) and lidocaine (17 µM). Use-dependent block was best explained by a significant slowing of recovery from fast and slow inactivation with a significant enhancement of slow inactivation in the presence of GS967. Furthermore, GS967 was found to exert these same effects on a prototypical long QT syndrome mutation (delKPQ). An engineered mutation at an interaction site for local anesthetic agents (F1760A) partially attenuated the effect of GS967 on UDB, but had no effect on tonic INaL block. We conclude that GS967 is a preferential inhibitor of INaL, but it also exerts previously unreported strong effects on slow inactivation and recovery from inactivation, resulting in substantial UDB that is not entirely dependent on a known interaction site for local anesthetic agents.
Objective: To perform functional characterization of a potentially pathogenic KCNB1 variant identified by clinical exome sequencing of a proband with a neurodevelopmental disorder that included epilepsy and centrotemporal spikes on EEG. Methods: Whole-exome sequencing identified the KCNB1 variant c.595A.T (p.Ile199Phe). Biochemical and electrophysiologic experiments were performed to determine whether this variant affected protein expression, trafficking, and channel functional properties. Results: Biochemical characterization of the variant suggested normal protein expression and trafficking. Functional characterization revealed biophysical channel defects in assembled homotetrameric and heterotetrameric channels. Conclusions: The identification of the KCNB1 variant c.595A.T (p.Ile199Phe) in a neurodevelopmental disorder that included epilepsy with centrotemporal spikes expands the phenotypic spectrum of epilepsies associated with KCNB1 variants. The KCNB1-I199F variant exhibited partial loss of function relative to the wild-type channel. This defect is arguably less severe than previously reported KCNB1 variants, suggesting the possibility that the degree of KCNB1 protein dysfunction may influence disease severity.
We have developed an automated patch clamp module for high-throughput ion channel screening, recording from 384 cells simultaneously. The module is incorporated into a laboratory pipetting robot and uses a 384-channel pipettor head for application of cells and compounds. The module contains 384 amplifier channels for fully parallel recordings using a digital amplifier. Success rates for completed experiments (1- to 4-point concentration–response curves for cells satisfying defined quality control parameters) of greater than 85% have been routinely achieved with, for example, HEK, CHO, and RBL cell lines expressing hNaV1.7, hERG, Kir2.1, GABA, or glutamate receptors. Pharmacology experiments are recorded and analyzed using specialized software, and the pharmacology of hNaV1.7 and hERG is described. Fast external solution exchange rates of 50 ms are demonstrated using Kir2.1. Short exposure times are achieved by stacking the external solutions inside the pipette of the robot to minimize exposure of the ligand on the receptor. This ensures that ligand-gated ion channels, for example, GABA and glutamate described in this report, can be reproducibly recorded. Stem cell–derived cardiomyocytes have also been used with success rates of 52% for cells that have a seal resistance of >200 MΩ, and recordings of voltage-gated Na+ and Ca2+ are shown.
Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers and new therapeutic targets are urgently needed. One of the hallmarks of cancer is changed pH-homeostasis and potentially pH-sensors may play an important role in cancer cell behavior. Two-pore potassium channels (K2P) are pH-regulated channels that conduct a background K+ current, which is involved in setting the plasma membrane potential (Vm). Some members of the K2P superfamily were reported as crucial players in driving tumor progression. The aim of this study was to investigate pH-regulated K+ currents in PDAC cells and determine possible effects on their pathological phenotype. Using a planar high-throughput patch-clamp system (SyncroPatch 384PE) we identified a pH-regulated K+ current in the PDAC cell line BxPC-3. The current was inhibited by extracellular acidification and intracellular alkalization. Exposure to a set of different K+ channel inhibitors, and the TREK-1 (K2P2.1)–specific activator BL1249, TREK-1 was identified as the main component of pH-regulated current. A voltage-sensor dye (VF2.1.Cl) was used to monitor effects of pH and BL1249 on Vm in more physiological conditions and TREK-1–mediated current was found as critical player in setting Vm. We assessed a possible role of TREK-1 in PDAC progression using cell proliferation and migration assays and observed similar trends with attenuated proliferation/migration rates in acidic (pH 7.0) and alkaline (pH > 7.4) conditions. Notably, BL1249 inhibited both PDAC cell proliferation and migration indicating that hyperpolarization of Vm attenuates cancer cell behavior. TREK-1 may therefore be a promising novel target for PDAC therapy.
Introduction From a drug discovery point of view, ion channels are very interesting and challenging targets. Over the past decade, great efforts have been made in developing platforms for patch clamp-based high-quality screening of ion channels in discovering new drug candidates as well for evaluating their safety profiles. Indeed, the automated patch clamp (APC) has recently reached the data throughput requirements of high-throughput screening (HTS) allowing for new screening strategies with ion channel active compounds. Areas covered This editorial article comments on the past and present developments of APC-based drug screening. Furthermore, it also looks at the implications of APC technology meeting HTS-standards as well as its use in compound safety evaluation. Expert opinion In the imminent future, we will see a paradigm shift in ion channel drug screening toward using APC-based platforms for primary drug library screens. This way, the redundancy of the drug discovery process and the risk of false-negatives could be drastically reduced. Furthermore, cardiac safety can be addressed early, avoiding late-phase withdrawals with promising drug candidates. It is our firm belief that APC-based ion channel HTS will facilitate the discovery of candidates, which otherwise would have not been found, and shorten the drug development cycle, saving time and cost.
Background An in vitro electrophysiological assay system, which can assess compound effects and thus show cardiotoxicity including arrhythmia risks of test drugs, is an essential method in the field of drug development and toxicology. Methods In this study, high-throughput electrophysiological recordings of human embryonic kidney (HEK 293) cells and Chinese hamster ovary (CHO) cells stably expressing human ether-a-go-go related gene (hERG) were performed utilizing an automated 384-well-patch-clamp system, which records up to 384 cells simultaneously. hERG channel inhibition, which is closely related to a drug-induced QT prolongation and is increasing the risk of sudden cardiac death, was investigated in the high-throughput screening patch-clamp system. Results In the automated patch-clamp measurements performed here, KV currents were investigated with high efficiency. Various hERG channel blockers showed concentration-dependent inhibition, the 50 % inhibitory concentrations (IC50) of those blockers were in good agreement with previous reports. Conclusions The high-throughput patch-clamp system has a high potential in the field of pharmacology, toxicology, and cardiac physiology, and will contribute to the acceleration of pharmaceutical drug development and drug safety testing.
Maria explains the solid supported membrane (SSM)-based methodology and gives a brief introduction on our two devices, the SURFE2R 96SE and the SURFE2R N1.
SSM-based electrophysiology helps to understand the mechanisms of different transporters. The technique was used to characterize and compare different sugar transporters and their transport deficient mutants. Proton-coupled (LacY, XylE, FucP), sodium-coupled (MelB) and loosely coupled (GlcP) sugar transporters were analyzed. A general transport model was concluded from the electrophysiological data. Here we present the most intriguing results for these transporters as well as our conclusions regarding the transport mechanism. We will discuss (1) substrate specifity, (2) protonation and coupling mechanisms, (3) the impact of different driving forces, (4) sugar binding kinetics and (5) the significance of specific amino acids for the transport cycle. All together SSM-based electrophysiology helped to conclude a detailed kinetic model for sugar transporters.
