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

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

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

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

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Targeting TRP channels for the treatment of eye diseases
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Mitochondrial Membrane Research
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2013 – Functional TRPV1 expression in human corneal fibroblasts
Port-a-Patch Publication in Experimental Eye Research (2013) Authors: Yang Y., Yang H., Wang Z., Mergler S., Wolosin JM., Reinach PS.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Poster PDF
2023 – Pharmacology of P2X3and P2X2/3receptors in cell lines and hiPSC-derived neurons: An automated patch clamp study
SyncroPatch 384, Patchliner, Port-a-Patch Poster
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2023 – Gating Of the Channel Pore of Ionotropic Glutamate Receptors with Bacterial Substrate Binding Proteins: Functional Coupling of the Ectoine Binding Protein Ehub to Glur0
Port-a-Patch Publication in Journal of Biotechnology and Biomedicine. (2023) Authors: Bernhard M., Laube B

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

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

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

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

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

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

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

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

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

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

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

Read more about the article here.

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

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

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

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

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

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

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

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

Read more about the article here.

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

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

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

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

Conference poster
2023- Pharmacology of transient receptor potential cation (TRP) channels using different activation stimuli
Poster presented at the 7th RSC-BMCS / SCI Symposium on Ion Channels as Therapeutic Targets
Poster PDF
2015 – Organellar Transporters and Ion Channels – How to access their electrophysiology by using the SURFE2R technology and Planar Patch Clamp
SURFE2R N1, SyncroPatch 96 (a predecessor model of the SyncroPatch 384) and Port-a-Patch poster, GRC - Organellar Channels and Transporters 2015
Poster PDF
2018 – Expression and pharmacology of GluA2-containing AMPA receptors in cell lines and stem cell-derived neurons
Port-a-Patch, Patchliner and SyncroPatch 384PE (a predecessor model of the SyncroPatch 384) poster, Europhysiology Meeting 2018
Poster PDF
2015 – The backstage pass to study your favorite TRP channel
Port-a-Patch, Patchliner and SyncroPatch 384PE (a predecessor model of the SyncroPatch 384) poster, TRP Meeting 2015
Poster PDF
2021 – Activation and inhibition of assay-ready TRPA1 and TRPV cells: an automated patch clamp study
Port-a-Patch, Patchliner and SyncroPatch 384i (a predecessor model of SyncroPatch 384), Biophysical Society Meeting 2021
Publication link
2020 – Electrophysiological characterization of transport across outer membrane channels from Gram‐negative bacteria in presence of lipopolysaccharides (LPS)
Port-a-Patch, Vesicle Prep Pro and Orbit mini Publication in Angewandte Chemie International Edition (2020) Authors: Wang J., Terrasse R., Bafna J.A., Benier L., Winterhalter M.

Multi‐drug resistance in Gram‐negative bacteria is often associated with low permeability of the outer membrane. To investigate the role of membrane channels in the uptake of antibiotics here, we show a robust approach using fusion of native outer membrane vesicles (OMV) into planar lipid bilayer, which moreover allows also characterization of membrane protein channels in their native environment. Two major membrane channels from E. coli , OmpF and OmpC, were overexpressed from the host and the corresponding OMVs were collected. Each OMV fusion revealed surprisingly single or only a few channel activities. The asymmetry of the OMV´s translates after fusion into the lipid membrane with the LPS dominantly present at the side of OMV addition. Compared to conventional reconstitution method, the channels fused from OMVs containing LPS have similar conductance but a much broader distribution and significantly lower permeation. We suggest using outer membrane vesicles as a fast and easy approach for functional and structural studies of membrane channels in the native membrane.

Publication link
2019 – Structure of the human ClC-1 chloride channel
Port-a-Patch, Vesicle Prep Pro and Orbit mini Publication in PLOS Biology (2019) Authors: Wang K., Preisler, SS, Zhang, L., Cui, Y., Missel, JW., Grønberg C., Gotfryd, K., Lindahl E., Andersson, M., Calloe, K., Egea P.F., Klaerke D.A., Pusch M., Pedersen P.A., Zhou, Z.H., Gourdon, P.

ClC-1 protein channels facilitate rapid passage of chloride ions across cellular membranes, thereby orchestrating skeletal muscle excitability. Malfunction of ClC-1 is associated with myotonia congenita, a disease impairing muscle relaxation. Here, we present the cryo-electron microscopy (cryo-EM) structure of human ClC-1, uncovering an architecture reminiscent of that of bovine ClC-K and CLC transporters. The chloride conducting pathway exhibits distinct features, including a central glutamate residue (“fast gate”) known to confer voltage-dependence (a mechanistic feature not present in ClC-K), linked to a somewhat rearranged central tyrosine and a narrower aperture of the pore toward the extracellular vestibule. These characteristics agree with the lower chloride flux of ClC-1 compared with ClCK and enable us to propose a model for chloride passage in voltage-dependent CLC channels. Comparison of structures derived from protein studied in different experimental conditions supports the notion that pH and adenine nucleotides regulate ClC-1 through interactions between the so-called cystathionine-β-synthase (CBS) domains and the intracellular vestibule (“slow gating”). The structure also provides a framework for analysis of mutations causing myotonia congenita and reveals a striking correlation between mutated residues and the phenotypic effect on voltage gating, opening avenues for rational design of therapies against ClC-1–related diseases. Author summary Chloride transporting CLC proteins are expressed in a wide range of organisms, and the family encompasses several members with numerous roles in human health and disease by allowing movement of chloride ions across the membranes that encapsulate cells and cellular organelles. Structurally, CLCs form dimers possessing a separate ion translocation pathway in each monomer, and they can operate as either channels or transporters that exchange chloride for protons. The CLC channel ClC-1 is critical to skeletal muscle excitability and has been proposed as a target to alleviate neuromuscular disorders. Here, we have analyzed the structure of human ClC-1 and revealed the high similarity of its ion conducting pathway to those observed in other CLC members, including prokaryotic and algal transporters. Our data suggest how ClC-1 is regulated by environmental cues to allow opening and closure, thereby permitting attenuation of muscle function. Our results help with understanding the principal determinants that govern CLC proteins and may guide downstream translational applications to combat muscle pathologies.

Application Note PDF
OmpF – “Lipid Bilayer recordings of OmpF reconstituted in Proteoliposomes “
Port-a-Patch and Vesicle Prep Pro application note:  

Solvent-free planar lipid bilayers were formed in an automatic manner by bursting of giant unilamellar vesicles (GUVs) positioned by suction on the apertures of our patch clamp chips made from borosilicate glass substrate. Incubation of GUVs with purified ion channel protein of interest yielded proteoliposomes. These proteoliposomes allow for immediate recording of channel activity after GUV sealing. This approach reduces the time consuming, laborious and sometimes difficult protein reconstitution processes normally performed after bilayer formation.

Application Note PDF
KcsA – “Lipid bilayer recordings of KcsA channels reconstituted in proteoliposomes”
Port-a-Patch and Vesicle Prep Pro application note:  

Solvent-free planar lipid bilayers were formed in an automated manner using suction to attract a giant unilamellar vesicle (GUV) to the patch clamp chip which subsequently bursts across the aperture. Incubation of GUV's with purified KcsA channel protein yielded proteoliposomes. These proteoliposomes allow for immediate recording of channel activity after GUV sealing. The rapid formation of protein-containing planar lipid bilayers is of potential use for the efficient electrophysiological characterization of KcsA as shown here and also other ion channel proteins of interest.

Application Note PDF
MscL – “Lipid bilayer recordings of a mechanosensitive channel, MscL, using Nanion’s pressure clampchannel, MscL, using Nanion’s pressure clamp”
Port-a-Patch and Vesicle Prep Pro application note:  

Solvent-free planar lipid bilayers were formed in an automated manner using suction to attract a giant unilamellar vesicle (GUV) to the patch clamp chip which subsequently bursts across the aperture. Incubation of GUVs with purified MscL channel protein yielded proteoliposomes. These proteoliposomes allow for immediate recording of channel activity after GUV sealing. The rapid formation of protein-containing planar lipid bilayers is of potential use for the efficient electrophysiological characterization of MscL as shown here and also other ion channel proteins of interest.In order to study the effect of pressure, the functional MscL purified was reconstituted in our system. The reconstitution was done in GUVs and then bilayers were formed on a chip (Kreir, Farre et al. 2008). The Port-a-Patch system has a pump controlled by a computer and could apply from +300 to -300 mBar and is controlled via software allowing accurate pressure control. All pressure applications could be visualized and recorded at the same time as the recordings.

Publication link
2022 – Temporin B Forms Hetero-Oligomers with Temporin L, Modifies Its Membrane Activity, and Increases the Cooperativity of Its Antibacterial Pharmacodynamic Profile
Port-a-Patch and Vesicle Prep Pro Publication in Biochemistry (2022)  Authors: Ferguson P.M., Clarke M., Manzo G., Hind C.K, Clifford M., Sutton J.M., Lorenz C.D., Phoenix D.A., Mason J.A.

The pharmacodynamic profile of antimicrobial peptides (AMPs) and their in vivo synergy are two factors that are thought to restrict resistance evolution and ensure their conservation. The frog Rana temporaria secretes a family of closely related AMPs, temporins A-L, as an effective chemical dermal defence. The antibacterial potency of temporin L has been shown to increase synergistically in combination with both temporins B and A but this is modest. Here we show that the less potent temporin B enhances the cooperativity of the in vitro antibacterial activity of the more potent temporin L against EMRSA-15 and that this may be associated with an altered interaction with the bacterial plasma membrane, a feature critical for the antibacterial activity of most AMPs. Addition of buforin II, a histone H2A fragment, can further increase the cooperativity. Molecular dynamics simulations indicate temporins B and L readily form hetero-oligomers in models of Gram-positive bacterial plasma membranes. Patch-clamp studies show transmembrane ion conductance is triggered with lower amounts of both peptides and more quickly, when used in combination, but conductance is of a lower amplitude and pores are smaller. Temporin B may therefore act by forming temporin L/B hetero-oligomers that are more effective than temporin L homo-oligomers at bacterial killing and/or by reducing the probability of the latter forming until a threshold concentration is reached. Exploration of the mechanism of synergy between AMPs isolated from the same organism may therefore yield antibiotic combinations with advantageous pharmacodynamic properties.

Publication link
2022 – The human TRPA1 intrinsic cold and heat sensitivity involves separate channel structures beyond the N-ARD domain
Vesicle Prep Pro and Port-a-Patch Publication in Nature Communications (2022) Authors: Moparthi L., Sinica V., Filipovic M., Vlachova V., Zygmunt P.M.

The human TRPA1 (hTRPA1) is an intrinsic thermosensitive ion channel responding to both cold and heat, depending on the redox environment. Here, we have studied purified hTRPA1 truncated proteins to gain further insight into the temperature gating of hTRPA1. We found in patch-clamp bilayer recordings that ∆1-688 hTRPA1, without the N-terminal ankyrin repeat domain (N-ARD), was more sensitive to cold and heat, whereas ∆1-854 hTRPA1 that is also lacking the S1-S4 voltage sensing-like domain (VSLD) gained sensitivity to cold but lost its heat sensitivity. The thiol reducing agent TCEP abolished the temperature sensitivity of both ∆1-688 hTRPA1 and ∆1-854 hTRPA1. Cold and heat activity of ∆1-688 hTRPA1 and ∆1-854 hTRPA1 were associated with different structural conformational changes as revealed by intrinsic tryptophan fluorescence measurements. Heat evoked major structural rearrangement of the VSLD as well as the C-terminus domain distal to the transmembrane pore domain S5-S6 (CTD), whereas cold only caused minor conformational changes. As shown for Δ1-854 hTRPA1, a sudden drop in tryptophan fluorescence occurred within 25-20°C indicating a transition between heat and cold conformations of the CTD, and thus it is proposed that the CTD contains a bidirectional temperature switch priming hTRPA1 for either cold or heat. In whole-cell patch clamp electrophysiology experiments, replacement of the cysteines 865, 1021 and 1025 with alanine modulated the cold sensitivity of hTRPA1 when heterologously expressed in HEK293T cells. It is proposed that the hTRPA1 CTD harbors cold and heat sensitive domains allosterically coupled to the S5-S6 pore region and the VSLD, respectively.

Publication link
2022 – Cannabinoid non-cannabidiol site modulation of TRPV2 structure and function
Port-a-Patch and Vesicle Prep Pro Publication in Nature Communications (2022) Authors: Zhang L., Simonsen C., Zimova L., Wang K., Moparthi L., Gaudet R., Ekoff M., Nilsson G., Hellmich U., Vlachova V., Gourdon P., Zygmunt P.

TRPV2 is a ligand-operated temperature sensor with poorly defined pharmacology. Here, we combine calcium imaging and patch-clamp electrophysiology with cryo-electron microscopy (cryo-EM) to explore how TRPV2 activity is modulated by the phytocannabinoid Δ9-tetrahydrocannabiorcol (C16) and by probenecid. C16 and probenecid act in concert to stimulate TRPV2 responses including histamine release from rat and human mast cells. Each ligand causes distinct conformational changes in TRPV2 as revealed by cryo-EM. Although the binding for probenecid remains elusive, C16 associates within the vanilloid pocket. As such, the C16 binding location is distinct from that of cannabidiol, partially overlapping with the binding site of the TRPV2 inhibitor piperlongumine. Taken together, we discover a new cannabinoid binding site in TRPV2 that is under the influence of allosteric control by probenecid. This molecular insight into ligand modulation enhances our understanding of TRPV2 in normal and pathophysiology.

Publication link
2022 – Recent advances in ion-channel probes for nanopore sensing: Insights into the probe architectures
Port-a-Patch and Vesicle Prep Pro Publication in Analytica Chimica Acta (2022) Authors: Hussein E.A., Rice B., White R.J.

This review introduces the recent advances in the nanopore sensing platform, ion channel probes (ICPs), with a particular focus on the different probe design (2011–2022). The use of ion channel proteins has emerged in different applications to understand the dynamics of many biological processes and characterize or detect biomolecules. The development of utilizing protein channels in nanopore sensing has led to diverse platforms in which the ion channels, or biological nanopores, can be embedded in a lipid membrane. Ion channel probes, where the ion channels are integrated at the tip of a solid probe, enable higher spatially-resolved detection of small molecules and extend the applications of ion channels to map different surfaces and perform chemical imaging. Different probe materials and designs have been exploited throughout the last decade, which opens the door for multiple probe architecture and applications. We provide more insights into the advances of ICP designs that render them well-suited for further applications.

Publication link
2022 – Balancing water solubility with membrane permeability in the design of a synthetic ionophore
Port-a-Patch and Vesicle Prep Pro Pre-Print in ChemRxiv (2022) Authors: Yang K., Boles J.E., White L.J., Hilton K.L.F., Lai H.Y., Long Y., Hiscock J.R., Haynes C.J.E.

Synthetic ionophores are promising therapeutic targets, yet current limitations associated with their lipophilicity and poor water solubility prevent the translation of this molecular technology into the clinic. In this work we report investigations into the cation transport ability of a series of antimicrobial supramolecular, self-associating amphiphiles (SSAs). We identify a member of this class of compounds to function as a K+ transporter in cooperative action with a known anionophore. This SSA is soluble in a range of organic solvents and in 100% water, retaining its transport activity when delivered from a purely aqueous solution – therefore overcoming current molecular delivery limitations. These findings shed light on a potential antimicrobial mechanism of action and inform the design of future therapeutic targets that can balance water solubility and membrane penetration

Publication link
2022 – Bolaamphiphile Analogues of 12-bis-THA Cl2 Are Potent Antimicrobial Therapeutics with Distinct Mechanisms of Action against Bacterial, Mycobacterial, and Fungal Pathogens
Port-a-Patch and Vesicle Prep Pro Publication in ASM Journals (2022)  Authors: Di Blasio S., Clarke M., Hind C., Asai M., Laurence L., Benvenuti A., Hassan M., Semenya D., Kwun-Wai D., Horrocks V., Manzo G., Van Der Lith S., Lam C., Gentile E., Annette C., Bosse J., Li Y., Panaretou B., Langford P., Robertson B., Lam J., Sutton J., McArthur M., Mason A.