Sodium–calcium exchangers (NCXs) are membrane transporters that play an important role in Ca2+ homeostasis and Ca2+ signaling. The recent crystal structure of NCX_Mj, a member of the NCX family from the archaebacterium Methanococcus jannaschii, provided insight into the atomistic details of sodium–calcium exchange. Here, we extend these findings by providing detailed functional data on purified NCX_Mj using solid supported membrane (SSM)–based electrophysiology, a powerful but unexploited tool for functional studies of electrogenic transporter proteins. We show that NCX_Mj is highly selective for Na+, whereas Ca2+ can be replaced by Mg2+ and Sr2+ and that NCX_Mj can be inhibited by divalent ions, particularly Cd2+. By directly comparing the apparent affinities of Na+ and Ca2+ for NCX_Mj with those for human NCX1, we show excellent agreement, indicating a strong functional similarity between NCX_Mj and its eukaryotic isoforms. We also provide detailed instructions to facilitate the adaption of this method to other electrogenic transporter proteins. Our findings demonstrate that NCX_Mj can serve as a model for the NCX family and highlight several possible applications for SSM-based electrophysiology.
Solid-supported membrane (SSM)-based electrophysiology allows functional characterization of transporters which are otherwise difficult to investigate due to their slow transport conductivity or intracellular localization. The "SURFE2R 96SE" platform makes this technology suitable for compound characterization at higher throughputs.
For this case study, we prepared membrane vesicles from a PepT1-expressing cell line and screened an ion-channel/transporter-focused library of 237 bioactive compounds for substrate and inhibitory activity. With the SURFE2R 96SE, the screen was completed in less than 8 hours with a success rate of 98%, demonstrating utility of the technology for compound screening.
On the downside, application of SSM-based electrophysiology is limited to targets with an electrogenic net transport, assay setup is rather complex, and cost of ownership is significant. This may be an obstacle for implementing SURFE2R technology in-house. In response, Assay.Works entered into a partnership with Nanion, providing SURFE2R assays as a service. Backed by Nanions extensive expertise, we utilize our assay development and screening capabilities, including cell and membrane production, compound libraries, and sample management, to help clients realizing proof-of-concept, compound characterization, or profiling studies.
The human peptide transporter PepT1 is an uptake transporter responsible for initial absorption and renal reabsorption of dietary oligopeptides. It is primarily located in the plasma membranes of enterocytes of the small intestine as well as the renal proximal tubular cells. PepT1 functions as a co-transporter, coupling the uphill peptide transport into the cells to the electrochemical proton gradient. Due to the movement of protons, PepT1 is an electrogenic transporter. PepT1 shows a very high capacity but a low affinity and substrate specificity. Its ability to transport a large range of compounds has enabled the rational design of drugs and pro-drugs (e.g. penicillins, ACE inhibitors) which have good oral bio-availability using delivery via PepT1. Designing pro-drugs with higher affinity for PepT1 is a successful strategy to increase the bio-availability of poorly absorbed drugs. Here we present electric real-time PepT1 activity measurements on the SURFE2R instruments using purified plasma membranes of CHO cells overexpressing PepT1. Peptide transport was activated on the SURFE2R N1 using a sensor with attached PepT1- containing membrane fragments which was inserted into the device. This was perfused with a buffer containing the dipeptide glycyl-glycine as the substrate. The data presented here show activation of PepT1 by glyclyglycine and inhibition by Lys[Z(No2)]-Val on the SURFE2R N1 and scale-up of the assay on the SURFE2R N96.
The excitatory amino acid transporter 3 (EAAT3; also known as EAAC1) is a sodium-dependent neuronal uptake transporter encoded by the slc1a1 gene. It plays amajor role in the reuptake of glutamate from the synaptic cleft, thereby maintaining a low extra- cellular concentration of glutamate and regulating the excitatory neurotransmission. EAAT3 is also involved in the uptake of aspartate and cysteine into the cells. The transporter is highly expressed in mature neurons, where it is distributed in somata and dendrites. EAAT3 functions as a cotransporter, coupling the uphill substrate transport into the cells to the electrochemical gradients of sodium and potassium. The stoichiometry of transport is 1 glutamate with 3 Na+ and 1 H+ moving into the cell to 1 K+ moving out of the cell. Therefore EAAT3 is an electrogenic transporter, generating a net charge flow.Dysfunction of glutamate transporters leads to increased extracellular glutamate levels, thereby causing neuro- toxicity and neurodegeneration. Regulatory mechanisms facilitating EAAT3 function are, therefore, interesting as targets for the treatment of neurodegenerative diseases.Here we present EAAT3 activity measurements on the SURFE2R N1 instrument using purified plasma membrane of CHO cells expressing EAAT3.
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. The GABA transporters belong to the family of neurotransmitter:sodium symporters referred to as the solute carrier 6 (SLC6) family in humans. GATs co-transport GABA, Na+ and Cl- with the proposed stiochiometry 1 GABA: 2 Na+: 1 Cl-, resulting in a net influx of 1 positive charge per cycle. So far, 4 GATs have been identified, GAT1, GAT2, GAT3 and BGT1.GAT1 is expressed throughout the brain in both GABAergic and non-GABAergic neurons, and is expressed in particularly high levels in the olfactory bulb, basal ganglia, cerebellum and retina. The physiological function of GAT1 is primarily to terminate synaptic transmission but also to ensure the fidelity of synaptic transmission by preventing the spread of neurotransmitter to neighbouring synapses. GABA transporters also play an important role in neurotransmitter reutilization. In certain circumstances, e.g. when the sodium gradient increases during ischemia or following seizures, GATs can act in reverse which may have a protective effect during seizures, by inhibiting electrical excitability. There is some evidence that GATs may play a role in neurodegenerative diseases such as Parkinson’s and Alzheimer’s and may provide a novel target for treating these conditions.Here we present human GAT1 activity measurements on the SURFE2R N1 instrument using purified plasma membranes from HEK cells. GABA affinity and effect of inhibitors were investigated.
ClCs are a family of chloride ion channels and transporters with important physiological roles including regulation of the membrane potential, transepithelial salt transport and ion homeostasis1,2. To date, 9 members of the ClC family have been identified in mammals1,2, the first 4 (ClC-1, ClC-2, ClC-Ka, and ClC-Kb) are located on the plasma membrane where they act as chloride ion channels whereas the remaining 5 are located in intracellular organelles (ClC-3-7) and are chloride-proton exchangers1-3. These transporters are important for endosome, lysosome and synaptic vesicle acidification1,2, and mutations in, e.g. ClC-5 underlie the rare chronic kidney disorder, Dent’s Disease, and mutations in ClC-7 underlie osteopetrosis 2,4, a rare inherited bone hardening disorder. Given their ubiquitous expression and importance in physiological processes, they are important potential drug targets.ClC from Escherichia coli (Ec-ClC or ClC-ec1) is closely ClC from Escherichia coli (Ec-ClC or ClC-ec1) is closely related to its mammalian counterparts and is a Cl-/H+ exchanger3,5. It transports 2 Cl- into the cell, coupled to the efflux of 1 H+3,5. Therefore Ec-ClC is an electrogenic transporter, generating a net charge flow. In E. coli, the Ec-ClC mediates acid resistance of enteric bacteria by promoting H+ extrusion1,5,6. Here we present Ec-ClC activity measurements on the SURFE2R N1 instrument using proteoliposomes reconstituted with purified Ec-ClC at different lipid-to-protein ratios.