12-Bis-THA Cl2 [12,12′-(dodecane-1,12-diyl)-bis-(9-amino-1,2,3,4-tetrahydroacridinium) chloride] is a cationic bolalipid adapted from dequalinium chloride (DQC), a bactericidal anti-infective indicated for bacterial vaginosis (BV). Here, we used a structure-activity-relationship study to show that the factors that determine effective killing of bacterial, fungal, and mycobacterial pathogens differ, to generate new analogues with a broader spectrum of activity, and to identify synergistic relationships, most notably with aminoglycosides against Acinetobacter baumannii and Pseudomonas aeruginosa, where the bactericidal killing rate was substantially increased. Like DQC, 12-bis-THA Cl2 and its analogues accumulate within bacteria and fungi. More hydrophobic analogues with larger headgroups show reduced potential for DNA binding but increased and broader spectrum antibacterial activity. In contrast, analogues with less bulky headgroups and stronger DNA binding affinity were more active against Candida spp. Shortening the interconnecting chain, from the most lipophilic twelve-carbon chain to six, improved the selectivity index against Mycobacterium tuberculosis in vitro, but only the longer chain analogue was therapeutic in a Galleria mellonella infection model, with the shorter chain analogue exacerbating the infection. In vivo therapy of Escherichia coli ATCC 25922 and epidemic methicillin-resistant Staphylococcus aureus 15 (EMRSA-15) infections in Galleria mellonella was also achieved with longer-chain analogues, as was therapy for an A. baumannii 17978 burn wound infection with a synergistic combination of bolaamphiphile and gentamicin. The present study shows how this class of bolalipids may be adapted further to enable a wider range of potential applications.

Publication link
2020 – The structure of the antimicrobial human cathelicidin LL-37 shows oligomerization and channel formation in the presence of membrane mimics
Port-a-Patch and Vesicle Prep Pro Publication in Scientific Reports (2020) Authors: Sancho-Vaello E., Gil-Carton D., François P., Bonetti E.J., Kreir M., Pothula K.R., Kleinekathöfer U., Zeth K.

The human cathelicidin LL-37 serves a critical role in the innate immune system defending bacterial infections. LL-37 can interact with molecules of the cell wall and perforate cytoplasmic membranes resulting in bacterial cell death. To test the interactions of LL-37 and bacterial cell wall components we crystallized LL-37 in the presence of detergents and obtained the structure of a narrow tetrameric channel with a strongly charged core. The formation of a tetramer was further studied by cross-linking in the presence of detergents and lipids. Using planar lipid membranes a small but defined conductivity of this channel could be demonstrated. Molecular dynamic simulations underline the stability of this channel in membranes and demonstrate pathways for the passage of water molecules. Time lapse studies of E. coli cells treated with LL-37 show membrane discontinuities in the outer membrane followed by cell wall damage and cell death. Collectively, our results open a venue to the understanding of a novel AMP killing mechanism and allows the rational design of LL-37 derivatives with enhanced bactericidal activity.

Publication link
2021 – Norfluoxetine inhibits TREK-2 K2P channels by multiple mechanisms including state-independent effects on the selectivity filter gate
Port-a-Patch and Vesicle Prep Pro Publication in Journal of General Physiology (2021) Authors: Proks P., Schewe M., Conrad L.J., Rao S., Rathje S., Rödström K.E.J., Carpenter E.P., Baukrowitz T., Tucker S.J.

The TREK subfamily of two-pore domain K+ (K2P) channels are inhibited by fluoxetine and its metabolite, norfluoxetine (NFx). Although not the principal targets of this antidepressant, TREK channel inhibition by NFx has provided important insights into the conformational changes associated with channel gating and highlighted the role of the selectivity filter in this process. However, despite the availability of TREK-2 crystal structures with NFx bound, the precise mechanisms underlying NFx inhibition remain elusive. NFx has previously been proposed to be a state-dependent inhibitor, but its binding site suggests many possible ways in which this positively charged drug might inhibit channel activity. Here we show that NFx exerts multiple effects on single-channel behavior that influence both the open and closed states of the channel and that the channel can become highly activated by 2-APB while remaining in the down conformation. We also show that the inhibitory effects of NFx are unrelated to its positive charge but can be influenced by agonists which alter filter stability, such as ML335, as well as by an intrinsic voltage-dependent gating process within the filter. NFx therefore not only inhibits channel activity by altering the equilibrium between up and down conformations but also can directly influence filter gating. These results provide further insight into the complex allosteric mechanisms that modulate filter gating in TREK K2P channels and highlight the different ways in which filter gating can be regulated to permit polymodal regulation.

Publication link
2020 – Ionophore constructed from non-covalent assembly of a G-quadruplex and liponucleoside transports K+-ion across biological membranes
Port-a-Patch and Vesicle Prep Pro Publication in Nature Communications (2020)   Authors: Debnath M., Chakraborty S., Kumar Y.P., Chaudhuri R., Jana B., Dash J.

The selective transport of ions across cell membranes, controlled by membrane proteins, is critical for a living organism. DNA-based systems have emerged as promising artificial ion transporters. However, the development of stable and selective artificial ion transporters remains a formidable task. We herein delineate the construction of an artificial ionophore using a telomeric DNA G-quadruplex (h-TELO) and a lipophilic guanosine (MG). MG stabilizes h-TELO by non-covalent interactions and, along with the lipophilic side chain, promotes the insertion of h-TELO within the hydrophobic lipid membrane. Fluorescence assays, electrophysiology measurements and molecular dynamics simulations reveal that MG/h-TELO preferentially transports K+-ions in a stimuli-responsive manner. The preferential K+-ion transport is presumably due to conformational changes of the ionophore in response to different ions. Moreover, the ionophore transports K+-ions across CHO and K-562 cell membranes. This study may serve as a design principle to generate selective DNA-based artificial transporters for therapeutic applications.

Publication link
2020 – Purification and initial characterization of Plasmodium falciparum K+ channels, PfKch1 and PfKch2 produced in Saccharomyces cerevisiae
Vesicle Prep Pro and Port-a-Patch Publication in Microbial Cell Factories (2020) Authors: Molbaek K., Tejada M., Ricke C.H., Scharff-Poulsen P., ElleKVist P., Helix-Nielsen C., Kumar N., Klaerke D.A., Pedersen P.A.

Resistance towards known antimalarial drugs poses a significant problem, urging for novel drugs that target vital proteins in the malaria parasite Plasmodium falciparum. However, recombinant production of malaria proteins is notoriously difficult. To address this, we have investigated two putative K+channels, PfKch1 and PfKch2, identified in the P.falciparum genome. We show that PfKch1 and PfKch2 and a C-terminally truncated version of PfKch1 (PfKch11−1094)could indeed be functionally expressed in vivo, since a K+-uptake deficient Saccharomyces cerevisiae strain was complemented by the P. falciparum cDNAs. PfKch11−1094-GFP and GFP-PfKch2 fusion proteins were overexpressedin yeast, purified and reconstituted in lipid bilayers to determine their electrophysiological activity. Single channel conductance amounted to 16 ± 1 pS for PfKch11−1094-GFP and 28 ± 2 pS for GFP-PfKch2. We predicted regulator of K+-conductance (RCK) domains in the C-terminals of both channels, and we accordingly measured channel activity in the presence of Ca2+.

Publication link
2020 – Hepatitis C Virus p7 Induces Membrane Permeabilization by Interacting with Phosphatidylserine
Port-a-Patch and Vesicle Prep Pro Publication in International Journal of Molecular Sciences (2020) Authors: Lee H-R., Lee G.Y., You D-G., Kim H.K., Yoo Y.D.

Hepatitis C virus (HCV) p7 is known to be a nonselective cation channel for HCV maturation. Because the interaction of HCV proteins with host lipids in the endoplasmic reticulum membrane is crucial for the budding process, the identification of p7–lipid interactions could be important for understanding the HCV life cycle. Here, we report that p7 interacts with phosphatidylserine (PS) to induce membrane permeabilization. The interaction of p7 with PS was not inhibited by Gd3+ ions, which have been known to interact with negatively charged lipids, but channel activity and p7-induced mitochondrial depolarization were inhibited by Gd3+ ions. From the present results, we suggest that the p7–PS interaction plays an essential role in regulating its ion channel function and could be a potential molecular target for anti-HCV therapy.

Publication link
2020 – Human TRPA1 is an inherently mechanosensitive bilayer-gated ion channel
Port-a-Patch and Vesicle Prep Pro Publication in Cell Calcium (2020) Authors: Moparthi L., Zygmunt P.M.

The role of mammalian Transient Receptor Potential Ankyrin 1 (TRPA1) as a mechanosensor is controversial. Here, we report that purified human TRPA1 (hTRPA1) with and without its N-terminal ankyrin repeat domain responded with pressure-dependent single-channel current activity when reconstituted into artificial lipid bilayers. The hTRPA1 activity was abolished by the thiol reducing agent TCEP. Thus, depending on its redox state, hTRPA1 is an inherent mechanosensitive ion channel gated by force-from-lipids.

Publication link
2020 – Calcium activates purified human TRPA1 with and without its N-terminal ankyrin repeat domain in the absence of calmodulin
Port-a-Patch and Vesicle Prep Pro Publication in Cell Calcium (2020) Authors: Moparthi L., Moparthi S.B., Wenger J., Zygmunt P.M.

Extracellular influx of calcium or release of calcium from intracellular stores have been shown to activate mammalian TRPA1 as well as to sensitize and desensitize TRPA1 electrophilic activation. Calcium binding sites on both intracellular N- and C-termini have been proposed. Here, we demonstrate based on Förster resonance energy transfer (FRET) and bilayer patch-clamp studies, a direct calmodulin-independent action of calcium on the purified human TRPA1 (hTRPA1), causing structural changes and activation without immediate subsequent desensitization of hTRPA1 with and without its N-terminal ankyrin repeat domain (N-ARD). Thus, calcium alone activates hTRPA1 by a direct interaction with binding sites outside the N-ARD.

Publication link
2020 – Conformational equilibrium shift underlies altered K+ channel gating as revealed by NMR
Port-a-Patch and Vesicle Prep Pro Publication in Nature Communications (2020)   Authors: Iwahasi Y., Toyama Y., Imai S., Itoh H., Osawa M., Inoue M., Shimada I.

The potassium ion (K+) channel plays a fundamental role in controlling K+ permeation across the cell membrane and regulating cellular excitabilities. Mutations in the transmembrane pore reportedly affect the gating transitions of K+ channels, and are associated with the onset of neural disorders. However, due to the lack of structural and dynamic insights into the functions of K+ channels, the structural mechanism by which these mutations cause K+ channel dysfunctions remains elusive. Here, we used nuclear magnetic resonance spectroscopy to investigate the structural mechanism underlying the decreased K+-permeation caused by disease-related mutations, using the prokaryotic K+ channel KcsA. We demonstrated that the conformational equilibrium in the transmembrane region is shifted toward the non-conductive state with the closed intracellular K+-gate in the disease-related mutant. We also demonstrated that this equilibrium shift is attributable to the additional steric contacts in the open-conductive structure, which are evoked by the increased side-chain bulkiness of the residues lining the transmembrane helix. Our results suggest that the alteration in the conformational equilibrium of the intracellular K+-gate is one of the fundamental mechanisms underlying the dysfunctions of K+ channels caused by disease-related mutations.

Publication link
2019 – Temporin L and aurein 2.5 have identical conformations but subtly distinct membrane and antibacterial activities
Port-a-Patch and Vesicle Prep Pro Publication in Nature Scientific Reports (2019) Authors: Manzo G., Ferguson P.M., Hind C.H., Clifford M., Gustilo V.B., Ali H., Bansal S.S., Bui T.T., Drake A.F., Atkinson R.A., Sutton J.M., Lorenz C.D., Phoenix D.A., Mason A.J.

Frogs such as Rana temporaria and Litoria aurea secrete numerous closely related antimicrobial peptides (AMPs) as an effective chemical dermal defence. Damage or penetration of the bacterial plasma membrane is considered essential for AMP activity and such properties are commonly ascribed to their ability to form secondary amphipathic, α-helix conformations in membrane mimicking milieu. Nevertheless, despite the high similarity in physical properties and preference for adopting such conformations, the spectrum of activity and potency of AMPs often varies considerably. Hence distinguishing apparently similar AMPs according to their behaviour in, and effects on, model membranes will inform understanding of primary-sequence-specific antimicrobial mechanisms. Here we use a combination of molecular dynamics simulations, circular dichroism and patch-clamp to investigate the basis for differing anti-bacterial activities in representative AMPs from each species; temporin L and aurein 2.5. Despite adopting near identical, α-helix conformations in the steady-state in a variety of membrane models, these two AMPs can be distinguished both in vitro and in silico based on their dynamic interactions with model membranes, notably their differing conformational flexibility at the N-terminus, ability to form higher order aggregates and the characteristics of induced ion conductance. Taken together, these differences provide an explanation of the greater potency and broader antibacterial spectrum of activity of temporin L over aurein 2.5. Consequently, while the secondary amphipathic, α-helix conformation is a key determinant of the ability of a cationic AMP to penetrate and disrupt the bacterial plasma membrane, the exact mechanism, potency and spectrum of activity is determined by precise structural and dynamic contributions from specific residues in each AMP sequence.

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2020 – A pleurocidin analogue with greater conformational flexibility, enhanced antimicrobial potency and in vivo therapeutic efficacy
Vesicle Prep Pro and Port-a-Patch Publication in Communications Biology (2020) Authors: Manzo G., Hind C.K., Ferguson P.M., Amison R.T., Hodgson-Casson A.C., Ciazynska K.A., Weller B.J., Clarke M., Lam C., Man R.C.H., O'Shaughnessy B.G., Clifford M., Bui T.T., Drake A.F., Atkinson R.A., Lam J.K.W., Pitchford S.C., Page C.P., Phoenix D.A., Lorenz C.D., Sutton J.M., Mason A.J.

Antimicrobial peptides (AMPs) are a potential alternative to classical antibiotics that are yet to achieve a therapeutic breakthrough for treatment of systemic infections. The antibacterial potency of pleurocidin, an AMP from Winter Flounder, is linked to its ability to cross bacterial plasma membranes and seek intracellular targets while also causing membrane damage. Here we describe modification strategies that generate pleurocidin analogues with substantially improved, broad spectrum, antibacterial properties, which are effective in murine models of bacterial lung infection. Increasing peptide–lipid intermolecular hydrogen bonding capabilities enhances conformational flexibility, associated with membrane translocation, but also membrane damage and potency, most notably against Gram-positive bacteria. This negates their ability to metabolically adapt to the AMP threat. An analogue comprising D-amino acids was well tolerated at an intravenous dose of 15 mg/kg and similarly effective as vancomycin in reducing EMRSA-15 lung CFU. This highlights the therapeutic potential of systemically delivered, bactericidal AMPs.

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2019 – Minor sequence modifications in temporin B cause drastic changes in antibacterial potency and selectivity by fundamentally altering membrane activity
Port-a-Patch and Vesicle Prep Pro Publication in Nature Scientific Reports (2019)    Authors: Manzo G., Ferguson P.M., Gustilo V.B., Ali H., Bui T.T., Drake A.F., Atkinson R.A., Batoni G., Lorenz C.D., Phoenix D.A., Mason A.J.

Antimicrobial peptides (AMPs) are a potential source of new molecules to counter the increase in antimicrobial resistant infections but a better understanding of their properties is required for effective translation as therapeutics. Details of the mechanism of their interaction with the bacterial plasma membrane are desired since damage or penetration of this structure is considered essential for AMP activity. Relatively modest modifications to AMP primary sequence can induce substantial changes in potency and/or spectrum of activity but, hitherto, have not been predicted to substantially alter the mechanism of interaction with the bacterial plasma membrane. Here we use a combination of molecular dynamics simulations, circular dichroism, liquid- and solid-state NMR and patch clamp to investigate the extent to which temporin B and its analogues can be distinguished both in vitro and in silico on the basis of their interactions with model membranes. Enhancing the hydrophobicity of the N-terminus and cationicity of the C-terminus in temporin B improves its membrane activity and potency against both Gram-negative and Gram-positive bacteria. In contrast, enhancing the cationicity of the N-terminus abrogates its ability to trigger channel conductance and renders it ineffective against Staphylococcus aureus while nevertheless enhancing its potency against Escherichia coli. Our findings suggest even closely related AMPs may target the same bacterium with fundamentally differing mechanisms of action.

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2019 – Molecular understanding of calcium permeation through the open Orai channel
Port-a Patch and Vesicle Prep Pro Publication in PLOS Computational Biology (2019)    Authors: Liu X., Wu G., Yu Y., Chen X., Ji R., Lu J., Li X., Zhang X., Yang X., Shen Y.