The concentrative nucleoside transporter 1 (CNT1) is a sodium-dependent uptake transporter encoded by the SLC28A1 gene. CNT1 functions as a co-transporter, coupling the uphill nucleoside transport into the cells to the electrochemical gradient of sodium. The stoichiometry of transport is proposed to be 1:1, but a stoichiometry of 2 Na+: 1 nucleoside has also been suggested. CNT1 is an electrogenic transporter, generating a net charge flow. It plays a major role in the uptake of pyrimidines, including uridine and cytidine, from the extracellular milieu into the cytoplasm in nucleoside salvage pathways which is the first step of nucleoside biosynthesis. The transporter is expressed in epithelial tissues including liver, kidney and small intestine where it is localized to the apical membrane. CNTs are important targets for many antiviral and anticancer agents, and CNT1 has been proposed to play a role in tumor biology via a mechanism beyond nucleoside transport. In fact, tumors expressing high levels of CNT1 can indicate a higher risk of relapse for breast cancer patients, suggesting that nucleoside salvage may interfere with chemosensitivity. On the other hand, high expression of the CNT1 protein could promote drug- induced cytotoxicity if patients were treated with suitable hCNT substrates. In any case, hCNT1 is an important mediator in the transport of anticancer and antiviral nucleoside drugs by mechanisms that require further study.Here we present CNT1 activity measurements on the SURFE2R N1 instrument using purified plasma membrane of CHO cells expressing CNT1.
The Na+/H+ exchanger SLC9B2, also known as NHA2, correlates with the long-sought-after Na+/Li+ exchanger linked to the pathogenesis of diabetes mellitus and essential hypertension in humans. Despite the functional importance of NHA2, structural information and the molecular basis for its ion-exchange mechanism have been lacking. Here we report the cryo-EM structures of bison NHA2 in detergent and in nanodiscs, at 3.0 and 3.5 Å resolution, respectively. The bison NHA2 structure, together with solid-state membrane-based electrophysiology, establishes the molecular basis for electroneutral ion exchange. NHA2 consists of 14 transmembrane (TM) segments, rather than the 13 TMs previously observed in mammalian Na+/H+ exchangers (NHEs) and related bacterial antiporters. The additional N-terminal helix in NHA2 forms a unique homodimer interface with a large intracellular gap between the protomers, which closes in the presence of phosphoinositol lipids. We propose that the additional N-terminal helix has evolved as a lipid-mediated remodeling switch for the regulation of NHA2 activity.
Auxins are hormones that have central roles and control nearly all aspects of growth and development in plants. The proteins in the PIN-FORMED (PIN) family (also known as the auxin efflux carrier family) are key participants in this process and control auxin export from the cytosol to the extracellular space. Owing to a lack of structural and biochemical data, the molecular mechanism of PIN-mediated auxin transport is not understood. Here we present biophysical analysis together with three structures of Arabidopsis thaliana PIN8: two outward-facing conformations with and without auxin, and one inward-facing conformation bound to the herbicide naphthylphthalamic acid. The structure forms a homodimer, with each monomer divided into a transport and scaffold domain with a clearly defined auxin binding site. Next to the binding site, a proline–proline crossover is a pivot point for structural changes associated with transport, which we show to be independent of proton and ion gradients and probably driven by the negative charge of the auxin. The structures and biochemical data reveal an elevator-type transport mechanism reminiscent of bile acid/sodium symporters, bicarbonate/sodium symporters and sodium/proton antiporters. Our results provide a comprehensive molecular model for auxin recognition and transport by PINs, link and expand on a well-known conceptual framework for transport, and explain a central mechanism of polar auxin transport, a core feature of plant physiology, growth and development.
Here, we present a solid-supported membrane (SSM)-based electrophysiological approach to study sugar binding and Na+/glucose cotransport by SGLT1 in membrane vesicles. SSM-based electrophysiology delivers a cumulative real-time current readout from numerous SGLT1 proteins simultaneously using a gold-coated sensor chip.In contrast to conventional techniques, which mainly operate with voltage steps, currents are triggered by sugar or sodium addition. Sugar concentration jumps in the presence of sodium lead to transport currents between 5 and 10 nA. Remarkably, in the absence of sodium (i.e. no transport), we observed fast pre-steady-state (PSS) currents with time constants between 3 and 10 ms. These PSS currents mainly originate from sugar binding. Sodium binding does not induce PSS currents. Due to high time resolution, PSS currents were distinguished from transport and eventually correlated with conformational transitions within the sugar translocation pathway.In addition, we analyzed the impact of driving forces on transport and binding currents, showing that membrane voltage and sodium concentration gradients lead to an increased transport rate without affecting sugar binding kinetics. We also compared Na+/sugar efflux with physiologically relevant influx and found similar transport rates, but lower affinity in efflux mode.SSM-based electrophysiology is a powerful technique, which overcomes bottlenecks for transport measurements observed in other techniques such as the requirement of labels or the lack of real-time data. Rapid solution exchange enables the observation of substrate-induced electrogenic events like conformational transitions, opening novel perspectives for in-depth functional studies of SGLT1 and other transporters.
Na+/H+ exchangers catalyse an ion-exchange activity that is carried out in most, if not all cells. SLC9B2, also known as NHA2, correlates with the long-sought after sodium/lithium (Na+/Li+) exchanger linked to the pathogenesis of diabetes mellitus and essential hypertension in humans. Despite its functional importance, structural information and the molecular basis of its ion-exchange mechanism have been lacking. Here, we report the cryo EM structures of bison NHA2 in detergent and in nanodiscs at 3.0 and 3.5 Å resolution, respectively. NHA2 shares closest structural similarity to the bacterial electrogenic Na+/H+ antiporter NapA, rather than other mammalian SLC9A members. Nevertheless, SSM-based electrophysiology results with NHA2 show the catalysis of electroneutral rather than electrogenic ion exchange, and the ion-binding site is quite distinctive, with a tryptophan-arginine- glutamate triad separated from the well-established ion-binding aspartates. These triad residues fine-tune ion binding specificity, as demonstrated by a salt-bridge swap mutant that converts NHA2 into a Li+-specific transporter. Strikingly, an additional N-terminal helix in NHA2 establishes a unique homodimer with a large ∼ 25 Å intracellular gap between protomers. In the presence of phosphatidylinositol lipids, the N-terminal helix rearranges and closes this gap. We confirm that dimerization of NHA2 is required for activity in vivo, and propose that the N- terminal helix has evolved as a lipid-mediated remodelling switch for regulation of transport activity.
Phosphocholine molecules decorating bacterial cell wall teichoic acids and outer-membrane lipopolysaccharide have fundamental roles in adhesion to host cells, immune evasion, and persistence. Bacteria carrying the operon that performs phosphocholine decoration synthesize phosphocholine after uptake of the choline precursor by LicB, a conserved transporter among divergent species. Streptococcus pneumoniae is a prominent pathogen where phosphocholine decoration plays a fundamental role in virulence. Here, we present cryo–electron microscopy and crystal structures of S. pneumoniae LicB, revealing distinct conformational states and describing architectural and mechanistic elements essential to choline import. Together with in vitro and in vivo functional characterization, we found that LicB displays proton-coupled import activity and promiscuous selectivity involved in adaptation to choline deprivation conditions, and describe LicB inhibition by synthetic nanobodies (sybodies). Our results provide previously unknown insights into the molecular mechanism of a key transporter involved in bacterial pathogenesis and establish a basis for inhibition of the phosphocholine modification pathway across bacterial phyla.