The Orai channel is characterized by voltage independence, low conductance, and high Ca2+ selectivity and plays an important role in Ca2+ influx through the plasma membrane (PM). How the channel is activated and promotes Ca2+ permeation is not well understood. Here, we report the crystal structure and cryo-electron microscopy (cryo-EM) reconstruction of a Drosophila melanogaster Orai (dOrai) mutant (P288L) channel that is constitutively active according to electrophysiology. The open state of the Orai channel showed a hexameric assembly in which 6 transmembrane 1 (TM1) helices in the center form the ion-conducting pore, and 6 TM4 helices in the periphery form extended long helices. Orai channel activation requires conformational transduction from TM4 to TM1 and eventually causes the basic section of TM1 to twist outward. The wider pore on the cytosolic side aggregates anions to increase the potential gradient across the membrane and thus facilitate Ca2+ permeation. The open-state structure of the Orai channel offers insights into channel assembly, channel activation, and Ca2+ permeation.

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2018 – Single channel recording of a mitochondrial calcium uniporter
Port-a-Patch and Vesicle Prep Pro Publication in BBRC (2018)   Authors: Wu G., Li S., Zong G., Liu, X., Fei S., Shen L., Guan X., Yanga X., Shen Y.

Mitochondrial calcium uniporter (MCU) is the pore-forming subunit of the entire uniporter complex and plays an important role in mitochondrial calcium uptake. However, the single channel recording of MCU remains controversial. Here, we expressed and purified different MCU proteins and then reconstituted them into planar lipid bilayers for single channel recording. We showed that MCU alone from Pyronema omphalodes (pMCU) is active with prominent single channel Ca2+ currents. In sharp contrast, MCU alone from Homo sapiens (hMCU) is inactive. The essential MCU regulator (EMRE) activates hMCU, and therefore, the complex (hMCU-hEMRE) shows prominent single channel Ca2+ currents. These single channel currents are sensitive to the specific MCU inhibitor Ruthenium Red. Our results clearly demonstrate that active MCU can conduct large amounts of calcium into the mitochondria.

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2019 – Golgi anti-apoptotic proteins are evolutionarily conserved ion channels that regulate cell death in plants
Port-a-Patch and Vesicle Prep Pro Pre-Publication in BioRxiv (2019)   Authors: Sierla M., Prole D.L., Saraiva N., Carrara G., Dinischiotu N., Vaattovaara A., Wrzaczek M., Taylor C.W., Smith F.L., Feys B.

Programmed cell death regulates developmental and stress responses in eukaryotes. Golgi anti-apoptotic proteins (GAAPs) are evolutionarily conserved cell death regulators. Human and viral GAAPs inhibit apoptosis and modulate intracellular Ca2+ fluxes, and viral GAAPs form cation selective channels. Although most mammalian cell death regulators are not conserved at the sequence level in plants, the GAAP gene family shows expansion, with five paralogues (AtGAAP1-5) in the Arabidopsis genome. We pursued molecular and physiological characterization of AtGAAPs making use of the advanced knowledge of their human and viral counterparts. Structural modeling of AtGAAPs predicted the presence of a channel-like pore, and electrophysiological recordings from purified AtGAAP3 reconstituted into lipid bilayers confirmed that plant GAAPs can function as ion channels. AtGAAP1 and AtGAAP4 localized exclusively to the Golgi within the plant cell, while AtGAAP2, AtGAAP3 and AtGAAP5 also showed tonoplastic localization. Gene expression analysis revealed differential spatial expression and abundance of transcript for AtGAAP paralogues in Arabidopsis tissues. We demonstrate that AtGAAP1-5 inhibit Bax-induced cell death in yeast. However, overexpression of AtGAAP1 induces cell death in Nicotiana benthamiana leaves and lesion mimic phenotype in Arabidopsis. We propose that AtGAAPs function as Golgi-localized ion channels that regulate cell death by affecting ionic homeostasis within the cell.

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2018 – Reconstitution and Electrophysiological Characterization of Ion Channels in Lipid Bilayers
Port-a-Patch and Vesicle Prep Pro Publication in Current Protocols in Pharmacology (2018)  Authors: Klaerke D.A., de los Angeles Tejada M., Grøsfjeld Christensen V., Lassen M., Amstrup Pedersen P., Calloe K.

Detergent‐solubilized purified ion channels can be reconstituted into lipid bilayers for electrophysiological analysis. Traditionally, ion channels were inserted into vesicles and subsequently fused with planar “black lipid membranes” formed from lipids dissolved in a hydrophobic solvent such as decane. Provided in this article is a step‐by‐step guide to reconstitute purified ion channel proteins into giant unilamellar vesicles (GUVs). This procedure results in the formation of proteoliposomes that can be used for planar bilayer formation and electrophysiological characterization of single‐channel currents. By using preformed GUVs it is possible to omit the membrane solvent. Compared to traditional preparations, the lipid bilayers formed from GUVs provide an environment that more closely resembles the native cell membrane. Also described is an alternate protocol that entails the production of planar lipid bilayers from GUVs onto which proteins in detergent are added.

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2018 – Romo1 is a mitochondrial nonselective cation channel with viroporin-like characteristics
Port-a-Patch and Vesicle Prep Pro Publication in Journal of Cell Biology (2018)   Authors: Lee G.Y., You D.G., Lee H.R., Hwang S.W., Lee C.J., Do Yoo Y.

Reactive oxygen species (ROS) modulator 1 (Romo1) is a nuclear-encoded mitochondrial inner membrane protein known to regulate mitochondrial ROS production and to act as an essential redox sensor in mitochondrial dynamics. Although its physiological roles have been studied for a decade, the biophysical mechanisms that explain these activities of Romo1 are unclear. In this study, we report that Romo1 is a unique mitochondrial ion channel that differs from currently identified eukaryotic ion channels. Romo1 is a highly conserved protein with structural features of class II viroporins, which are virus-encoded nonselective cation channels. Indeed, Romo1 forms a nonselective cation channel with its amphipathic helical transmembrane domain necessary for pore-forming activity. Notably, channel activity was specifically inhibited by Fe2+ ions, an essential transition metal ion in ROS metabolism. Using structural bioinformatics, we designed an experimental data-guided structural model of Romo1 with a rational hexameric structure. We propose that Romo1 establishes a new category of viroporin-like nonselective cation channel in eukaryotes.

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2018 – Parallel evolution of frog antimicrobial peptides produces identical conformations but subtly distinct membrane and antibacterial activities
Port-a-Patch and Vesicle Prep Pro pre-Publication in bioRxiv (2018)   Authors: Manzo G., Ferguson P.M., Hind C., Clifford M., Gustilo V.B., Ali H., Bansal S.S., Bui T.T., Drake A.F., Atkinson R.A., Sutton M.J., Lorenz C.D., Phoenix D.A., Mason A.J.

Frogs such as Rana temporaria and Litoria aurea secrete numerous closely related antimicrobial peptides (AMPs) as an effective chemical dermal defence. Despite the high similarity in physical properties and preference for adopting secondary amphipathic, α-helix conformations in membrane mimicking milieu, their spectrum of activity and potency often varies considerably. Damage or penetration of the bacterial plasma membrane is considered essential for AMP activity and hence distinguishing apparently similar AMPs according to their behaviour in, and effects on, model membranes will inform understanding of species specific effective antimicrobial mechanisms. Here we use a combination of molecular dynamics simulations, circular dichroism and patch-clamp to investigate the basis for differing anti-bacterial activities in representative AMPs from each species; temporin L and aurein 2.5. Despite adopting near identical, α-helix conformations in the steady-state in a variety of membrane models, these two AMPs can be distinguished both in vitro and in silico based on their dynamic interactions with model membranes; the greater conformational flexibility and the higher amplitude channel conductance induced offers a rationale for the greater potency and broader spectrum of activity of temporin L over aurein 2.5. Specific contributions from individual residues are identified that define the mechanisms of action of each AMP. Our findings suggest AMPs in frogs are examples of parallel evolution whose utility is based on apparently similar but subtly distinct mechanisms of action.

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2018 – Quantifying Permeation of Small Charged Molecules across Channels: Electrophysiology in Small Volumes
Port-a-Patch and Vesicle Prep Pro Publication in ACS Omega (2018)    Authors: Wang J., Benier L., Winterhalter M.

A major bottleneck in the development of small molecule antibiotics is to achieve good permeability across the outer membrane in Gram-negative bacteria. Optimization with respect to permeability surprisingly lacks appropriate methods. Recently we proposed to use the diffusion potential for charged molecules created by their difference in electrophoretic mobility while crossing the outer membrane channel under a concentration gradient. The latter provides semi-quantitative values but the current available setups require large volumes and thus exclude several classes of molecules. Here we propose a simple approach capturing proteoliposomes at aperture of glass surface (planar aperture or conical glass capillary) decreasing the necessary volume below 50 µL. We measured the transport of two charged molecules sulbactam and ceftazidime across the two major porins in E.coli. Both molecules permeate through these porins were observed with sulbactam owes higher permeability.

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2018 – An aromatic cluster in Lysinibacillus sphaericus BinB involved in toxicity and proper in-membrane folding
Port-a-Patch and Vesicle Prep Pro Publication in Archives of Biochemistry and Biophysics (2018)   Authors: Chooduang A., Surya W., Torres J., Boonserm P.

The binary toxin from Lysinibacillus sphaericus has been successfully used for controlling mosquito-transmitted diseases. Based on structural alignments with other toxins, an aromatic cluster in the C-terminal domain of BinB (termed here BC) has been proposed to be important for toxicity. We tested this experimentally using BinB mutants bearing single mutations in this aromatic cluster. Consistent with the hypothesis, two of these mutations, F311A and F315A, were not toxic to Culex quinquefasciatus larvae and were unable to permeabilize liposomes or elicit ion channel activity, in contrast to wild-type BinB. Despite these effects, none of these mutations altered significantly the interaction between the activated forms of the two subunits in solution. These results indicate that these aromatic residues on the C-terminal domain of BinB are critical for toxin insertion in membranes. The latter can be by direct contact of these residues with the membrane surface, or by facilitating the formation a membrane-inserting oligomer.

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2018 – Molecular mechanism of the Orai channel activation
Port-a-Patch and Vesicle Prep Pro Pre-publicatin in bioRxiv (2018)   Authors: Liu, X., Wu, G., Yu, Y., Chen, X., Ji, R., Lu, J., Li, X., Zhang, X., Yang, X., Shen, Y.

The Orai channel is characterized by voltage independence, low conductance and high Ca2+ selectivity and plays an important role in Ca2+ influx through the plasma membrane. How the channel is activated and promotes Ca2+ permeation are not well understood. Here, we report the crystal structure and cryo-electron microscopy reconstruction of a Drosophila melanogaster Orai mutant (P288L) channel that is constitutively active according to electrophysiology. The open state of the Orai channel showed a hexameric assembly in which six TM1 helices in the center form the ion-conducting pore, and six TM4 helices in the periphery form extended long helices. Orai channel activation requires conformational transduction from TM4 to TM1 and eventually causes the basic section of TM1 to twist outward. The wider pore on the cytosolic side aggregates anions to increase the potential gradient across the membrane and thus facilitate Ca2+ permeation. The open-state structure of the Orai channel offers insights into channel assembly, channel activation and Ca2+ permeation.

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2017 – Polydim-I antimicrobial activity against MDR bacteria and its model membrane interaction
Port-a-Patch and  Vesicle Prep Pro Publication in PLoS ONE (2017)  Authors: Rangel M., Fernandes dos Santos Castro F., Mota-Lima L.D., Clissa P.B., Martins D.B., Perez dos Santos Cabrera M., Mortari M.R.

The rapid spread of multi-drug resistant pathogens represents a serious threat to public health, considering factors such as high mortality rates, treatment restrictions and high prevalence of multi-drug resistant bacteria in the hospital environment. Antimicrobial peptides (AMPs) may exhibit powerful antimicrobial activity against different and diverse microorganisms, also presenting the advantage of absence or low toxicity towards animal cells. In this study, the evaluation of the antimicrobial activity against multi-drug resistant bacteria of a recently described AMP from wasp, Polydim-I, was performed. Polydim-I presented activity against standard strains (non-carriers of multi-resistant genes) that are susceptible to commercial antimicrobials, and also against multi-drug resistant strains at concentrations bellow 1μg/ml (0.41 μM). This is a rather low concentration among those reported for AMPs. At this concentration we found out that Polydim-I inhibits almost 100% of the tested pathogens growth, while with the ATCC strains the minimum inhibitory concentration (MIC100) is 400 times higher. Also, in relation to in vitro activity of conventional drugs against multi-drug resistant bacteria strains, Polydim-I is almost 10 times more efficient and with broader spectrum. Cationic AMPs are known as multi-target compounds and specially for targeting the phospholipid matrix of bacterial membranes. Exploring the interactions of Polydim-I with lipid bilayers, we have confirmed that this interaction is involved in the mechanism of action. Circular dichroism experiments showed that Polydim-I undergoes a conformational transition from random coil to a mostly helical conformation in the presence of membrane mimetic environments. Zeta potential measurements confirmed the binding and partial charge neutralization of anionic asolectin vesicles, and also suggested a possible aggregation of peptide molecules. FTIR experiments confirmed that some peptide aggregation occurs, which is minimized in the presence of strongly anionic micelles of sodium dodecyl sulfate. Also, Polydim-I induced channel-like structures formation to asolectin lipid bilayers, as demonstrated in the electrophysiology experiments. We suggest that cationic Polydim-I targets the membrane lipids due to electrostatic attraction, partially accumulates, neutralizing the opposite charges and induces pore formation. Similar mechanism of action has already been suggested for other peptides from wasp venoms, especially mastoparans.

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2017 – The Rotavirus NSP4 Viroporin Domain is a Calcium-conducting Ion Channel
Port-a-Patch and Vesicle Prep Pro Publication in Nature Scientific Reports    Authors: Pham T., Perry J.L., Dosey T.L., Delcour L.H.,Hyser J.M.

Viroporins are small virus-encoded ion channel proteins. Most viroporins are monovalent selective cation channels, with few showing the ability to conduct divalent cations, like calcium (Ca2+). Nevertheless, some viroporins are known to disrupt host cell Ca2+ homeostasis, which is critical for virus replication and pathogenesis. Rotavirus nonstructural protein 4 (NSP4) is an endoplasmic reticulum transmembrane glycoprotein that has a viroporin domain (VPD), and NSP4 viroporin activity elevates cytosolic Ca2+ in mammalian cells. The goal of this study was to demonstrate that the NSP4 VPD forms an ion channel and determine whether the channel can conduct Ca2+. Using planar lipid bilayer and liposome patch clamp electrophysiology, we show that a synthetic peptide of the NSP4 VPD has ion channel activity. The NSP4 VPD was selective for cations over anions and channel activity was observed to have both well-defined “square top” openings as well as fast current fluctuations, similar to other viroporins. Importantly, the NSP4 VPD showed similar conductance of divalent cations (Ca2+ and Ba2+) as monovalent cations (K+), but a viroporin defective mutant lacked Ca2+ conductivity. These data demonstrate that the NSP4 VPD is a Ca2+-conducting viroporin and establish the mechanism by which NSP4 disturbs host cell Ca2+ homeostasis.

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2017 – Evaluation of the antimicrobial activity of the mastoparan Polybia-MPII isolated from venom of the social wasp Pseudopolybia vespiceps testacea (Vespidae, Hymenoptera)
Port-a-Patch and Vesicle Prep Pro Publication in International Journal of Antimicrobial Agents (2017)   Authors: Silva J.C., Neto L.M., Neves R.C., Gonçalves J.C., Trentini M.M., Mucury-Filho R., Smidt K.S., Fensterseifer I.C., Silva O.N., Lima L.D., Clissa P.B., Vilela N., Guilhelmelli F., Silva L.P., Rangel M., Kipnis A., Silva-Pereira I., Franco O.L., Junqueira-Kipnis A.P., Bocca A.L., Mortari M.R.

Mastoparans, a class of peptides found in wasp venom, have significant effects following a sting as well as useful applications in clinical practice. Among these is their potential use in the control of micro-organisms that cause infectious diseases with a significant impact on society. Thus, the present study describes the isolation and identification of a mastoparan peptide from the venom of the social wasp Pseudopolybia vespiceps and evaluated its antimicrobial profile against bacteria (Staphylococcus aureus and Mycobacterium abscessus subsp. massiliense), fungi (Candida albicans and Cryptococcus neoformans) and in vivo S. aureus infection. The membrane pore-forming ability was also assessed. The mastoparan reduced in vitro and ex vivo mycobacterial growth by 80% at 12.5 µM in infected peritoneal macrophages but did not affect the shape of bacterial cells at the dose tested (6.25 µM). The peptide also showed potent action against S. aureus in vitro (EC50 and EC90 values of 1.83 µM and 2.90 µM, respectively) and reduced the in vivo bacterial load after 6 days of topical treatment (5 mg/kg). Antifungal activity was significant, with EC50 and EC90 values of 12.9 µM and 15.3 µM, respectively, for C. albicans, and 11 µM and 22.70 µM, respectively, for C. neoformans. Peptides are currently attracting interest for their potential in the design of antimicrobial drugs, particularly due to the difficulty of micro-organisms in developing resistance to them. In this respect, Polybia-MPII proved to be highly effective, with a lower haemolysis rate compared with peptides of the same family.

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2017 – Organelle membrane derived patches: reshaping classical methods for new targets
Port-a-Patch and Vesicle Prep Pro Publication in Nature Scientific Reports (2017)    Authors: Shapovalov G., Ritaine A., Bidaux G., Slomianny, C., Borowiec A-S., Gordienko D., Bultynck G., Skryma R., Prevarskaya N.

Intracellular ion channels are involved in multiple signaling processes, including such crucial ones as regulation of cellular motility and fate. With 95% of the cellular membrane belonging to intracellular organelles, it is hard to overestimate the importance of intracellular ion channels. Multiple studies have been performed on these channels over the years, however, a unified approach allowing not only to characterize their activity but also to study their regulation by partner proteins, analogous to the patch clamp “golden standard”, is lacking. Here, we present a universal approach that combines the extraction of intracellular membrane fractions with the preparation of patchable substrates that allows to characterize these channels in endogenous protein environment and to study their regulation by partner proteins. We validate this method by characterizing activity of multiple intracellular ion channels localized to different organelles and by providing detailed electrophysiological characterization of the regulation of IP3R activity by endogenous Bcl-2. Thus, after synthesis and reshaping of the well-established approaches, organelle membrane derived patch clamp provides the means to assess ion channels from arbitrary cellular membranes at the single channel level.

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2017 – Asymmetric mechanosensitivity in a eukaryotic ion channel
Port-a-Patch and Vesicle Prep Pro Publication in PNAS (2017) Authors: Clausen M.V., Jarerattanachata V., Carpenterc E.P., Mark S. , Sansomb P., Tucker S.J.

Living organisms perceive and respond to a diverse range of mechanical stimuli. A variety of mechanosensitive ion channels have evolved to facilitate these responses, but the molecular mechanisms underlying their exquisite sensitivity to different forces within the membrane remains unclear. TREK-2 is a mammalian two-pore domain (K2P) K+ channel important for mechanosensation, and recent studies have shown how increased membrane tension favors a more expanded conformation of the channel within the membrane. These channels respond to a complex range of mechanical stimuli, however, and it is uncertain how differences in tension between the inner and outer leaflets of the membrane contribute to this process. To examine this, we have combined computational approaches with functional studies of oppositely oriented single channels within the same lipid bilayer. Our results reveal how the asymmetric structure of TREK-2 allows it to distinguish a broad profile of forces within the membrane, and illustrate the mechanisms that eukaryotic mechanosensitive ion channels may use to detect and fine-tune their responses to different mechanical stimuli. Significance: One important way in which living organisms are able to detect and respond to their environment is via the conversion of mechanical forces into electrical signals. However, the molecular mechanisms that enable mammalian “mechanosensitive” ion channels to detect a wide profile of forces within the membrane remain unclear. By studying the functional activity of individual TREK-2 K2P channels inserted in different directions into a lipid bilayer, we are now able to describe how the asymmetric structure of this channel enables it to sense such a broad profile of forces. These results help us understand how eukaryotic ion channels respond to a rich variety of sensory stimuli.

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2017 – Bilayer-Mediated Structural Transitions Control Mechanosensitivity of the TREK-2 K2P Channel
Port-a-Patch and Vesicle Prep Pro Publication in Structure (2017)   Authors: Arya P, Jarerattanachat V., Clausen M.V., Schewe M., McClenaghan C., Argent L., Conrad L.J., Dong Y.Y., Pike A.C.W., Carpenter E.P., Baukrowitz T., Sansom M.S.P., Tucker S.J.

The mechanosensitive two-pore domain (K2P) K+ channels (TREK-1, TREK-2, and TRAAK) are important for mechanical and thermal nociception. However, the mechanisms underlying their gating by membrane stretch remain controversial. Here we use molecular dynamics simulations to examine their behavior in a lipid bilayer. We show that TREK-2 moves from the “down” to “up” conformation in direct response to membrane stretch, and examine the role of the transmembrane pressure profile in this process. Furthermore, we show how state-dependent interactions with lipids affect the movement of TREK-2, and how stretch influences both the inner pore and selectivity filter. Finally, we present functional studies that demonstrate why direct pore block by lipid tails does not represent the principal mechanism of mechanogating. Overall, this study provides a dynamic structural insight into K2P channel mechanosensitivity and illustrates how the structure of a eukaryotic mechanosensitive ion channel responds to changes in forces within the bilayer.

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2016 – Structural and functional characterization of a calcium-activated cation channel from Tsukamurella paurometabola
Port-a-Patch and Vesicle Prep Pro Publication in Nature Communications (2016) Authors: Dhakshnamoorthy B., Rohaim A., Rui H., Blachowicz L., Roux B.

The selectivity filter is an essential functional element of K+ channels that is highly conserved both in terms of its primary sequence and its three-dimensional structure. Here, we investigate the properties of an ion channel from the Gram-positive bacterium Tsukamurella paurometabola with a selectivity filter formed by an uncommon proline-rich sequence. Electrophysiological recordings show that it is a non-selective cation channel and that its activity depends on Ca2+ concentration. In the crystal structure, the selectivity filter adopts a novel conformation with Ca2+ ions bound within the filter near the pore helix where they are coordinated by backbone oxygen atoms, a recurrent motif found in multiple proteins. The binding of Ca2+ ion in the selectivity filter controls the widening of the pore as shown in crystal structures and in molecular dynamics simulations. The structural, functional and computational data provide a characterization of this calcium-gated cationic channel.

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2016 – The N-terminal Ankyrin Repeat Domain Is Not Required for Electrophile and Heat Activation of the Purified Mosquito TRPA1 Receptor
Port-a-Patch and Vesicle Prep Pro Publication in Nature (2016) Authors: Survery S., Moparthi L., Kjellbom P., Högestätt E.D., Zygmunt P.M., Johanson U.

Temperature sensors are crucial for animals to optimize living conditions. The temperature response of the ion channel transient receptor potential A1 (TRPA1) is intriguing, some orthologs have been reported to be activated by cold and others by heat, but the molecular mechanisms responsible for its activation remain elusive. Single-channel electrophysiological recordings of heterologously expressed and purified Anopheles gambiae TRPA1 (AgTRPA1), with and without the N-terminal ankyrin repeat domain, demonstrate that both proteins are functional as they responded to the electrophilic compounds allyl isothiocyanate (AITC) and cinnamaldehyde as well as heat. The proteins similar intrinsic fluorescence properties and corresponding quenching when activated by AITC or heat, suggest lipid bilayer-independent conformational changes outside the N-terminal domain. The results show that AgTRPA1 is an inherent temperature- and chemoreceptor, and analogous to what has been reported for the human TRPA1 ortholog the N-terminal domain may tune the response but is not required for the activation by these stimuli.

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2016 – Photosensitization in Porphyrias and Photodynamic Therapy Involves TRPA1 and TRPV1
Port-a-Patch and Vesicle Prep Pro Publication in The Journal of Neuroscience (2016) Authors: Babes A., Sauer S.K., Moparthi L., Kichko T.I., Neacsu C., Namer B., Filipovic M., Zygmunt P.M., Reeh P.W., Fischer M.J.

Photosensitization, an exaggerated sensitivity to harmless light, occurs genetically in rare diseases, such as porphyrias, and in photodynamic therapy where short-term toxicity is intended. A common feature is the experience of pain from bright light. In human subjects, skin exposure to 405 nm light induced moderate pain, which was intensified by pretreatment with aminolevulinic acid. In heterologous expression systems and cultured sensory neurons, exposure to blue light activated TRPA1 and, to a lesser extent, TRPV1 channels in the absence of additional photosensitization. Pretreatment with aminolevulinic acid or with protoporphyrin IX dramatically increased the light sensitivity of both TRPA1 and TRPV1 via generation of reactive oxygen species. Artificial lipid bilayers equipped with purified human TRPA1 showed substantial single-channel activity only in the presence of protoporphyrin IX and blue light. Photosensitivity and photosensitization could be demonstrated in freshly isolated mouse tissues and led to TRP channel-dependent release of proinflammatory neuropeptides upon illumination. With antagonists in clinical development, these findings may help to alleviate pain during photodynamic therapy and also allow for disease modification in porphyria patients. Significance Statement: Cutaneous porphyria patients suffer from burning pain upon exposure to sunlight and other patients undergoing photodynamic therapy experience similar pain, which can limit the therapeutic efforts. This study elucidates the underlying molecular transduction mechanism and identifies potential targets of therapy. Ultraviolet and blue light generates singlet oxygen, which oxidizes and activates the ion channels TRPA1 and TRPV1. The disease and the therapeutic options could be reproduced in models ranging from isolated ion channels to human subjects, applying protoporphyrin IX or its precursor aminolevulinic acid. There is an unmet medical need, and our results suggest a therapeutic use of the pertinent antagonists in clinical development.

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2016 – Pore architecture of TRIC channels and insights into their gating mechanism
Port-a-Patch and Vesicle Prep Pro Publication in Nature (2016) Authors: Yang H., Hu M., Guo J., Ou X., Cai T., Liu Z.

Intracellular Ca2+ signalling processes are fundamental to muscle contraction, neurotransmitter release, cell growth and apoptosis. Release of Ca2+ from the intracellular stores is supported by a series of ion channels in sarcoplasmic or endoplasmic reticulum (SR/ER). Among them, two isoforms of the trimeric intracellular cation (TRIC) channel family, named TRIC-A and TRIC-B, modulate the release of Ca2+ through the ryanodine receptor or inositol triphosphate receptor, and maintain the homeostasis of ions within SR/ER lumen. Genetic ablations or mutations of TRIC channels are associated with hypertension, heart disease, respiratory defects and brittle bone disease. Despite the pivotal function of TRIC channels in Ca2+ signalling, their pore architectures and gating mechanisms remain unknown. Here we present the structures of TRIC-B1 and TRIC-B2 channels from Caenorhabditis elegans in complex with endogenous phosphatidylinositol-4,5-biphosphate (PtdIns(4,5)P2, also known as PIP2) lipid molecules. The TRIC-B1/B2 proteins and PIP2 assemble into a symmetrical homotrimeric complex. Each monomer contains an hourglass-shaped hydrophilic pore contained within a seven-transmembrane-helix domain. Structural and functional analyses unravel the central role of PIP2 in stabilizing the cytoplasmic gate of the ion permeation pathway and reveal a marked Ca2+-induced conformational change in a cytoplasmic loop above the gate. A mechanistic model has been proposed to account for the complex gating mechanism of TRIC channels.

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2016 – Functional characterization of p7 viroporin from hepatitis C virus produced in a cell-free expression system
Port-a-Patch and Vesicle Prep Pro Publication in Protein Expression and Purification (2016) Authors: Soranzo T., Cortès S., Gilde F., Kreir M., Picart C., Lenormand J.-L.

Using a cell-free expression system we produced the p7 viroporin embedded into a lipid bilayer in a single-step manner. The protein quality was assessed using different methods. We examined the channel forming activity of p7 and verified its inhibition by 5-(N,N-Hexamethylene) amiloride (HMA). Fourier transformed infrared spectroscopy (FTIR) experiments further showed that when p7 was inserted into synthetic liposomes, the protein displayed a native-like conformation similar to p7 obtained from other sources. Photoactivatable amino acid analogs used for p7 protein synthesis enabled oligomerization state analysis in liposomes by cross-linking. Therefore, these findings emphasize the quality of the cell-free produced p7 proteoliposomes which can benefit the field of the hepatitis C virus (HCV) protein production and characterization and also provide tools for the development of new inhibitors to reinforce our therapeutic arsenal against HCV.

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2016 – Human TRPA1 is a heat sensor displaying intrinsic U-shaped thermosensitivity
Port-a-Patch and Vesicle Prep Pro Publication in Scientific Reports (2016) Authors: Moparthi L., Kichko T. I., Eberhardt M., Högestätt E. D., Kjellbom P., Johanson U., Reeh P., W., Leffler A., Filipovic M. R., Zygmunt P. M.

Thermosensitive Transient Receptor Potential (TRP) channels are believed to respond to either cold or heat. In the case of TRP subtype A1 (TRPA1), there seems to be a species-dependent divergence in temperature sensation as non-mammalian TRPA1 is heat-sensitive whereas mammalian TRPA1 is sensitive to cold. It has been speculated but never experimentally proven that TRPA1 and other temperature-sensitive ion channels have the inherent capability of responding to both cold and heat. Here we show that redox modification and ligands affect human TRPA1 (hTRPA1) cold and heat sensing properties in lipid bilayer and whole-cell patch-clamp recordings as well as heat-evoked TRPA1-dependent calcitonin gene-related peptide (CGRP) release from mouse trachea. Studies of purified hTRPA1 intrinsic tryptophan fluorescence, in the absence of lipid bilayer, consolidate hTRPA1 as an intrinsic bidirectional thermosensor that is modified by the redox state and ligands. Thus, the heat sensing property of TRPA1 is conserved in mammalians, in which TRPA1 may contribute to sensing warmth and uncomfortable heat in addition to noxious cold.

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2015 – Quantification of Fluoroquinolone Uptake through the Outer Membrane Channel OmpF of Escherichia coli
Port-a-Patch and Vesicle Prep Pro Publication in Journal of the American Chemical Society (2015) Authors: Cama J., Bajaj H., Pagliara S., Maier T., Braun Y., Winterhalter M., Keyser U.F.

Decreased drug accumulation is a common cause of antibiotic resistance in microorganisms. However, there are few reliable general techniques capable of quantifying drug uptake through bacterial membranes. We present a semiquantitative optofluidic assay for studying the uptake of autofluorescent drug molecules in single liposomes. We studied the effect of the Escherichia coli outer membrane channel OmpF on the accumulation of the fluoroquinolone antibiotic, norfloxacin, in proteoliposomes. Measurements were performed at pH 5 and pH 7, corresponding to two different charge states of norfloxacin that bacteria are likely to encounter in the human gastrointestinal tract. At both pH values, the porins significantly enhance drug permeation across the proteoliposome membranes. At pH 5, where norfloxacin permeability across pure phospholipid membranes is low, the porins increase drug permeability by 50-fold on average. We estimate a flux of about 10 norfloxacin molecules per second per OmpF trimer in the presence of a 1 mM concentration gradient of norfloxacin. We also performed single channel electrophysiology measurements and found that the application of transmembrane voltages causes an electric field driven uptake in addition to concentration driven diffusion. We use our results to propose a physical mechanism for the pH mediated change in bacterial susceptibility to fluoroquinolone antibiotics.

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2016 – Conductance and Capacity of Plain Lipid Membranes under Conditions of Variable Gravity
Port-a-Patch and Vesicle Prep Pro Publication in Journal of Biomedical Science and Engineering (2016) Authors: Sieber M., Kaltenbach S., Hanke W. and Kohn F.P.M

Biological cell membranes are complex structures containing mainly lipids and proteins. Functional aspects of such membranes are usually attributed to membrane integral proteins. However, it is well established that parameters of the lipid matrix are modifying the function of proteins. Additionally, electrical capacity and conductance of the plain lipid matrix of membranes are contributing directly to cellular functions as there is, for example, the propagation of action potentials. Accordingly the dependence of these parameters on changes of gravity might be important in the field of life sciences under space conditions. In this study consequently we have performed experiments in parabolic flight campaigns utilizing the patch-clamp technology to investigate conductance and capacity of plain lipid vesicle membranes under conditions of changing gravity. Both capacity and conductance were found to be gravity dependent. The changes in capacity could be contributed to changes in membrane geometry. Significant permeability in plain lipid membranes could be only observed at high potentials, where spontaneous current fluctuations occurred. The probability of these fluctuations was gravity dependent.

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2015 – Different Ligands of the TRPV3 Cation Channel Cause Distinct Conformational Changes As Revealed by Intrinsic Tryptophan Fluorescence Quenching
Port-a-Patch and Vesicle Prep Pro Publication in Journal of Biological Chemistry (2015) Authors: Billen B., Brams M., Debaveye S., Remeeva A., Alpizar Y.A., Waelkens E., Kreir M., Brüggemann A., Talavera K., Nilius B., Voets T., Ulens C.