Phosphocholine molecules decorating bacterial cell wall teichoic acids and outer-membrane lipopolysaccharide have significant roles in adhesion to host cells, immune evasion, and persistence. Bacteria carrying the operon that performs phosphocholine decoration, synthesize phosphocholine after uptake of the choline precursor by LicB, a conserved transporter among divergent species. Streptococcus pneumoniae is a prominent pathogen where phosphocholine decoration plays a fundamental role in virulence. Here we present cryo-electron microscopy and crystal structures of S. pneumoniae LicB, revealing distinct conformational states and describing architectural and mechanistic elements essential to choline import. Together with in vitro and in vivo functional characterization, we found that LicB displays proton-coupled import activity and promiscuous selectivity involved in adaptation to choline deprivation conditions, and describe LicB inhibition by synthetic nanobodies (sybodies) and hemicholinium-3. Our results provide novel insights into the molecular mechanism of a key transporter involved in bacterial pathogenesis and establish a basis for inhibition of the phosphocholine modification pathway across bacterial phyla.
ATP8A2 is a mammalian P4-ATPase (flippase) that translocates the negatively charged lipid substrate phosphatidylserine from the exoplasmic leaflet to the cytoplasmic leaflet of cellular membranes. Using an electrophysiological method based on solid supported membranes, we investigated the electrogenicity of specific reaction steps of ATP8A2 and explored a potential phospholipid translocation pathway involving residues with positively charged side chains. Changes to the current signals caused by mutations show that the main electrogenic event occurs in connection with release of the bound phosphatidylserine to the cytoplasmic leaflet and support the hypothesis that the phospholipid interacts with specific lysine and arginine residues near the cytoplasmic border of the lipid bilayer during the translocation and/or reorientation required for insertion into the cytoplasmic leaflet.
The human peptide transporter hPEPT1 (SLC15A1) is responsible for uptake of dietary di- and tripeptides and a number of drugs from the small intestine by utilizing the proton electrochemical gradient, and hence an important target for peptide-like drug design and drug delivery. hPEPT1 belongs to the ubiquitous major facilitator superfamily that all contain a 12TM core structure, with global conformational changes occurring during the transport cycle. Several bacterial homologues of these transporters have been characterized, providing valuable insight into the transport mechanism of this family. Here we report the overexpression and purification of recombinant hPEPT1 in a detergent-solubilized state. Thermostability profiling of hPEPT1 at different pH values revealed that hPEPT1 is more stable at pH 6 as compared to pH 7 and 8. Micro-scale thermophoresis (MST) confirmed that the purified hPEPT1 was able to bind di- and tripeptides respectively. To assess the in-solution oligomeric state of hPEPT1, negative stain electron microscopy was performed, demonstrating a predominantly monomeric state.
Ammonium translocation through biological membranes, by the ubiquitous Amt-Mep-Rh family of transporters, plays a key role in all domains of life. Two highly conserved histidine residues protrude into the lumen of the pore of these transporters, forming the family’s characteristic Twin-His motif. It has been hypothesized that the motif is essential to confer the selectivity of the transport mechanism. Here, using a combination of in vitro electrophysiology on Escherichia coli AmtB, in silico molecular dynamics simulations, and in vivo yeast functional complementation assays, we demonstrate that variations in the Twin-His motif trigger a mechanistic switch between a specific transporter, depending on ammonium deprotonation, to an unspecific ion channel activity. We therefore propose that there is no selective filter that governs specificity in Amt-Mep-Rh transporters, but the inherent mechanism of translocation, dependent on the fragmentation of the substrate, ensures the high specificity of the translocation. We show that coexistence of both mechanisms in single Twin-His variants of yeast Mep2 transceptors disrupts the signaling function and so impairs fungal filamentation. These data support a signaling process driven by the transport mechanism of the fungal Mep2 transceptors.
Proteins from the bacterial small multidrug resistance (SMR) family are proton-coupled exporters of diverse antiseptics and antimicrobials, including polyaromatic cations and quaternary ammonium compounds. The transport mechanism of the Escherichia coli transporter, EmrE, has been studied extensively, but a lack of high-resolution structural information has impeded a structural description of its molecular mechanism. Here we apply a novel approach, multipurpose crystallization chaperones, to solve several structures of EmrE, including a 2.9 Å structure at low pH without substrate. We report five additional structures in complex with structurally diverse transported substrates, including quaternary phosphonium, quaternary ammonium, and planar polyaromatic compounds. These structures show that binding site tryptophan and glutamate residues adopt different rotamers to conform to disparate structures without requiring major rearrangements of the backbone structure. Structural and functional comparison to Gdx-Clo, an SMR protein that transports a much narrower spectrum of substrates, suggests that in EmrE, a relatively sparse hydrogen bond network among binding site residues permits increased sidechain flexibility.
The dimeric transporter, EmrE, effluxes polyaromatic cationic drugs in a proton-coupled manner to confer multidrug resistance in bacteria. Although the protein is known to adopt an antiparallel asymmetric topology, its high-resolution drug-bound structure is so far unknown, limiting our understanding of the molecular basis of promiscuous transport. Here we report an experimental structure of drug-bound EmrE in phospholipid bilayers, determined using 19F and 1H solid-state NMR and a fluorinated substrate, tetra(4-fluorophenyl) phosphonium (F4-TPP+). The drug-binding site, constrained by 214 protein-substrate distances, is dominated by aromatic residues such as W63 and Y60, but is sufficiently spacious for the tetrahedral drug to reorient at physiological temperature. F4-TPP+ lies closer to the proton-binding residue E14 in subunit A than in subunit B, explaining the asymmetric protonation of the protein. The structure gives insight into the molecular mechanism of multidrug recognition by EmrE and establishes the basis for future design of substrate inhibitors to combat antibiotic resistance.
Copper (Cu)-transporting P1B-type ATPases are ubiquitous metal transporters and crucial for maintaining Cu homeostasis in all domains of life. In bacteria, the P1B-type ATPase CopA is required for Cu-detoxification and exports excess Cu(I) in an ATP-dependent reaction from the cytosol into the periplasm. CopA is a member of the CopA1-type ATPase family and has been biochemically and structurally characterized in detail. In contrast, less is known about members of the CopA2-type ATPase family, which are predicted to transport Cu(I) into the periplasm for cuproprotein maturation. One example is CcoI, which is required for the maturation of cbb3-type cytochrome oxidase (cbb3-Cox) in different species. Here, we reconstituted purified CcoI of Rhodobacter capsulatus into liposomes and determined Cu transport using solid-supported membrane electrophysiology. The data demonstrate ATP-dependent Cu(I) translocation by CcoI, while no transport is observed in the presence of a non-hydrolysable ATP analog. CcoI contains two cytosolically exposed N-terminal metal binding sites (N-MBSs), which are both important, but not essential for Cu delivery to cbb3-Cox. CcoI and cbb3-Cox activity assays in the presence of different Cu concentrations suggest that the glutaredoxin-like N-MBS1 is primarily involved in regulating the ATPase activity of CcoI, while the CopZ-like N-MBS2 is involved in Cu(I) acquisition. The interaction of CcoI with periplasmic Cu chaperones was analyzed by genetically fusing CcoI to the chaperone SenC. The CcoI-SenC fusion protein was fully functional in vivo and sufficient to provide Cu for cbb3-Cox maturation. In summary, our data demonstrate that CcoI provides the link between the cytosolic and periplasmic Cu chaperone networks during cbb3-Cox assembly.