TRPV3 is a thermosensitive ion channel primarily expressed in epithelial tissues of the skin, nose, and tongue. The channel has been implicated in environmental thermosensation, hyperalgesia in inflamed tissues, skin sensitization, and hair growth. Although transient receptor potential (TRP) channel research has vastly increased our understanding of the physiological mechanisms of nociception and thermosensation, the molecular mechanics of these ion channels are still largely elusive. In order to better comprehend the functional properties and the mechanism of action in TRP channels, high-resolution three-dimensional structures are indispensable, because they will yield the necessary insights into architectural intimacies at the atomic level. However, structural studies of membrane protein c are currently hampered by difficulties in protein purification and in establishing suitable crystallization conditions. In this report, we present a novel protocol for the purification of membrane proteins, which takes advantage of a C-terminal GFP fusion. Using this protocol, we purified human TRPV3. We show that the purified protein is a fully functional ion channel with properties akin to the native channel using planar patch clamp on reconstituted channels and intrinsic tryptophan fluorescence spectroscopy. Using intrinsic tryptophan fluorescence spectroscopy, we reveal clear distinctions in the molecular interaction of different ligands with the channel. Altogether, this study provides powerful tools to broaden our understanding of ligand interaction with TRPV channels, and the availability of purified human TRPV3 opens up perspectives for further structural and functional studies.

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2015 – Golgi anti-apoptotic proteins are highly conserved ion channels that affect apoptosis and cell migration
Port-a-Patch and Vesicle Prep Pro Publication in Journal of Biological Chemistry (2015) Authors: Carrara G., Saraiva N., Parsons M., Byrne B., Prole D.L., Taylor C.W., Smith G.L.

Golgi anti-apoptotic proteins (GAAPs) are multitransmembrane proteins that are expressed in the Golgi apparatus and are able to homo-oligomerize. They are highly conserved throughout eukaryotes and are present in some prokaryotes and orthopoxviruses. Within eukaryotes, GAAPs regulate the Ca(2+) content of intracellular stores, inhibit apoptosis, and promote cell adhesion and migration. Data presented here demonstrate that purified viral GAAPs (vGAAPs) and human Bax inhibitor 1 form ion channels and that vGAAP from camelpox virus is selective for cations. Mutagenesis of vGAAP, including some residues conserved in the recently solved structure of a related bacterial protein, BsYetJ, altered the conductance (E207Q and D219N) and ion selectivity (E207Q) of the channel. Mutation of residue Glu-207 or -178 reduced the effects of GAAP on cell migration and adhesion without affecting protection from apoptosis. In contrast, mutation of Asp-219 abrogated the anti-apoptotic activity of GAAP but not its effects on cell migration and adhesion. These results demonstrate that GAAPs are ion channels and define residues that contribute to the ion-conducting pore and affect apoptosis, cell adhesion, and migration independently.

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2014 – Temperature-sensitive gating of hCx26: high-resolution Raman spectroscopy sheds light on conformational changes
Port-a-Patch and Vesicle Prep Pro Publication in Biomedical Optics Express (2014) Authors: Kniggendorf A.-K., Meinhardt-Wollweber M., Yuan X., Roth B., Seifert A., Fertig N., Zeilinger C.

The temperature-sensitive gating of human Connexin 26 (hCx26) was analyzed with confocal Raman microscopy. High-resolution Raman spectra covering the spectral range between 400 and 1500 rel. cm−1 with a spectral resolution of 1 cm−1 were fully annotated, revealing notable differences between the spectrum recorded from solubilized hCx26 in Ca2+-buffered POPC at 10°C and any other set of protein conditions (temperature, Ca2+ presence, POPC presence). Spectral components originating from specific amino acids show that the TM1/EL1 parahelix and probably the TM4 trans-membrane helix and the plug domain are involved in the gating process responsible for fully closing the hemichannel.

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2015 – Cell-free expression of a functional pore-only sodium channel
Port-a-Patch and Vesicle Prep Pro Publication in Protein Expression and Purification (2015) Authors: Kovácsová G, Gustavsson E, Wang J, Kreir M, Peuker S, Westenhoff S.

Voltage-gated sodium channels participate in the propagation of action potentials in excitable cells. Eukaryotic NaVs are pseudo homotetrameric polypeptides, comprising four repeats of six transmembrane segments (S1–S6). The first four segments form the voltage-sensing domain and S5 and S6 create the pore domain with the selectivity filter. Prokaryotic NaVs resemble these characteristics, but are truly tetrameric. They can typically be efficiently synthesized in bacteria, but production in vitro with cell-free synthesis has not been demonstrated. Here we report the cell-free expression and purification of a prokaryotic tetrameric pore-only sodium channel. We produced milligram quantities of the functional channel protein as characterized by size-exclusion chromatography, infrared spectroscopy and electrophysiological recordings. Cell-free expression enables advanced site-directed labelling, post-translational modifications, and special solubilization schemes. This enables next-generation biophysical experiments to study the principle of sodium ion selectivity and transport in sodium channels.

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2014 – Structure of a Conserved Golgi Complex-targeting Signal in CoroNaVirus Envelope Proteins
Port-a-Patch and Vesicle Prep Pro Publication in Journal of Biological Chemistry (2014) Authors: Li Y., Surya W., Claudine S., Torres J.

CoroNaVirus envelope (CoV E) proteins are ~100-residue polypeptides with at least one channel-forming α-helical transmembrane (TM) domain. The extramembrane C terminal tail contains a completely conserved proline, at the center of a predicted β coil β motif. This hydrophobic motif has been reported to constitute a Golgi-targeting signal, or a second TM domain. However, no structural data for this, or other extramembrane domains in CoV E proteins, is available. Herein, we show that the E protein in the severe acute respiratory syndrome (SARS) virus has only one TM domain in micelles, whereas the predicted β coil β motif forms a short membrane-bound α helix connected by a disordered loop to the TM domain. However, complementary results suggest that this motif is potentially poised for conformational change, or in dynamic exchange with other conformations.

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2014 – Studying mechanosensitive ion channels with an automated patch clamp
Port-a-Patch and Vesicle Prep Pro Publication in European Biophysics Journal (2014) Authors: Barthmes M.,  Mac Donald F.J., Birkner J.P., Brüggemann A., Wahl-Schott C., Koçer A.

Patch clamp electrophysiology is the main technique to study mechanosensitive ion channels (MSCs), however, conventional patch clamping is laborious and success and output depends on the skills of the operator. Even though automated patch systems solve these problems for other ion channels, they could not be applied to MSCs. Here, we report on activation and single channel analysis of a bacterial mechanosensitive ion channel using an automated patch clamp system. With the automated system, we could patch not only giant unilamellar liposomes but also giant Escherichia coli (E. coli) spheroplasts. The tension sensitivity and channel kinetics data obtained in the automated system were in good agreement with that obtained from the conventional patch clamp. The findings will pave the way to high throughput fundamental and drug screening studies on mechanosensitive ion channels.

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2014 – Human TRPA1 is intrinsically cold- and chemosensitive with and without its N-terminal ankyrin repeat domain
Port-a-Patch and Vesicle Prep Pro Publication in Proceedings of the National Academy of Sciences of the United States of America (2014)   Authors: Moparthi L., Survery S., Kreir M., Simonsen C., Kjellbom P., Högestätt E.D., Johanson U., Zygmunt P.M.

We have purified and reconstituted human transient receptor potential (TRP) subtype A1 (hTRPA1) into lipid bilayers and recorded single-channel currents to understand its inherent thermo- and chemosensory properties as well as the role of the ankyrin repeat domain (ARD) of the N terminus in channel behavior. We report that hTRPA1 with and without its N-terminal ARD (Δ1–688 hTRPA1) is intrinsically cold-sensitive, and thus, cold-sensing properties of hTRPA1 reside outside the N-terminal ARD. We show activation of hTRPA1 by the thiol oxidant 2-((biotinoyl)amino)ethyl methanethiosulfonate (MTSEA-biotin) and that electrophilic compounds activate hTRPA1 in the presence and absence of the N-terminal ARD. The nonelectrophilic compounds menthol and the cannabinoid Δ9-tetrahydrocannabiorcol (C16) directly activate hTRPA1 at different sites independent of the N-terminal ARD. The TRPA1 antagonist HC030031 inhibited cold and chemical activation of hTRPA1 and Δ1–688 hTRPA1, supporting a direct interaction with hTRPA1 outside the N-terminal ARD. These findings show that hTRPA1 is an intrinsically cold- and chemosensitive ion channel. Thus, second messengers, including Ca2+, or accessory proteins are not needed for hTRPA1 responses to cold or chemical activators. We suggest that conformational changes outside the N-terminal ARD by cold, electrophiles, and nonelectrophiles are important in hTRPA1 channel gating and that targeting chemical interaction sites outside the N-terminal ARD provides possibilities to fine tune TRPA1-based drug therapies (e.g., for treatment of pain associated with cold hypersensitivity and cardiovascular disease).

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2014 – Membrane assembly of the functional KcsA potassium channel in a vesicle-based eukaryotic cell-free translation system
Port-a-Patch and Vesicle Prep Pro Publication in Biosensors and Bioelectronics (2014)   Authors: Dondapati S.K., Kreir M., Quast R.B., Wüstenhagen D.A., Brüggemann A., Fertig N., Kubick S.

The potassium channel KcsA was heterologously expressed in a eukaryotic cell-free system. Both, the expression yields and functional analysis of the protein were reported. Qualitative and quantitative analyses of KcsA expression were performed by using 14C-labeled leucine as one of the amino acids supplemented in the cell-free reaction mixture. There was a time dependent increase in the protein yield as well as the intensity of the native tetramer band in insect cell derived microsomes. Electrophysiology measurements demonstrated the functional activity of the microsomes harboring KcsA showing single-channel currents with the typical biophysical characteristics of the ion channel. The channel behavior was asymmetric and showed positive rectification with larger currents towards positive voltages. KcsA channel currents were effectively blocked by potassium selective barium (Ba2+). This functional demonstration of an ion channel in eukaryotic cell-free system has a large potential for future applications including drug screening, diagnostic applications and functional assessment of complex membrane proteins like GPCRs by coupling them to ion channels in cell-free systems. Furthermore, membrane proteins can be expressed directly from linear DNA templates within 90 min, eliminating the need for additional cloning steps, which makes this cell-free system fast and efficient.

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2012 – Green Fluorescent Protein Changes the Conductance of Connexin 43 (Cx43) Hemichannels Reconstituted in Planar Lipid Bilayer
Port-a-Patch and Vesicle Prep Pro Publication in The Journal of Biological Chemistry (2012) Authors: Carnarius C., Kreir M., Krick M., Methfessel C., Moehrle V., Valerius O., Brüggemann A., Steinem C., Fertig N.

In mammalian tissues, connexin 43 (Cx43) is the most prominent member of the connexin family. In a single lipid bilayer, six connexin subunits assemble into a hemichannel (connexon). Direct communication of apposing cells is realized by two adjacent hemichannels, which can form gap junction channels. Here, we established an expression system in Pichia pastoris to recombinantly produce and purify Cx43 as well as Cx43 fused to green fluorescent protein (GFP). Proteins were isolated from crude cell membrane fractions via affinity chromatography. Cx43 and Cx43-GFP hemichannels were reconstituted in giant unilamellar vesicles as proven by fluorescence microscopy, and their electrophysiological behavior was analyzed on the single channel level by planar patch clamping. Cx43 and Cx43-GFP both showed an ohmic behavior and a voltage-dependent open probability. Cx43 hemichannels exhibited one major mean conductance of 224 ± 26 picosiemens (pS). In addition, a subconductance state at 124 ± 5 pS was identified. In contrast, the analysis of Cx43-GFP single channels revealed 10 distinct conductance states in the range of 15 to 250 pS, with a larger open probability at 0 mV as compared with Cx43, which suggests that intermolecular interactions between the GFP molecules alter the electrophysiology of the protein.

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2014 – An outer membrane channel protein of Mycobacterium tuberculosis with exotoxin activity
Port-a-Patch and Vesicle Prep Pro Publication in Proceedings of the National Academy of Sciences of the United States of America (2014) Authors: Danilchanka A., Sun J., Pavlenok M., Maueröder C., Speer A., Siroy A., Marrero J., Trujillo C., Mayhew D.L., Doornbos K.S., Muñoz 'L.E., HerrmannM., EhrtS., Berens C., Niederweisa M.

The ability to control the timing and mode of host cell death plays a pivotal role in microbial infections. Many bacteria use toxins to kill host cells and evade immune responses. Such toxins are unknown in Mycobacterium tuberculosis. Virulent M. tuberculosis strains induce necrotic cell death in macrophages by an obscure molecular mechanism. Here we show that the M. tuberculosis protein Rv3903c (channel protein with necrosis-inducing toxin, CpnT) consists of an N-terminal channel domain that is used for uptake of nutrients across the outer membrane and a secreted toxic C-terminal domain. Infection experiments revealed that CpnT is required for survival and cytotoxicity of M. tuberculosis in macrophages. Furthermore, we demonstrate that the C-terminal domain of CpnT causes necrotic cell death in eukaryotic cells. Thus, CpnT has a dual function in uptake of nutrients and induction of host cell death by M. tuberculosis.

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2011 – Voltage-gated sodium channel (NaV) protein dissection creates a set of functional pore-only proteins
Port-a-Patch and Vesicle Prep Pro Publication in Proceedings of the National Academy of Sciences of the United States (2011) Authors: Shaya D., Kreir M., Robbins R.A., Wong S., Hammon J., Brüggemann A., Minor Jr. D.L.

Many voltage-gated ion channel (VGIC) superfamily members contain six-transmembrane segments in which the first four form a voltage-sensing domain (VSD) and the last two form the pore domain (PD). Studies of potassium channels from the VGIC superfamily together with identification of voltage-sensor only proteins have suggested that the VSD and the PD can fold independently. Whether such transmembrane modularity is common to other VGIC superfamily members has remained untested. Here we show, using protein dissection, that the Silicibacter pomeroyi voltage-gated sodium channel (NaVSp1) PD forms a stand-alone, ion selective pore (NaVSp1p) that is tetrameric, α-helical, and that forms functional, sodium-selective channels when reconstituted into lipid bilayers. Mutation of the NaVSp1p selectivity filter from LESWSM to LDDWSD, a change similar to that previously shown to alter ion selectivity of the bacterial sodium channel NaVBh1 (NaChBac), creates a calcium-selective pore-only channel, CaVSp1p. We further show that production of PDs can be generalized by making pore-only proteins from two other extremophile NaVs: one from the hydrocarbon degrader Alcanivorax borkumensis (NaVAb1p), and one from the arsenite oxidizer Alkalilimnicola ehrlichei (NaVAe1p). Together, our data establish a family of active pore-only ion channels that should be excellent model systems for study of the factors that govern both sodium and calcium selectivity and permeability. Further, our findings suggest that similar dissection approaches may be applicable to a wide range of VGICs and, thus, serve as a means to simplify and accelerate biophysical, structural, and drug development efforts.

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2012 – Design, synthesis and functional analysis of dansylated polytheonamide mimic: an artificial Peptide ion channel
Port-a-Patch and Vesicle Prep Pro Publication in Journal of the American Chemical Society (2012) Authors: Itoh H., Matsuoka S., Kreir M., Inoue M.

We report herein the design, total synthesis, and functional analysis of a novel artificial ion channel molecule, designated as dansylated polytheonamide mimic (3). The channel 3 was designed based on an exceptionally potent cytotoxin, polytheonamide B (1). Our strategy for the development of synthetic ion channels, which could be easily derivatized for various functions, involved two key features. First, the structure of 1 was simplified by replacing many of nonproteinogenic amino acid residues which required multistep synthesis by commercially available amino acids while retaining those residues necessary for folding. It significantly reduced the number of synthetic steps and facilitated a practical chemical construction of 3. Second, the introduction of propargyl glycine at residue 44 enabled facile installation of dansyl group as a reporter of the membrane localization of 3. Application of a newly designed protective group strategy provided efficient construction of the 37 amino acid sequence of residues 12–48 through one automatic solid-phase peptide synthesis. After peptide cleavage from the resin, 3 was synthesized via dansyl group introduction and one fragment-coupling reaction with residues 1–11, followed by the global deprotection. The simplified mimic 3 exhibited potent cytotoxicity toward p388 mouse leukemia cells (IC50 = 12 nM), effectively induced ion transport across the lipid bilayers of liposomes, and displayed H+ and Na+ ion channel activities. Because of its simplified yet functional scaffold structure with a potential for diversification, our rationally designed ion channel molecule should be useful as a novel platform for developing various cytotoxic channel molecules with additional desired functions.