The sarco(endo)plasmic reticulum Ca2+−ATPase (SERCA) hydrolyzes ATP to transport Ca2+ from the cytoplasm to the sarcoplasmic reticulum (SR) lumen, thereby inducing muscle relaxation. Dysfunctional SERCA has been related to various diseases. The identification of small‐molecule drugs that can activate SERCA may offer a therapeutic approach to treat pathologies connected with SERCA malfunction. Herein, we propose a method to study the mechanism of interaction between SERCA and novel SERCA activators, i. e. CDN1163, using a solid supported membrane (SSM) biosensing approach. Native SR vesicles or reconstituted proteoliposomes containing SERCA were adsorbed on the SSM and activated by ATP concentration jumps. We observed that CDN1163 reversibly interacts with SERCA and enhances ATP‐dependent Ca2+ translocation. The concentration dependence of the CDN1163 effect provided an EC50=6.0±0.3 μM. CDN1163 was shown to act directly on SERCA and to exert its stimulatory effect under physiological Ca2+ concentrations. These results suggest that CDN1163 interaction with SERCA can promote a protein conformational state that favors Ca2+ release into the SR lumen.
KdpFABC is an oligomeric K+ transport complex in prokaryotes that maintains ionic homeostasis under stress conditions. The complex comprises a channel-like subunit (KdpA) from the Superfamily of K+ Transporters and a pump-like subunit (KdpB) from the superfamily of P-type ATPases. Recent structural work has defined the architecture and generated contradictory hypotheses for the transport mechanism. Here, we use substrate analogs to stabilize four key intermediates in the reaction cycle and determine the corresponding structures by cryo-EM. We find that KdpB undergoes conformational changes consistent with other representatives from the P-type superfamily, whereas KdpA, KdpC and KdpF remain static. We observe a series of spherical densities that we assign as K+ or water and which define a pathway for K+ transport. This pathway runs through an intramembrane tunnel in KdpA and delivers ions to sites in the membrane domain of KdpB. Our structures suggest a mechanism where ATP hydrolysis is coupled to K+ transfer between alternative sites in the membrane domain of KdpB, ultimately reaching a low-affinity site where a water-filled pathway allows release of K+ to the cytoplasm.
Here, we present a protocol for the functional characterization of the H+-coupled human peptide transporter PepT1 and sufficient notes to transfer the protocol to the Na+-coupled sugar transporter SGLT1, the organic cation transporter OCT2, the Na+/Ca2+ exchanger NCX, and the neuronal glutamate transporter EAAT3.The assay was developed for the commercially available SURFE2R N1 instrument (Nanion Technologies GmbH) which applies solid supported membrane (SSM)-based electrophysiology. This technique is widely used for the functional characterization of membrane transporters with more than 100 different transporters characterized so far. The technique is cost-effective, easy to use, and capable of high-throughput measurements.SSM-based electrophysiology utilizes SSM-coated gold sensors to physically adsorb membrane vesicles containing the protein of interest. A fast solution exchange provides the substrate and activates transport. For the measurement of PepT1 activity, we applied a peptide concentration jump to activate H+/peptide symport. Proton influx charges the sensor. A capacitive current is measured reflecting the transport activity of PepT1. Multiple measurements on the same sensor allow for comparison of transport activity under different conditions. Here, we determine EC50 for PepT1-mediated glycylglycine transport and perform an inhibition experiment using the specific peptide inhibitor Lys[Z(NO2)]-Val.
Single domain antibodies (nanobodies) have been extensively used in mechanistic and structural studies of proteins and they pose an enormous potential as tools for developing clinical therapies, many of which depend on the inhibition of membrane proteins such as transporters. However, most of the methods used to determine the inhibition of transport activity are difficult to perform in high-throughput routines and depend on labeled substrates availability thereby complicating the screening of large nanobody libraries. Solid-supported membrane (SSM) electrophysiology is a high-throughput method, used for characterizing electrogenic transporters and measuring their transport kinetics and inhibition. Here we show the implementation of SSM-based electrophysiology to select inhibitory and non-inhibitory nanobodies targeting an electrogenic secondary transporter and to calculate nanobodies inhibitory constants. This technique may be especially useful for selecting inhibitory nanobodies targeting transporters for which labeled substrates are not available.
Human excitatory amino acid transporter 3 (hEAAT3) mediates glutamate uptake in neurons, intestine, and kidney. Here, we report cryo-EM structures of hEAAT3 in several functional states where the transporter is empty, bound to coupled sodium ions only, or fully loaded with three sodium ions, a proton, and the substrate aspartate. The structures suggest that hEAAT3 operates by an elevator mechanism involving three functionally independent subunits. When the substrate-binding site is near the cytoplasm, it has a remarkably low affinity for the substrate, perhaps facilitating its release and allowing the rapid transport turnover. The mechanism of the coupled uptake of the sodium ions and the substrate is conserved across evolutionarily distant families and is augmented by coupling to protons in EAATs. The structures further suggest a mechanism by which a conserved glutamate residue mediates proton symport.
Novel approaches in synthetic biology focus on the bottom-up modular assembly of natural, modified natural or artificial components into molecular systems with functionalities not found in nature. A possible application for such techniques is the bioremediation of natural water sources contaminated with small organic molecules (e.g., drugs and pesticides). A simple molecular system to actively accumulate and degrade pollutants could be a bionanoreactor composed of a liposome or polymersome scaffold combined with energizing- (e.g., light-driven proton pump), transporting- (e.g., proton-driven transporter) and degrading modules (e.g., enzyme). This work focuses on the engineering of a transport module specific for β-lactam antibiotics. We previously solved the crystal structure of a bacterial peptide transporter, which allowed us to improve the affinity for certain β-lactam antibiotics using structure-based mutagenesis combined with a bacterial uptake assay. We were able to identify specific mutations, which enhanced the affinity of the transporter for antibiotics containing certain structural features. Screening of potential compounds allowed for the identification of a β-lactam antibiotic ligand with relatively high affinity. Transport of antibiotics was evaluated using a solid-supported membrane electrophysiology assay. In summary, we have engineered a proton-driven β-lactam antibiotic translocation module, contributing to the growing toolset for bionanotechnological applications.
Ammonium translocation through biological membranes by the ubiquitous Amt-Mep-Rh family of transporters plays a key role in all domains of life. Two highly conserved histidine residues protrude into the lumen of these transporters, forming the family’s characteristic Twin-His motif. It has been hypothesized that the motif is essential to confer the selectivity of the transport mechanism. Here, using a combination of in vitro electrophysiology, in vivo yeast functional complementation and in silico molecular dynamics simulations, we demonstrate that variations in the Twin-His motif trigger a mechanistic switch between a specific transporter, depending on ammonium deprotonation, to an unspecific ion channel activity. We therefore propose that there is no selective filter that governs the specificity in Amt-Mep transporters but the inherent mechanism of translocation, dependent on the fragmentation of the substrate, ensures the high specificity of the translocation. We further show that both mechanisms coexist in fungal Mep2 Twin-His variants, disrupting the transceptor function and so inhibiting the filamentation process. These data strongly support a transport mechanism-mediated signalling process in the long-standing debate on the sensory function of Mep2-like transporters.