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2010 – Permeation of antibiotics through escherichia coli OmpF and OmpC porins: screening for influx on a single-molecule level
Port-a-Patch and Vesicle Prep Pro Publication in Journal of Biomolecular Screening (2010) Authors: Mahendran K.R., Kreir M., Weingart H., Fertig N., Winterhalter M.

A chip-based automated patch-clamp technique provides an attractive biophysical tool to quantify solute permeation through membrane channels. Proteo–giant unilamellar vesicles (proteo-GUVs) were used to form a stable lipid bilayer across a micrometer-sized hole. Because of the small size and hence low capacitance of the bilayer, single-channel recordings were achieved with very low background noise. The latter allowed the characterization of the influx of 2 major classes of antibiotics—cephalosporins and fluoroquinolones—through the major Escherichia coli porins OmpF and OmpC. Analyzing the ion current fluctuations in the presence of antibiotics revealed transport properties that allowed the authors to determine the mode of permeation. The chip-based setup allows rapid solution exchange and efficient quantification of antibiotic permeation through bacterial porins on a single-molecule level.

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2010 – Studying mechanosensitive ion channels using liposomes
Port-a-Patch and Vesicle Prep Pro Publication in Methods in Molecular Biology (2010) Authors: Martinac B., Rohde P.R., Battle A. R., Petrov E., Pal P, Alexander Foo A.FW, Vásquez V., Huynh T. and Kloda A.

Mechanosensitive (MS) ion channels are the primary molecular transducers of mechanical force into electrical and/or chemical intracellular signals in living cells. They have been implicated in innumerable mechanosensory physiological processes including touch and pain sensation, hearing, blood pressure control, micturition, cell volume regulation, tissue growth, or cellular turgor control. Much of what we know about the basic physical principles underlying the conversion of mechanical force acting upon membranes of living cells into conformational changes of MS channels comes from studies of MS channels reconstituted into artificial liposomes. Using bacterial MS channels as a model, we have shown by reconstituting these channels into liposomes that there is a close relationship between the physico-chemical properties of the lipid bilayer and structural dynamics bringing about the function of these channels.

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2009 – Amyloid-beta-Induced Ion Flux in Artificial Lipid Bilayers and Neuronal Cells: Resolving a Controversy
Port-a-Patch and Vesicle Prep Pro Publication in Neurotoxicity Research (2009) Authors: Capone R., Quiroz F.G., Prangkio P., Saluja I., Sauer A.M., Bautista M.R., Turner R.S., Yang J., Mayer M.

Understanding the pathogenicity of amyloid-beta (Aβ) peptides constitutes a major goal in research on Alzheimer’s disease (AD). One hypothesis entails that Aβ peptides induce uncontrolled, neurotoxic ion flux through cellular membranes. The exact biophysical mechanism of this ion flux is, however, a subject of an ongoing controversy which has attenuated progress toward understanding the importance of Aβ-induced ion flux in AD. The work presented here addresses two prevalent controversies regarding the nature of transmembrane ion flux induced by Αβ peptides. First, the results clarify that Αβ can induce stepwise ion flux across planar lipid bilayers as opposed to a gradual increase in transmembrane current; they show that the previously reported gradual thinning of membranes with concomitant increase in transmembrane current arises from residues of the solvent hexafluoroisopropanol, which is commonly used for the preparation of amyloid samples. Second, the results provide additional evidence suggesting that Aβ peptides can induce ion channel-like ion flux in cellular membranes that is independent from the postulated ability of Αβ to modulate intrinsic cellular ion channels or transporter proteins.

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2009 – The M34A mutant of Connexin26 reveals active conductance states in pore-suspending membranes
Port-a-Patch and Vesicle Prep Pro Publication in Journal of Structural Biology (2009) Authors: Gaßmann O., Kreir M., Ambrosi C., Pranskevich J., Oshima A, Röling C., Sosinsky G., Fertig N., Steinem C.

Connexin26 (Cx26) is a member of the connexin family, the building blocks for gap junction intercellular channels. These dodecameric assemblies are involved in gap junction-mediated cell–cell communication allowing the passage of ions and small molecules between two neighboring cells. Mutations in Cx26 lead to the disruption of gap junction-mediated intercellular communication with consequences such as hearing loss and skin disorders. We show here that a mutant of Cx26, M34A, forms an active hemichannel in lipid bilayer experiments. A comparison with the Cx26 wild-type is presented. Two different techniques using micro/nano-structured substrates for the formation of pore-suspending lipid membranes are used. We reconstituted the Cx26 wild-type and Cx26M34A into artificial lipid bilayers and observed single channel activity for each technique, with conductance levels of around 35, 70 and 165 pS for the wild-type. The conductance levels of Cx26M34A were found at around 45 and 70 pS.

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2006 – High-resolution electrophysiology on a chip: Transient dynamics of alamethicin channel formation
Port-a-Patch and Vesicle Prep Pro Publication in Biochimica et Biophysica Acta - Biomembranes (2006) Authors: Sondermann M., George M., Fertig N., Behrends J.C.

Microstructured planar substrates have been shown to be suitable for patch clamp recording from both whole cells and isolated patches of membrane, as well as for measurements from planar lipid bilayers. Here, we further explore this technology with respect to high-resolution, low noise single-channel recording. Using solvent-free lipid bilayers from giant unilamellar vesicles obtained by electro-swelling, we recorded channels formed by the peptaibol alamethicin, a well-studied model system for voltage-dependent channels, focusing on the transient dynamics of single-channel formation upon application of a voltage step. With our setup, we were able to distinctly resolve dwell times well below 100 mus and to perform a thorough statistical analysis of alamethicin gating. Our results show good agreement with models that do not rely on the existence of non-conducting preaggregate states. Microstructured apertures in glass substrates appear promising with respect to future experiments on cellular ion channels reconstituted in suspended lipid membranes.

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2008 – Rapid screening of membrane protein activity: electrophysiological analysis of OmpF reconstituted in proteoliposomes
Port-a-Patch and Vesicle Prep Pro Publication in Lab-on-a-chip (2008) Authors: Kreir M., Farre C., Beckler M., George M., Fertig N.

Solvent-free planar lipid bilayers were formed in an automatic manner by bursting of giant unilamellar vesicles (GUVs) after gentle suction application through micron-sized apertures in a borosilicate glass substrate. Incubation of GUVs with the purified ion channel protein of interest yielded proteoliposomes. These proteoliposomes allow for immediate recording of channel activity after GUV sealing. This approach reduces the time-consuming, laborious and sometimes difficult protein reconstitution processes normally performed after bilayer formation. Bilayer recordings are attractive for investigations of membrane proteins not accessible to patch clamp analysis, like e.g. proteins from organelles. In the presented work, we show the example of the outer membrane protein OmpF from Escherichiacoli. We reconstituted OmpF in proteoliposomes and observed the characteristic trimeric conductance levels and the typical gating induced by pH and transmembrane voltage. Moreover, OmpF is the main entrance for beta-lactam antibiotics and we investigated translocation processes of antibiotics and modulation of OmpF by spermine. We suggest that the rapid formation of porin containing lipid bilayers is of potential for the efficient electrophysiological characterization of the OmpF protein, for studying membrane permeation processes and for the rapid screening of antibiotics.

Application Note PDF
CiPA- “Identification of cardiac liability in drug discovery using the Port-a-Patch”
Port-a-Patch application note:   Cells were kindly provided by NEXEL and Charles River, USA.  

In 2013 the Cardiac Safety Research Consortium (CSRC), the Health and Environmental Sciences Institute (HESI), and the US Food and Drug Administration (FDA) proposed a paradigm to improve assessment of the proarrhythmic risk of therapeutic compounds. This paradigm, the Comprehensive In-vitro Proarrhythmia Assay (CiPA), was introduced to provide a more complete assessment of proarrhythmic risk by evaluating and implementing currently available high throughput methods. An important part of this is the electrophysiological evaluation of hERG, and also other cardiac channels including NaV1.5 and CaV1.2. The Q&A draft from August 2020 describes how nonclinical assays such as patch clamp can be used as a part of an integrated risk assessment prior to first-in-human studies, and in later stages of clinical development.Following up on hERG and NaV1.5 best practices and calibration standards which have been published recently on automated patch clamp devices, we show here cardiac ion channel recordings from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) or overexpressing cell lines generated with the world’s smallest patch clamp setups: Port-a-Patch and Port-a-Patch mini. Recordings at RT or physiological temperature of hERG recorded from HEK cells, and peak or late INa current recorded from iPSC-CMs or CHO cells are shown. INa-Late was activated by ATX-II and blocked by ranolazine, INa-Peak was blocked by tetracaine in a concentration-dependent manner, and hERG was blocked by increasing concentrations of dofetilide.

Webinar
07.09.2021 | Webinar: Automated patch clamp assay development for the study of red blood cells (RBCs) in health and disease
Presenters: Nicoletta Murciano (PhD Candidate - Nanion Technologies ), Dr. Lars Kaestner (Professor - Saarland University)

Calcium (Ca2+) is a universal signalling molecule and is critically important in regulating many physiological functions and survival of RBCs. Amongst others, intracellular Ca2+ controls cell volume and deformability. This process plays a substantial role in RBCs since their volume needs to adapt when passing blood vessel constrictions during the flow. Excessive Ca2+ uptake also leads to accelerated cell clearance causing anaemia.

Therefore, studying Ca2+ regulation is crucial to understand RBC diseases. Piezo1, KCa3.1 (Gardos channel) and NMDA receptors are three channels present in the RBC membrane and critical for Ca2+ regulation.

We developed functional assays to measure these channels in healthy and diseased RBCs populations using electrophysiological tools, contributing to the characterization of RBC diseases.

User meeting video
14.10.2020 | Webinar: Measurement of Transient receptor potential cation (TRP) channels using the Patchliner and Port-a-Patch
Dr. András Horváth (Nanion Technologies; Germany) This is an on-demand webinar from Nan]i[on and Friends 2020.

CHO cells expressing transient receptor potential cation channel V (TRPV) members 1, 3 and 4 and subfamily M member 8 were studied using our automated patch clamp systems, the Patchliner Octo (PL) and Port-a-Patch Perfusion (PaPP). During the recordings, heat, cold and/or ligand activation was performed. A classical ramp pulse-protocol (–100 mV to 100 mV) was applied.

Heat activation of TRPV1, 3, 4 channels was performed repeatedly by the heated pipetted (37-45 °C) of the PL. Interestingly ruthenium red (RR, 50 and 200 µM) was not able to prevent heat activation. Experiments involving TRPV4 were also performed on the PaPP. The cannel could be activated by heat and only partially blocked by RR. Ligand activation could be also performed on the PL (10 µM Capsaicin – TRPV1, 200 µM 2-APB – TRPV3, 100 nM GSK1016790 – TRPV4) and TRPV4 on the PaPP. In all cases the effect could be inhibited using blockers. TRPM8 channel could be repetitively activated using solution at 10°C on the PaPP at 10 °C. Capsazepine (10 µM) was used to block the activated current.

Both the PL and PaPP are powerful tools to study TRP channel physiology (both using heat activation and ligand activation) and could be used to find compounds which block the temperature and ligand response separately.

Webinar
20.11.2018 | Webinar: The RELEVANCE of ion channel interplay – Voltage-activated channels in non-excitable cells
RELEVANCE is an EU funded innovative training network and investigates in five scientific work packages different aspects, were the characterisation of red blood cells has an societal importance, such as in transfusion medicine, anaemias, diagnostics or in sports medicine. This will unavoidably result in the investigation of ion channels. The webinar is a joined presentation by a principle investigator of RELEVANCE, Prof. Lars Kaestner (Saarland University) and an early stage researcher, Maria Giustina Rotordam (Nanion Technologies, Munich).

Piezo1, KCa3.1 (Gardos channel) and CaV2.1 are three channels present in the red blood cell membrane. We will highlight the role of these channels in Hereditary Xerocytosis as well as in the Gardos Channelopathy using electrophysiological tools. Since red blood cells are everything but under suspicion to be excitable cells, we will take these cells as an example to show that KCa3.1, CaV2.1 and Piezo1 present an intimate interplay providing evidence that voltage-activated channels can well play a substantial role in non-excitable cells.

Publication link
2018 – Functional Analysis of Membrane Proteins Produced by Cell-Free Translation
Port-a-Patch and Orbit mini article in Protein Engineering (2018)   Authors: Dondapati S.K., Wüstenhagen D.A., Kubick S.

Cell-free production is a valuable and alternative method for the synthesis of membrane proteins. This system offers openness allowing the researchers to modify the reaction conditions without any boundaries. Additionally, the cell-free reactions are scalable from 20 μL up to several mL, faster and suitable for the high-throughput protein production. Here, we present two cell-free systems derived from Escherichia coli (E. coli) and Spodoptera frugiperda (Sf21) lysates. In the case of the E. coli cell-free system, nanodiscs are used for the solubilization and purification of membrane proteins. In the case of the Sf21 system, endogenous microsomes with an active translocon complex are present within the lysates which facilitate the incorporation of the bacterial potassium channel KcsA within the microsomal membranes. Following cell-free synthesis, these microsomes are directly used for the functional analysis of membrane proteins.

Publication link
2022 – Prediction and verification of potential lead analgesic and antiarrhythmic components in Corydalis yanhusuo W. T. Wang based on voltage-gated sodium channel proteins
Port-a-Patch and Buffer Solution Publication in International Journal of Biological Macromolecules (2022) Authors: Sun J., Liu X., Zhao S., Zhang S., Yang L., Zhang J., Zhao M., Xu Y.

Corydalis yanhusuo W. T. Wang, a traditional Chinese herbal medicine, has been used as an analgesic for thousands of years and it also promotes blood circulation. In this study, 33 Corydalis yanhusuo alkaloid active components were acquired from Traditional Chinese Medicine Database and Analysis Platform (TCMSP). A total of 543 pain-related targets, 1774 arrhythmia targets, and 642 potential targets of these active components were obtained using Swiss Target Prediction, GeneCards, Open Target Platform, and Therapeutic Target Database. Fifty intersecting targets were visualized through a Venn diagram, KEGG and GO pathway enrichment analysis. The analysis proposed that sodium ion channels are likely potential targets of Corydalis yanhusuo active components as analgesia and anti-arrhythmia agents. Molecular docking showed that the 33 components could bind to NaV1.7 and NaV1.5 (two subtypes of ion channel proteins) with different binding energies. In a patch clamp study, dihydrosanguinarine and dihydrochelerythrine, two monomers with the strongest binding effects, could inhibit the peak currents and promote both activation and inactivation phases of NaV1.5. Meanwhile, dihydrosanguinarine and dihydrochelerythrine could also inhibit peak currents and promote the activation phase of NaV1.7. Therefore, the findings from this study provide valuable information for future uses of traditional Chinese medicines to treat pain and cardiovascular disease.

Publication link
2021 – Active components of Bupleurum chinense and Angelica biserrata showed analgesic effects in formalin induced pain by acting on NaV1.7
Port-a-Patch and Buffer Solution Publication in Journal of Ethnopharmacology (2021) Authors: Xu Y., Yu Y., Wang Q., Li W., Zhang S., Liao X., Liu Y., Su Y., Zhao M., Zhang J.

Ethnopharmacology relevance - Pain is an unpleasant sensory and emotional experience, often accompanied by the occurrence of a variety of diseases. More than 800 kinds of traditional Chinese medicines (TCM) has now been reported for pain relief and several monomers have been developed into novel analgesic drugs. Bupleurum chinense and Angelica biserrata were representatives of the TCM that are currently available for the treatment of pain.

Webinar
14.10.2020 | Webinar: TRP channels in human corneal cells and their role in ophthalmology
Prof. Dr. Stefan Mergler (Charité -Universitätsmedizin Berlin; Germany) This is an on-demand webinar from Nan]i[on and Friends 2020.
Product Sheet PDF
Port-a-Patch – Product Sheet
Webinar
07.10.2015 | Webinar: Patch Clamp Made Easy, Fast Track to Excellence
This webinar shows applications that go way beyond possibilities of conventional patch-clamping, where the Port-a-Patch facilitates completely novel scientific directions.

The Port-a-Patch platform, used in the scientific work presented in this webinar, is a highly versatile patch clamp platform for high quality recordings form cells, organelles and artificial membranes.
The Port-a-Patch is a semi-automated patch clamp device supporting stable giga-seals and excellent voltage-control of the membrane.
The Port-a-Patch replaces a classical patch clamp rig, is easy to learn, still with the same quality and accuracy as known from conventional gold standard patch clamp. However, the planar geometry, compact size and versatile add-ons, allows an unprecedented experimental freedom of this platform.

This webinar shows applications that go way beyond possibilities of conventional patch-clamping, where the Port-a-Patch facilitates completely novel scientific directions. The high scientific impact of the Port-a-Patch is illustrated by its vast publications list, including high rank journals such as Science, Nature, PNAS.