Transport stoichiometry determination can provide great insight into the mechanism and function of ion-coupled transporters. Traditional reversal potential assays are a reliable, general method for determining the transport stoichiometry of ion-coupled transporters, but the time and material costs of this technique hinder investigations of transporter behavior under multiple experimental conditions. Solid supported membrane electrophysiology (SSME) allows multiple recordings of liposomal or membrane samples adsorbed onto a sensor, and is sensitive enough to detect transport currents from moderate-flux transporters that are inaccessible to traditional electrophysiology techniques. Here, we use SSME to develop a new method for measuring transport stoichiometry with greatly improved throughput. Using this technique, we were able to verify the recent report of a fixed 2:1 stoichiometry for the proton:guanidinium antiporter Gdx, reproduce the 1H+:2Cl- antiport stoichiometry of CLC-ec1, and confirm loose proton:nitrate coupling for CLC-ec1. Furthermore, we were able to demonstrate quantitative exchange of internal contents of liposomes adsorbed onto SSME sensors to allow multiple experimental conditions to be tested on a single sample. Our SSME method provides a fast, easy, general method for measuring transport stoichiometry, which will facilitate future mechanistic and functional studies of ion-coupled transporters.
By providing broad resistance to environmental biocides, transporters from the small multidrug resistance (SMR) family drive the spread of multidrug resistance cassettes among bacterial populations. A fundamental understanding of substrate selectivity by SMR transporters is needed to identify the types of selective pressures that contribute to this process. Using solid-supported membrane electrophysiology, we find that promiscuous transport of hydrophobic substituted cations is a general feature of SMR transporters. To understand the molecular basis for promiscuity, we solved X-ray crystal structures of a SMR transporter Gdx-Clo in complex with substrates to a maximum resolution of 2.3 Å. These structures confirm the family’s extremely rare dual topology architecture and reveal a cleft between two helices that provides accommodation in the membrane for the hydrophobic substituents of transported drug-like cations.
Transport stoichiometry provides insight into the mechanism and function of ion-coupled transporters, but measuring transport stoichiometry is time-consuming and technically difficult. With the increasing evidence that many ion-coupled transporters employ multiple transport stoichiometries under different conditions, improved methods to determine transport stoichiometry are required to accurately characterize transporter activity. Reversal potential was previously shown to be a reliable, general method for determining the transport stoichiometry of ion-coupled transporters (Fitzgerald & Mindell, 2017). Here, we develop a new technique for measuring transport stoichiometry with greatly improved throughput using solid supported membrane electrophysiology (SSME). Using this technique, we are able to verify the recent report of a fixed 2:1 stoichiometry for the proton:guanidinium antiporter Gdx. Our SSME method requires only small amounts of transporter and provides a fast, easy, general method for measuring transport stoichiometry, which will facilitate future mechanistic and functional studies of ion-coupled transporters.
Transition metals are essential trace elements and their high-affinity uptake is required for many organisms. Metal transporters are often characterised using metal-sensitive fluorescent dyes, limiting the metals and experimental conditions that can be studied. Here, we have tested whether metal transport by Enterococcus faecalis MntH2 can be measured with an electrophysiology method that is based on the solid-supported membrane technology. E. faecalis MntH2 belongs to the Natural Resistance-Associated Macrophage Protein (Nramp) family of proton-coupled transporters, which transport divalent transition metals and do not transport the earth metals. Electrophysiology confirms transport of Mn(II), Co(II), Zn(II) and Cd(II) by MntH2. However, no uptake responses for Cu(II), Fe(II) and Ni(II) were observed, while the presence of these metals abolishes the uptake signals for Mn(II). Fluorescence assays confirm that Ni(II) is transported. The data are discussed with respect to properties and structures of Nramp-type family members and the ability of electrophysiology to measure charge transport and not directly substrate transport.
Those of a certain age may remember (and their younger colleagues can read) accounts of the vivid debate in the 1970s surrounding the coupling mechanism involved in oxidative and photo phosphorylation. By that time, Mitchell's chemiosmotic hypothesis had already gained credence, and the debated issue was how a transmembrane H+ potential difference drives ATP synthesis by Ftype ATP synthases. The major mechanisms that were considered assumed that the membrane (Fo) and peripheral (F1) parts were functionally connected in different ways. Peter Mitchell proposed a “direct coupling” mechanism in which protons are translocated through Fo into the catalytic site of F1, where they participate directly in ADP phosphorylation and form water as the second product (Mitchell, 1974). Paul Boyer, the proponent of the main competing mechanism, advocated an “indirect coupling” mechanism (successively termed “alternating site”, “binding change”, or “rotational”) that implied that protons transfer their energy to the catalytic site indirectly, via distant conformational strain (Boyer, 1997). The debate was resolved in favor of Boyer's mechanism when it became clear that the alternative mechanism is inconsistent with H+ /ATP stoichiometry and, finally, when the three-dimensional structure of the F-ATPase was determined (Abrahams et al., 1994).
Significance: Phospholipid flippases constitute the largest subfamily of P-type ATPases and have in eukaryotic organisms evolved as a central transport system for selective translocation of phospholipids across biological membranes to generate membrane lipid asymmetry, a property essential for numerous cellular processes. The importance of flippases is highlighted by severe neurological disorders and liver diseases caused by flippase dysfunction in humans. The electrogenicity of phospholipid transport by flippases has not previously been explored. We demonstrated that phosphatidylserine translocation by the flippase ATP8A2 generates electrical current, resulting from specific steps in the flippase reaction cycle moving the charged lipid head group between the membrane bilayer leaflets, and that no charged substrate is being countertransported. These findings unravel key features of phospholipid flippases. Abstract: Phospholipid flippases (P4-ATPases) utilize ATP to translocate specific phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of biological membranes, thus generating and maintaining transmembrane lipid asymmetry essential for a variety of cellular processes. P4-ATPases belong to the P-type ATPase protein family, which also encompasses the ion transporting P2-ATPases: Ca2+-ATPase, Na+/K+-ATPase, and H+,K+-ATPase. In comparison with the P2-ATPases, understanding of P4-ATPases is still very limited. The electrogenicity of P4-ATPases has not been explored, and it is not known whether lipid transfer between membrane bilayer leaflets can lead to displacement of charge across the membrane. A related question is whether P4-ATPases countertransport ions or other substrates in the opposite direction, similar to the P2-ATPases. Using an electrophysiological method based on solid supported membranes, we observed the generation of a transient electrical current by the mammalian P4-ATPase ATP8A2 in the presence of ATP and the negatively charged lipid substrate phosphatidylserine, whereas only a diminutive current was generated with the lipid substrate phosphatidylethanolamine, which carries no or little charge under the conditions of the measurement. The current transient seen with phosphatidylserine was abolished by the mutation E198Q, which blocks dephosphorylation. Likewise, mutation I364M, which causes the neurological disorder cerebellar ataxia, mental retardation, and disequilibrium (CAMRQ) syndrome, strongly interfered with the electrogenic lipid translocation. It is concluded that the electrogenicity is associated with a step in the ATPase reaction cycle directly involved in translocation of the lipid. These measurements also showed that no charged substrate is being countertransported, thereby distinguishing the P4-ATPase from P2-ATPases.