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Port-a-Patch Product Flyer – Internal Perfusion
Flyer PDF
Port-a-Patch Product Flyer – Suction Control Pro
Application Note PDF
TRPV4 – Activation and inhibition of TRPV4 using different stimuli on the Port-a-Patch
Port-a-Patch application note:   

TRPV4 is a member of the transient receptor potential TRPV4 is a member of the transient receptor potential channel (TRP) family. Transient receptor potential vanilloidtype 4 (TRPV4) shares approximately 40% identity with TRPV1 and TRPV2 and is a Ca2+-permeable non-selectivecation channel1-3 expressed in a wide range of tissues including neurons of the central and peripheral nervous systems, and in non-neuronal tissue including human T cells, corneal and retinal epithelial cells, endothelial cells of the eye, liver, heart, kidney, synoviocytes, epitheliallining of trachea and lung airways, stellate cells of thepancreas, and many more.

Flyer PDF
Port-a-Patch Accessories Catalog
Application Note PDF
TRPM8 – Cold activation of TRPM8 using the External Perfusion System and the Port-a-Patch
Port-a-Patch application note:   

TRPM8 is a member of the transient receptor potential channel (TRP) family. TRPM8 is known to be a thermosensitive channel, activated by cold temperatures (below ~25˚C) and ligands such as menthol, Eucalyptol and icilin1-4. It belongs to the melastatin subfamily of TRP channels5 and shows an outward rectification with a relatively high permeability for calcium ions and little selectivity between monovalent cations. Menthol, a secondary alcohol produced by the peppermint herb, Mentha piperita, is widely used in the food and pharmaceutical industries as a cooling/soothing compound and odorant.Here we present data of hTRPM8 collected on the Port-a-Patch using cooled solution via the External Perfusion System. Cold activated hTRPM8 was blocked by capsazepine with an IC50 in good agreement with the literature.Here we present data of hTRPM8 collected on the Port-a-Patch using cooled solution via the External Perfusion System. Cold activated hTRPM8 was blocked by capsazepine with an IC50 in good agreement with the literature.

Application Note PDF
TRPM8 / TRPV1 – “TRPV1 and TRPM8 recorded on Nanion’s Port-a-Patch”
Port-a-Patch application note:  

TRPV1 and TRPM8 are members of the transient receptor potential channel (TRP) family. This family was designated TRP because of a spontaneously occurring Drosophila mutant lacking TRP that responded to a continuous light with a transient receptor potential. TRPV1 is mainly expressed in sensory nerves. Its presence is essential for transduction of nociception as well as inflammatory and hypothermic effects of vanniloid compounds. It also contributes to acute thermal nociception and thermal hyperalgesia following tissue injury. TRPV1 forms a relatively Ca2+-selective ion channel with outwardly rectifying properties. It is activated by vanilloids such as capsaicin, by protons, increased temperatures, lipoxygenase products, as well as anandamide (Huang et al., 2002). Menthol, a secondary alcohol produced by the peppermint herb, Mentha piperita, is widely used in the food and pharmaceutical industries as a cooling/soothing compound and odorant. It induces Ca2+ influx in a subset of sensory neurons from dorsal root and trigeminal ganglia, due to activation of TRPM8, a Ca2+-permeable, cold-activated member of the TRP superfamily of cation channels (McKemy et al., 2002; Peier et al., 2002). Here we present data of TRPV1 and TRPM8 collected on the Port-a-Patch. Channel activation with capsaicin as well as menthol are shown.

Application Note PDF
TRPA1 – “TRPA1 activation by allyl isothiocyanate recorded on the Port-a-Patch”
Port-a-Patch application note:  

Transient receptor potential (TRP) channels are an important class of receptors found widely distributed throughout the mammalian central and peripheral nervous systems. They have been shown to be activated by many stimuli including temperature, mechano-stimulation, divalent cations and pH, amongst others. TRP channels are receiving much attention as potential targets for the treatment of, for example, pain, respiratory diseases such as asthma, cancer and immune disorders (for review see ref. 1). The TRPA1 receptor was first cloned from cultured human lung fibroblasts but has subsequently been found to be expressed in sensory neurones and is often found co-localised with TRPV1. TRPA1 is activated by a number of chemical stimuli including allyl isothiocyanate (mustard oil), cinnamaldehyde (the active ingredient of cinnamon), chlorobenzylidene malononitrile (CS tear gas), hydrogen peroxide and hyperchlorite (chlorine gas). It is thought that TRPA1, together with TRPV1, may contribute to chemical hypersensitivity, chronic cough, and airway inflammation in asthma. Here we present data recorded on the Port-aPatch with external perfusion showing recordings of human TRPA1 (hTRPA1) activated by increasing concentrations of allyl isothiocyanate (AITC). hTRPA1 could be repetitively stimulated using low concentrations of AITC but was desensitized by high concentrations of AITC (>10 µM).

Application Note PDF
TRPM7 – “TRPM7 activation by internal sequestering of Mg2+ ions recorded on the Port-a-Patch”
Port-a-Patch application note:  

Transient receptor potential (TRP) channels are an important class of receptors found widely distributed throughout the mammalian central and peripheral nervous systems. They have been shown to be activated by many stimuli including temperature, mechano-stimulation, divalent cations and pH, amongst others. TRP channels are receiving much attention as potential targets for the treatment of, for example, pain, respiratory diseases such as asthma, cancer and immune disorders. The TRPM7 receptor is thought to play a role in magnesium homeostasis. A role for TRPM7 in intracellular pH sensing, the pathological response to blood vessel wall injury and cell adhesion has also been suggested. In electrophysiological studies TRPM7 can be recorded using a voltage ramp protocol. It displays a characteristic large outward current with little inward current and can be blocked by the presence of internal Mg2+ ions. Here we present data recorded on the Port-a-Patch with internal perfusion showing recordings of mouse TRPM7 (mTRPM7) and block of this channel by the internal perfusion of Mg2+ ions.

Application Note PDF
Mitochondria – “Recordings from mitochondria and mitoplasts”
Port-a-Patch application note:   

Mitochondria are often referred to as the “power house” of the cell since they are responsible for making most of the cell’s energy supply in the form of adenosine triphosphate (ATP). In addition to providing the cell with energy, mitochondria are thought to have roles in cell signalling, cellular differentiation and apoptosis. They have also been implicated in the pathophysiology of neurodegenerative disorders such as Parkinson’s Disease , and may also play a role in diabetes and in the ageing process. Mitochondria are usually rod shaped and range in size from approximately 1 - 10 µm. They have an outer membrane and a highly folded inner membrane. The outer membrane is highly permeable and contains one of the most well studied mitochondrial proteins, the voltage-dependent anion channel (VDAC). The inner membrane contains many ion channels, including the Ca2+ uniporter, a KATP channel, the Ca2+-activated K+ channel (KCa) and the inner membrane anion channel (IMAC). To study the mitochondrial inner membrane using the patch clamp technique, mitoplasts were formed. This is a process whereby the mitochondria are swelled, thus rupturing the outer membrane and exposing the inner membrane.

Application Note PDF
NaV1.7 – “Patch clamp recordings of hNaV1.7 on Nanion’s Port-a-Patch”
Port-a-Patch application note: Cells were kindly provided by Anaxon

The NaV1.7 gene (SCN9A) encodes a voltage-gated sodium (NaV) channel, primarily expressed in the peripheral nervous system and 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 role 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 voltagegated ion channels, a number of compounds display a  higher affinity for the inactivated state of the channel. For this reason, it is important to be able to reliably record both activation and inactivation kinetics of the channel. In this Application Note we present data using the Port-a-Patch characterizing CHO cells stably expressing hNaV1.7. The hNaV1.7 activation and inactivation properties and TTX sensitivity are consistent with those reported in the literature.

Application Note PDF
KV1.3 – “Automated internal perfusion of Jurkat cells expressing KV1.3 channels”
Port-a-Patch application note:   

KV1.3 is a voltage-gated potassium channel which plays a role in human T cell activation and proliferation, cell-mediated cytotoxicity and volume regulation. It is therefore an important target for the therapeutic control of T cellresponses. The KV1.3 channel is endogenously expressed in Jurkat cells, an immortalised T lymphocyte cell line. The channel is activated by membrane depolarisation at voltages typically more positive than -40 mV with rapid activation kinetics and slower inactivation. In common with many other voltage-gated potassium channels, KV1.3 can be blocked by replacing K+ with Cs+ ions in the pipette solution. Using Nanion’s Internal Perfusion System, we were able to record stable KV1.3 currents from Jurkat cells and reliably block this potassium current by perfusing the internal side of the chip with a Cs+-containing solution. The KV1.3 mediated current could also be completely recovered by perfusing the inside of the chip with K+-containing solution. KV1.3 mediated currents can also be blocked by internal perfusion with tetraethylammonium (TEA). We present data here showing block of KV1.3 by increasing concentrationsof TEA using the Internal Perfusion System. The IC50 that we obtained for TEA applied internally was in good agreement with the literature.

Application Note PDF
Mitochondria – “Mitoplasts from HEK cells on Nanion‘s Port-a-Patch”
Port-a-Patch application note:   

Mitochondria play an important role in metabolism by providing the cell with ATP due to oxidative phosphorylation. But they are also supposed to be involved in apoptosis and cytoprotection. The mitochondrial membranes contain a large number of ion channels, transporters and pores. However the physiological role of mitochondrial channels is largely unknown. The main channel of the outer membrane is VDAC, a well described anion conductance. In the inner membrane most prominent are the permeability transition pore (PTP) and the inner membrane anion channel IMAC. Different potassium channels are described as well (mitoBK, mitoKATP and Kv1.3) and a number of calcium conducting channels and receptors (for example MCU). To study the channels of the inner mitochondrial membrane the outer membrane can be stripped of by osmotic swelling. This method was used for the examples of electrophysiological measurement with the Port-a-Patch shown below. 

Application Note PDF
hERG – “Pharmacology of hERG recorded on Nanion´s Port-a-Patch”
Port-a-Patch application note:   Cells were kindly provided by Millipore.  

The hERG gene (KCNH2) encodes a potassium ion channel responsible for the repolarizing IKr current in the cardiac action potential (Sanguinetti et al., 1995). Abnormalities in this channel may lead to either Long QT syndrome (LQT2) (with loss-of-function mutations) or Short QT syndrome (with gain-of-function mutations), both potentially fatal cardiac arrhythmia, due to repolarization disturbances of the cardiac action potential. Given the importance of this channel in maintaining cardiac function, 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. These include antihistamines (e.g. Terfenadine), gastrointestinal prokinetic agents (e.g. Cisapride) and others. Here we present data collected on the Port-a-Patch. Astemizole, Terfenadine, Cisapride and Flunarizine dose-response curves on hERG expressed in CHO cells are shown. The mean current amplitude in these cells was 1076 ± 79 pA (n= 88) at -40 mV.

Application Note PDF
hERG – “Temperature controlled hERG recordings on the Port-a-Patch”
Port-a-Patch application note:   Cells were kindly provided by Millipore.  

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 cause 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 anti-arrhythmias and anti-psychotics, it has become an important target in compound safety screening. It is a desirable option to study this channel at physiological temperature since compounds can display different actions or potencies at physiological temperature. Here, we present data collected on the Port-a-Patch using the External Perfusion System coupled with Nanion’s Temperature Control. Cells were captured and sealed at room temperature and control recordings made before raising the temperature to 35°C so that parameters such as current peak amplitude could be compared at the two different temperatures. Furthermore, the hERG active compound, quinidine, was used at physiological temperature and a full dose response curve was achieved. This demonstrates the stability of the recordings on the Port-a-Patch at this temperature. The concentration response curve generated an IC50 similar to that obtained at room temperature, and similar to that published in the literature.

Application Note PDF
hERG – “Characterization of hERG (CHO) on Nanion’s Port-a-Patch”
Port-a-Patch application note:   Cells were kindly provided by Millipore.  

The hERG gene (KCNH2) encodes a potassium ion channel responsible for the repolarizing IKr current in the cardiac action potential (Sanguinetti et al., 1995). Abnormalities in this channel may lead to either Long QT Syndrome (LQT2) (with loss-of-function mutations) or Short QT syndrome (with gain-of-function mutations), both potentially fatal cardiac arrhythmia, due to repolarization disturbances of the cardiac action potential. Given the importance of this channel in maintaining cardiac function, 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. These include antihistamines (e.g. Terfenadine), gastrointestinal prokinetic agents (e.g. Cisapride) and others. In this report we present data that were collected on the Port-a-Patch. Cells (CHO permanently expressing hERG, supplied by Millipore) were tested. Current amplitudes, IVs and cisapride as well as quinidine dose response curves were analyzed.

Application Note PDF
hERG – “Characterization of hERG (HEK293) on Nanion’s Port-a-Patch”
Port-a-Patch application note:   Cells were kindly provided by Millipore.  

The hERG gene (KCNH2) encodes a potassium ion channel responsible for the repolarizing IKr current in the cardiac action potential (Sanguinetti et al., 1995). Abnormalities in this channel may lead to either Long QT Syndrome (LQT2) (with loss-of-function mutations) or Short QT syndrome (with gain-of-function mutations), both potentially fatal cardiac arrhythmia, due to repolarization disturbances of the cardiac action potential. Given the importance of this channel in maintaining cardiac function, 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. These include antihistamines (e.g. terfenadine), gastrointestinal prokinetic agents (e.g. cisapride) and others. In this report we present data that were collected on the Port-a-Patch. Cells (HEK293 stably expressing hERG, supplied by Millipore) were tested. Current amplitudes, IVs and cisapride as well as quinidine dose-response curves were analyzed.

Application Note PDF
CaV3.2 – “Characterization of CaV3.2 on Nanion’s Port-a-Patch”
Port-a-Patch application note:   Cells were kindly provided by Millipore.  

The gene CACNA1H encodes the α1H subunit of the voltage-gated calcium channel CaV3.2. It belongs to the low voltage-activated T-type calcium channels. CaV3.2 displays the typical characteristics of the T-type channels: activation at low depolarization of the membrane and transient kinetics. T-type Ca2+ channels are involved in diverse, mainly rhythmic processes like e.g. pacemaking and generation of thalamocortical rhythms in sleep or epilepsy. CaV3.2 is expressed in a wide variety of cells. Amongst others it has been found in kidney, smooth muscle, brain, adrenal and cardiac cells. It seems to be involved in contraction of smooth muscle and the secretion of the adrenal hormones aldosterone and cortisol. Pharmacological block of T-type channels may lead to new drugs for the treatment of hypertension and epilepsy. The biophysical and pharmacological properties of the cells are presented in this Application Note.

Application Note PDF
GABAA a5b3g2 – “Activation and inhibition of GABAA a5b3g2 receptors on the Port-a-Patch”
Port-a-Patch application note:   Cells were kindly provided by Merck Millipore.  

Gamma aminobutyric acid type A (GABAA) receptors are the most important inhibitory neurotransmitter receptors in the mammalian central nervous system (CNS). They are opened by GABA allowing the passage of chloride ions across the membrane. GABAA channels are modulated by a variety of different drugs including benzodiazepines, barbiturates, neuroactive steroids, anesthetics, and convulsants. The receptors are heteropentameric and depending on the subunit combination, they exhibit different electrophysiological and pharmacological properties. Six a-, three b-, three g-, one d-, one e-, one p-, one θ- and three r-subunits have been cloned, including splice variants of some of these subunits. Functional GABAA receptors typically assemble with two a, two b, and one g subunit, with alternating a and b subunits connected by a g subunit. GABAA receptors play a critical role in regulating excitability of the brain, anxiety, vigilance, as well as learning and memory. The a5 subunit is highly expressed in the hippocampus and olfactory bulb and expressed in low levels in other brain regions including the cortex, subiculum, hypothalamus, sympathetic preganglionic neurons, and amygdala. GABAA receptors containing the a5 subunit cluster at both extrasynaptic sites as well as synaptic sites thus contributing to tonic currents and synaptic GABA-ergic neurotransmission. a5 -containing receptors exhibit unique physiology and pharmacology and they are potential pharmacological targets for the treatment of neurodevelopmental disorders, depression, schizophrenia, and mild cognitive impairment. The Port-a-Patch with External Perfusion System was used to record a5b3g2 receptors expressed in HEK293 cells.

Application Note PDF
Cardiomyocytes – “Action Potentials in Mouse ES Cell-Derived Cor.At Cardiomyocytes on Nanion´s Port-a-Patch”
Port-a-Patch application note:   Cells were kindly provided by Axiogenesis.