The transport of charged molecules across biological membranes faces the dual problem of accommodating charges in a highly hydrophobic environment while maintaining selective substrate translocation. This has been the subject of a particular controversy for the exchange of ammonium across cellular membranes, an essential process in all domains of life. Ammonium transport is mediated by the ubiquitous Amt/Mep/Rh transporters that includes the human Rhesus factors. Here, using a combination of electrophysiology, yeast functional complementation and extended molecular dynamics simulations, we reveal a unique two-lane pathway for electrogenic NH4+ transport in two archetypal members of the family, the transporters AmtB from Escherichia coli and Rh50 from Nitrosomonas europaea. The pathway underpins a mechanism by which charged H+ and neutral NH3 are carried separately across the membrane after NH4+ deprotonation. This mechanism defines a new principle of achieving transport selectivity against competing ions in a biological transport process.
The movement of ammonium across biological membranes is a fundamental process in all living organisms and is mediated by the ubiquitous Amt/Mep/Rh family of transporters. Recent structural analysis and coupled mass spectrometry studies have shown that the Escherichia coli ammonium transporter, AmtB, specifically binds phosphatidylglycerol (PG). Upon PG binding, several residues of AmtB undergo a small conformational change, which stabilizes the protein against unfolding. However, no studies have so far been conducted to explore if PG binding to AmtB has functional consequences. Here, we used an in vitro experimental assay with purified components together with molecular dynamics simulations to characterise the relation between PG binding and AmtB activity. Firstly, our results indicate that the function of Amt in archaebacteria and eubacteria may differ. Secondly, we show that PG is an essential cofactor for AmtB activity and that in the absence of PG AmtB cannot complete the full translocation cycle. Furthermore, our simulations reveal previously undiscovered PG binding sites on the intracellular side of the lipid bilayer between the AmtB subunits. The possible molecular mechanisms explaining the functional role of PG are discussed.
Bacterial NhaB Na+/H+ exchangers belonging to the Ion Transporter superfamily are poorly characterized in contrast to Na+/H+ exchangers of the Cation Proton Antiporter superfamily which have NhaA from Escherichia coli as a prominent member. For a more detailed understanding of the intricacies of the exchanger’s transport mechanism, mutational studies are essential. Therefore, we mutated two protonatable residues present in the putative transmembrane region of NhaB from Klebsiella pneumoniae (KpNhaB), which could serve as substrate binding sites, Asp146 and Asp404, to either glutamate or alanine and analyzed transport function and stability of the mutants using electrophysiological and fluorimetric techniques. While mutation of either Asp residue to Glu only had slight to moderate effects on the transport activity of the exchanger, the mutations D404A and D146A, in particular, had more profound effects on the transport function. Furthermore, a double mutant, D146A/D404A, exhibited a remarkable behavior at alkaline pH, where recorded electrical currents changed polarity, showing steady-state transport with a stoichiometry of H+:Na+ 1, as opposed to the H+:Na+ > 1 stoichiometry of the WT. Thus, we showed that Asp146 and Asp404 are part of the substrate binding site(s) of KpNhaB and engineered a Na+/H+ exchanger with a variable stoichiometry.
Membrane-integral pyrophosphatases (mPPases) couple the hydrolysis of pyrophosphate (PPi) to the pumping of Na+, H+, or both these ions across a membrane. Recently solved structures of the Na+-pumping Thermotoga maritima mPPase (TmPPase) and H+-pumping Vigna radiata mPPase revealed the basis of ion selectivity between these enzymes and provided evidence for the mechanisms of substrate hydrolysis and ion-pumping. Our atomistic molecular dynamics (MD) simulations of TmPPase demonstrate that loop 5–6 is mobile in the absence of the substrate or substrate-analogue bound to the active site, explaining the lack of electron density for this loop in resting state structures. Furthermore, creating an apo model of TmPPase by removing ligands from the TmPPase:IDP:Na structure in MD simulations resulted in increased dynamics in loop 5–6, which results in this loop moving to uncover the active site, suggesting that interactions between loop 5–6 and the imidodiphosphate and its associated Mg2+ are important for holding a loop-closed conformation. We also provide further evidence for the transport-before-hydrolysis mechanism by showing that the non-hydrolyzable substrate analogue, methylene diphosphonate, induces low levels of proton pumping by VrPPase.
ATP7A and ATP7B are Cu+ -transporting ATPases of subclass IB and play a fundamental role in intracellular copper homeostasis. ATP7A/B transfer Cu+ ions across the membrane from delivery to acceptor proteins without establishing a free Cu+ gradient. Transfer of copper across the membrane is coupled to ATP hydrolysis. Current measurements on solid supported membranes (SSM) were performed to investigate the mechanism of copper-related charge transfer across ATP7A and ATP7B. SSM measurements demonstrated that electrogenic copper displacement occurs within ATP7A/B following addition of ATP and formation of the phosphorylated intermediate. Comparison of the time constants for cation displacement in ATP7A/B and sarcoplasmic reticulum Ca2+ -ATPase is consistent with the slower phosphoenzyme formation in copper ATPases. Moreover, ATP-dependent copper transfer in ATP7A/B is not affected by varying the pH, suggesting that net proton counter-transport may not occur in copper ATPases. Platinum anticancer drugs activate ATP7A/B and are subjected to ATP-dependent vectorial displacement with a mechanism analogous to that of copper.
Cholinergic hypofunction is associated with decreased attention and cognitive deficits in the central nervous system in addition to compromised motor function. Consequently, stimulation of cholinergic neurotransmission is a rational therapeutic approach for the potential treatment of a variety of neurological conditions. High affinity choline uptake (HACU) into acetylcholine (ACh)-synthesizing neurons is critically mediated by the sodium- and pH-dependent high-affinity choline transporter (CHT, encoded by the SLC5A7 gene). This transporter is comparatively well-characterized but otherwise unexplored as a potential drug target. We therefore sought to identify small molecules that would enable testing of the hypothesis that positive modulation of CHT mediated transport would enhance activity-dependent cholinergic signaling. We utilized existing and novel screening techniques for their ability to reveal both positive and negative modulation of CHT using literature tools. A screening campaign was initiated with a bespoke compound library comprising both the Pfizer Chemogenomic Library (CGL) of 2,753 molecules designed specifically to help enable the elucidation of new mechanisms in phenotypic screens and 887 compounds from a virtual screening campaign to select molecules with field-based similarities to reported negative and positive allosteric modulators. We identified a number of previously unknown active and structurally distinct molecules that could be used as tools to further explore CHT biology or as a starting point for further medicinal chemistry.
Cisplatin (cis-diamminedichlorido-Pt(ii)) is extensively used as a chemotherapeutic agent against various types of tumors. However, cisplatin administration causes serious side effects, including nephrotoxicity, ototoxicity and neurotoxicity. It has been shown that cisplatin can interact with P-type ATPases, e.g., Cu+-ATPases (ATP7A and ATP7B) and Na+/K+-ATPase. Cisplatin-induced inhibition of Na+/K+-ATPase has been related to the nephrotoxic effect of the drug. To investigate the inhibitory effects of cisplatin on the pumping activity of PII-type ATPases, electrical measurements were performed on sarcoplasmic reticulum Ca2+-ATPase (SERCA) and Na+/K+-ATPase embedded in vesicles/membrane fragments adsorbed on a solid-supported membrane. We found that cisplatin inhibits SERCA and Na+/K+-ATPase only when administered without a physiological reducing agent (GSH); in contrast, inhibition was also observed in the case of Cu+-ATPases in the presence of 1 mM GSH. Our results indicate that cisplatin is a much stronger inhibitor of SERCA (with an IC50 value of 1.3 μM) than of Na+/K+-ATPase (with an IC50 value of 11.1 μM); moreover, cisplatin inhibition of Na+/K+-ATPase is reversible, whereas it is irreversible in the case of SERCA. In the absence of a physiological substrate, while Cu+-ATPases are able to translocate cisplatin, SERCA and Na+/K+-ATPase do not perform ATP-dependent cisplatin displacement.
Na+/H+ exchange is essential for survival of all organisms, having a role in the regulation of the intracellular Na+ concentration, pH and cell volume. Furthermore, Na+/H+ exchangers were shown to be involved in the virulence of the bacterium Yersinia pestis, indicating they might be potential targets for novel antibiotic treatments. The model system for Na+/H+ exchangers is the NhaA transporter from Escherichia coli, EcNhaA. Therefore, the general transport mechanism of NhaA exchangers is currently well characterized. However, much less is known about NhaB exchangers, with only a limited number of studies available. The pathogen Klebsiella pneumoniae, which is a major source of nosocomial infection, possesses three electrogenic Na+/H+ exchangers, KpNhaA1, KpNhaA2 and KpNhaB, none of which have been previously investigated. Our aim in this study was to functionally characterize KpNhaB using solid supported membrane-based electrophysiology as the main investigation technique, and thus provide the first electrophysiological investigation of an NhaB Na+/H+ exchanger. We found that NhaB can be described by the same competition-based mechanism that was shown to be valid for electrogenic NhaA and NapA, and for electroneutral NhaP Na+/H+ exchangers. For comparison we also characterized the activity of KpNhaA1 and KpNhaA2 and found that the three exchangers have complementary activity profiles, which is likely a survival advantage for K. pneumoniae when faced with environments of different salinity and pH. This underlines their importance as potential antibiotic drug targets.
The sarcoplasmic reticulum Ca2+-ATPase SERCA promotes muscle relaxation by pumping calcium ions from the cytoplasm into the sarcoplasmic reticulum. SERCA activity is regulated by a variety of small transmembrane peptides, most notably by phospholamban in cardiac muscle and sarcolipin in skeletal muscle. However, how phospholamban and sarcolipin regulate SERCA is not fully understood. In the present study, we evaluated the effects of phospholamban and sarcolipin on calcium translocation and ATP hydrolysis by SERCA under conditions that mimic environments in sarcoplasmic reticulum membranes. For pre-steady-state current measurements, proteoliposomes containing SERCA and phospholamban or sarcolipin were adsorbed to a solid-supported membrane and activated by substrate concentration jumps. We observed that phospholamban altered ATP-dependent calcium translocation by SERCA within the first transport cycle, whereas sarcolipin did not. Using pre-steady-state charge (calcium) translocation and steady-state ATPase activity under substrate conditions (various calcium and/or ATP concentrations) promoting particular conformational states of SERCA, we found that the effect of phospholamban on SERCA depends on substrate preincubation conditions. Our results also indicated that phospholamban can establish an inhibitory interaction with multiple SERCA conformational states with distinct effects on SERCA's kinetic properties. Moreover, we noted multiple modes of interaction between SERCA and phospholamban and observed that once a particular mode of association is engaged it persists throughout the SERCA transport cycle and multiple turnover events. These observations are consistent with conformational memory in the interaction between SERCA and phospholamban, thus providing insights into the physiological role of phospholamban and its regulatory effect on SERCA transport activity.
Membrane-bound pyrophosphatases (M-PPases), which couple proton/sodium ion transport to pyrophosphate synthesis/hydrolysis, are important in abiotic stress resistance and in the infectivity of protozoan parasites. Here, three M-PPase structures in different catalytic states show that closure of the substrate-binding pocket by helices 5–6 affects helix 13 in the dimer interface and causes helix 12 to move down. This springs a ‘molecular mousetrap’, repositioning a conserved aspartate and activating the nucleophilic water. Corkscrew motion at helices 6 and 16 rearranges the key ionic gate residues and leads to ion pumping. The pumped ion is above the ion gate in one of the ion-bound structures, but below it in the other. Electrometric measurements show a single-turnover event with a non-hydrolysable inhibitor, supporting our model that ion pumping precedes hydrolysis. We propose a complete catalytic cycle for both proton and sodium-pumping M-PPases, and one that also explains the basis for ion specificity.
Bacterial sugar symporters in the Major Facilitator Superfamily (MFS) use the H+ (and in a few cases Na+) electrochemical gradients to achieve active transport of sugar into the cell. Because a number of structures of MFS sugar symporters have been solved recently, molecular insight into the transport mechanism is possible from detailed functional analysis. We present here a comparative electrophysiological study of the lactose permease (LacY), the fucose permease (FucP) and the xylose permease (XylE), which reveals common mechanistic principles and differences. In all three symporters energetically downhill electrogenic sugar/H+ symport is observed. Comparison of the pH dependence of symport at symmetrical pH exhibits broad bell-shaped pH profiles extending over 3 to 6 pH units and a decrease at extremely alkaline pH ≥ 9.4 and at acidic to neutral pH = 4.6-7.5. The pH dependence can be described by an acidic to neutral apparent pK (pKapp) and an alkaline pKapp. Experimental evidence suggests that the alkaline pKapp is due to H+ depletion at the protonation site, while the acidic pKapp is due to inhibition of deprotonation. Since previous studies suggest that a single carboxyl group in LacY (Glu325) may be the only side chain directly involved in H+ translocation and a carboxyl side chain with similar properties has been identified in FucP (Asp46) and XylE (Asp27), the present results imply that the pK of this residue is switched during H+/sugar symport in all three symporters.
Na+/H+ antiporters in the CPA1 branch of the cation proton antiporter family drive the electroneutral exchange of H+ against Na+ ions and ensure pH homeostasis in eukaryotic and prokaryotic organisms. Although their transport cycle is overall electroneutral, specific partial reactions are electrogenic. Here, we present an electrophysiological study of the PaNhaP Na+/H+ antiporter from Pyrococcus abyssi reconstituted into liposomes. Positive transient currents were recorded upon addition of Na+ to PaNhaP proteoliposomes, indicating a reaction where positive charge is rapidly displaced into the proteoliposomes with a rate constant of k >200 s-1 We attribute the recorded currents to an electrogenic reaction that includes Na+ binding and possibly occlusion. Subsequently, positive charge is transported out of the cell associated with H+ binding, so that the overall reaction is electroneutral. We show that the differences in pH profile and Na+ affinity of PaNhaP and the related MjNhaP1 from Methanocaldococcus jannaschii can be attributed to an additional negatively charged glutamate residue in PaNhaP. The results are discussed in the context of the physiological function of PaNhaP and other microbial Na+/H+ exchangers. We propose that both, electroneutral and electrogenic Na+/H+ antiporters, represent a carefully tuned self-regulatory system, which drives th