To provide scientists in basic or applied cardiology and toxicology with a standardized and pure cardiac myocyte model with functional expression of all essential cardiac ion channels, Axiogenesis has developed Cor.At® cardiomyocytes. These mouse embryonic stem cell derived cardiomyocytes are ready to use and 99.9 % pure without contamination by other cell types. Using the Porta-Patch®, Cor.At® cardiomyocytes have experimentally been shown to functionally express at least three essential cardiac currents INa, ICa, and IK, and to exhibit typical cardiac action potentials.

Application Note PDF
CaV2.2 – “Characterization of CaV2.2 on Nanion’s Port-a-Patch”
Port-a-Patch application note:   Cells were kindly provided by Millipore.  

The voltage gated N-type calcium channel (CaV2.2) is encoded by the gene CACNA1B. CaV2.2 is a high voltage activated calcium channel. CaV2.2 is found mainly in the brain, where it mediates neurotransmitter release at the  synapse. The strong depolarization of neuronal action potentials causes the opening of the channel. Calcium can then enter the cell and initiates the fusion of the neurotransmitter vesicles with the membrane. CaV2.2 is inhibited by w-conotoxin, a neurotoxin of the fish hunting snail, with high specificity. CaV2.2 has been implicated in the transmission of pain. Pharmacological block of CaV2.2 by compounds based on w-conotoxin has been shown to be effective against strong  chronical pain. The biophysical and pharmacological properties of the cells are presented in this Application Note.

Publication link
2022 – Structural basis of the negative allosteric modulation of 5-BDBD at human P2X4 receptors
Port-a-Patch Publication in Molecular Pharmacology (2022) Authors: Bidula S., Nadzirin I.B., Cominetti M.MD., Hickey H., Cullum S. A., Searcey M., Schmid R., Fountain S.

The P2X4 receptor is a ligand-gated ion channel activated by extracellular ATP. P2X4 activity is associated with neuropathic pain, vasodilation, and pulmonary secretion and is therefore of therapeutic interest. The structure-activity relationship of P2X4 antagonists is poorly understood. Here we elucidate the structure-activity of 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) at human P2X4 by combining pharmacology, electrophysiology, molecular modeling, and medicinal chemistry. 5-BDBD antagonized P2X4 in a noncompetitive manner but lacked effect at human P2X2. Molecular modeling and site-directed mutagenesis suggested an allosteric binding site for 5-BDBD located between two subunits in the body region of P2X4, with M109, F178, Y300, and I312 on one subunit and R301 on the neighboring subunit as key residues involved in antagonist binding. The bromine group of 5-BDBD was redundant for the antagonist activity of 5-BDBD, although an interaction between the carbonyl group of 5-BDBD and R301 in P2X4 was associated with 5-BDBD activity. 5-BDBD could inhibit the closed channel but poorly inhibited the channel in the open/desensitizing state. We hypothesize that this is due to constriction of the allosteric site after transition from closed to open channel state. We propose that M109, F178, Y300, R301, and I312 are key residues for 5-BDBD binding; provide a structural explanation of how they contribute to 5-BDBD antagonism; and highlight that the limited action of 5-BDBD on open versus closed channels is due to a conformational change in the allosteric site.

Application Note PDF
Bacterial Spheroplasts – “Preparation and recordings on the Port-a-Patch of native E. Coli spheroplasts”
Port-a-Patch application note:   

Bacterial membranes are not easy to patch clamp. Since bacterial ion channels are of increasing interest, we started to optimize the protocols for patch clamping bacterial spheroplasts with the Port-a-Patch. Bacterial spheroaplasts can be prepared up to a size of 5 µm. They consist of the inner bacterial membrane. This technique was first used for patch clamp experiments by Boris Matrinac in 1987 and let to the discovery of mechanosensitive channels in E. coli. Here we describe the preparation of Spheroplasts out of E. coli and show ion channel currents recorded with the Port-a-Patch.

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2022 – A chemically mediated artificial neuron
Port-a-Patch Publication in Nature Electronics (2022) Authors: Wang T., Wang M., Wang J., Yang L., Ren X., Song G., Chen S., Yuan Y., Liu R., Pan L., Li Z., Leow W.R., Luo Y., Ji S., Cui Z., He K., Zhang F., Lv F., Tian Y., Cai K., Yang B., Niu J., Zou H., Liu S., Xu G., Fan X., Hu B., Loh X.J., Wang L. Chen X.

Brain–machine interfaces typically rely on electrophysiological signals to interpret and transmit neurological information. In biological systems, however, neurotransmitters are chemical-based interneuron messengers. This mismatch can potentially lead to incorrect interpretation of the transmitted neuron information. Here we report a chemically mediated artificial neuron that can receive and release the neurotransmitter dopamine. The artificial neuron detects dopamine using a carbon-based electrochemical sensor and then processes the sensory signals using a memristor with synaptic plasticity, before stimulating dopamine release through a heat-responsive hydrogel. The system responds to dopamine exocytosis from rat pheochromocytoma cells and also releases dopamine to activate pheochromocytoma cells, forming a chemical communication loop similar to interneurons. To illustrate the potential of this approach, we show that the artificial neuron can trigger the controllable movement of a mouse leg and robotic hand.

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2022 – Mutagenicity and safety pharmacology of a standardized antidiabetic polyherbal formulation
Port-a-Patch Publication in Nature Scientific Reports (2022) Authors: Majid F.A.A., Fadhlina A., Ismail H.F., Zainol S.N., Mamillapalli A.K., Venkatesan V., Eswarappa R., Pillai R.

Synacinn is a standardized polyherbal extract formulated for the treatment of diabetes mellitus and its complications. This study aims to assess the mutagenicity potential of Synacinn by Ames assay and in vivo bone marrow micronucleus (MN) test on Sprague Dawley rat. Human ether-a-go-go-related gene (hERG) assay and Functional Observation Battery (FOB) were done for the safety pharmacology tests. In the Ames assay, Dose Range Finding (DRF) study and mutagenicity assays (+/− S9) were carried out. For the MN test, a preliminary and definitive study were conducted. In-life observations and number of immature and mature erythrocytes in the bone marrow cells were recorded. The hERG assay was conducted to determine the inhibitory effect on hERG potassium channel current expressed in human embryonic kidney cells (HEK293). FOB tests were performed orally (250, 750, and 2000 mg/kg) on Sprague Dawley rats. Synacinn is non-mutagenic against all tested strains of Salmonella typhimurium and did not induce any clastogenicity in the rat bone marrow. Synacinn also did not produce any significant inhibition (p ≤ 0.05) on hERG potassium current. Synacinn did not cause any neurobehavioural changes in rats up to 2000 mg/kg. Thus, no mutagenicity, cardiotoxicity and neurotoxicity effects of Synacinn were observed in this study.

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2021 – The TRPV2 channel mediates Ca2+ influx and the Δ9-THC-dependent decrease in osmotic fragility in red blood cells
Port-a-Patch Publication in Haematologica (2021) Authors: Belkacemi A., Fecher-Trost C., Tinschert R., Flormann D., Malihpour M., Wagner C., Meyer M.R., Beck A., and Flockerzi V.

Water and ionic homeostasis of red blood cells (RBC) is regulated by various active and passive transport mechanisms in the RBC membrane, including channels like aquaporins, the mechanically activated non-selective cation channel Piezo1 and the Ca2+-activated potassium channel KCa3.1. The human genome contains 27 genes that code for transient receptor potential (TRP) channels. The only TRP channel protein that has been detected in circulating mouse RBC is TRPC6, which might be associated with basal Ca2+ leakage and stress-stimulated Ca2+ entry. TRPC2 and TRPC3 are expressed by murine erythroid precursors and splenic erythroblasts, and in these cells, erythropoietin stimulates an increase in intracellular calcium concentration via TRPC2 and TRPC3. In this study we identified the TRP vanilloid (TRPV) 2 channel protein in mouse and human RBC by specific antibodies and mass spectrometry. TRPV2-dependent currents and Ca2+ entry were activated by the TRPV2 agonists cannabidiol (CBD) and Δ9-tetrahydrocannabinol (Δ9-THC) resulting in a leftshift of the hypotonicity-dependent hemolysis curve. This effect was reversed in the presence of the KCa3.1 inhibitor TRAM-34, whereas the knockout of Trpv2 right-shifted the hemolysis curve to higher tonicities. 

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2021 – Translational Studies on Anti-Atrial Fibrillatory Action of Oseltamivir by its in vivo and in vitro Electropharmacological Analyses
Port-a-Patch Publication in Frontiers in Pharmacology (2021) Authors: Kambayashi R.,  Izumi-Nakaseko H., Goto A., Tsurudome K., Ohshiro H., Izumi T.,  Hagiwara-Nagasawa M., Chiba K., Nishiyama R., Oyama S., Nunoi Y., Takei Y., Matsumoto A., Sugiyama A.

Oseltamivir has been shown to prolong the atrial conduction time and effective refractory period, and to suppress the onset of burst pacing-induced atrial fibrillation in vitro. To better predict its potential clinical benefit as an anti-atrial fibrillatory drug, we performed translational studies by assessing in vivo anti-atrial fibrillatory effect along with in vivo and in vitro electropharmacological analyses. Oseltamivir in intravenous doses of 3 (n = 6) and 30 mg/kg (n = 7) was administered in conscious state to the persistent atrial fibrillation model dogs to confirm its anti-atrial fibrillatory action. The model was prepared by tachypacing to the atria of chronic atrioventricular block dogs for > 6 weeks. Next, oseltamivir in doses of 0.3, 3 and 30 mg/kg was intravenously administered to the halothane-anesthetized intact dogs to analyze its in vivo electrophysiological actions (n = 4). Finally, its in vitro effects of 10–1,000 μM on IK,ACh, IKur, IKr, INa and ICaL were analyzed by using cell lines stably expressing Kir3.1/3.4, KV1.5, hERG, NaV1.5 or CaV1.2, respectively (n = 3 for IK,ACh and IKr or n = 6 for IKr, INa and ICaL). Oseltamivir in doses of 3 and 30 mg/kg terminated the atrial fibrillation in 1 out of 6 and in 6 out of 7 atrial fibrillation model dogs, respectively without inducing any lethal ventricular arrhythmia. Its 3 and 30 mg/kg delayed inter-atrial conduction in a frequency-dependent manner, whereas they prolonged atrial effective refractory period in a reverse frequency-dependent manner in the intact dogs. The current assay indicated that IC50 values for IK,ACh and IKr were 160 and 231 μM, respectively, but 1,000 µM inhibited INa, ICaL and IKur by 22, 19 and 13%, respectively. The extent of INa blockade was enhanced at faster beating rate and more depolarized resting membrane potential. Oseltamivir effectively terminated the persistent atrial fibrillation, which may be largely due to the prolongation of the atrial effective refractory period and inter-atrial conduction time induced by IK,ACh and IKr inhibitions along with INa suppression. Thus, oseltamivir can exert a powerful anti-atrial fibrillatory action through its ideal multi-channel blocking property; and oseltamivir would become a promising seed compound for developing efficacious and safe anti-atrial fibrillatory drugs. 

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2021 – Taspine is a natural product that suppresses P2X4 receptor activity via phosphoinositide 3-kinase inhibition
Port-a-Patch Publication in British Journal of Pharmacology (2021) Authors: Bin Nadzirin I., Fortuny-Gomez A., Ngum N., Richards D., Ali S., Searcey M., Fountain S.J.

Background & purpose: P2X4 is a ligand-gated cation channel activated by extracellular ATP, involved in neuropathic pain, inflammation and arterial tone.
Experimental approach: Natural products were screened against human or mouse P2X4 activity using fura-2 loaded 1321N1 cells for measurement of intracellular Ca2+ responses; whole-cell currents were measured by patch clamp electrophysiological. Human primary macrophage chemokine release was used to assess effect of taspine on inflammatory cell function. An enzymatic assay was performed to assess the effect of taspine on recombinant PI3-kinase.
Key Results: A natural product screen identified taspine as an inhibitor of humanP2X4 activity. Taspine inhibits human and mouse P2X4-mediated Ca2+ influx in 1321N1 cells expressing receptors but lacked activity at human P2X2, P2X3, P2X2/3 and P2X7 receptors. Taspine inhibited the maximal response at human and mouse P2X4 but effective on ATP potency. Taspine has a slow onset rate (∼15 min for half-maximal inhibition), irreversible over 30 min of washout. Taspine inhibitsP2X4-mediated Ca2+ signalling in mouse BV-2 microglia cells and human primary macrophage. Taspine inhibited P2X4-mediated CXCL5 secretion in human primary macrophage. Taspine reversed ivermectin-induced potentiation of P2X4 currents in1321N1 stably expressing cells. The PI3-kinase inhibitor LY294002 mimicked the properties of taspine on P2X4-mediated Ca2+ influx and whole-cell currents. Taspine directly inhibited the enzymatic activity of recombinant PI3-kinase in a competitive manner.
Conclusion and Implications: Taspine is a novel natural product P2X4 receptor inhib-itor, mediating its effect through PI3-kinase inhibition rather than receptor antagonism. Taspine can inhibit the pro-inflammatory signalling by P2X4 in human primary macrophage.

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2021 – The mechanism of non-blocking inhibition of sodium channels revealed by conformation-selective photolabeling
Port-a-Patch Publication in British Journal of Pharmacology (2021) Authors: Földi M.C., Pesti K., Zboray K., Toth A.V., Hegedűs T., Málnási‐Csizmadia A., Lukacs P., Mike A.

Background and Purpose: Sodium channel inhibitors can be used to treat hyperexcitability‐related diseases, including epilepsies, pain syndromes, neuromuscular disorders and cardiac arrhythmias. The applicability of these drugs is limited by their nonspecific effect on physiological function. They act mainly by sodium channel block and in addition by modulation of channel kinetics. While channel block inhibits healthy and pathological tissue equally, modulation can preferentially inhibit pathological activity. An ideal drug designed to target the sodium channels of pathological tissue would act predominantly by modulation. Thus far, no such drug has been described.Experimental Approach: Patch‐clamp experiments with ultra‐fast solution exchange and photolabeling‐coupled electrophysiology were applied to describe the unique mechanism of riluzole on NaV1.4 sodium channels. In silico docking experiments were used to study the molecular details of binding.Key Results: We present evidence that riluzole acts predominantly by non‐blocking modulation. We propose that, being a relatively small molecule, riluzole is able to stay bound to the binding site, but nonetheless stay off the conduction pathway, by residing in one of the fenestrations. We demonstrate how this mechanism can be recognized.Conclusions and Implications: Our results identify riluzole as the prototype of this new class of sodium channel inhibitors. Drugs of this class are expected to selectively prevent hyperexcitability, while having minimal effect on cells firing at a normal rate from a normal resting potential.

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2021 – Structure, gating and interactions of the voltage-dependent anion channel
Port-a-Patch Publication in European Biophysics Journal (2021) Authors: Najbauer E.E., Becker S., Giller K., Zweckstetter M., Lange A., Steinem C., de Groot B.L., Griesinger C., Andreas L.B.

The voltage-dependent anion channel (VDAC) is one of the most highly abundant proteins found in the outer mitochondrial membrane, and was one of the earliest discovered. Here we review progress in understanding VDAC function with a focus on its structure, discussing various models proposed for voltage gating as well as potential drug targets to modulate the channel’s function. In addition, we explore the sensitivity of VDAC structure to variations in the membrane environment, comparing DMPC-only, DMPC with cholesterol, and near-native lipid compositions, and use magic-angle spinning NMR spectroscopy to locate cholesterol on the outside of the β-barrel. We find that the VDAC protein structure remains unchanged in different membrane compositions, including conditions with cholesterol.

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2021 – Study of Anti-Inflammatory and Analgesic Activity of Scorpion Toxins DKK-SP1/2 from Scorpion Buthus martensii Karsch (BmK)
Port-a-Patch Publication in Toxins (2021) Authors: Liu Y., Li Y., Zhu Y., Zhang L., Ji J., Gui M., Li C., Song Y.

Buthus martensii Karsch (BmK), is a kind of traditional Chinese medicine, which has been used for a long history for the treatment of many diseases, such as inflammation, pain and cancer. In this study, DKK-SP1/2/3 genes were screened and extracted from the cDNA library of BmK.The DKK-SP1/2/3 were expressed by using plasmid pSYPU-1b in E. coli BL21, and recombinant proteins were obtained by column chromatography. In the xylene-induced mouse ear swelling and carrageenan-induced rat paw swelling model, DKK-SP1 exerted a significant anti-inflammatory effect by inhibiting the expression of NaV1.8 channel. Meanwhile, the release of pro-inflammatory cytokines(COX-2, IL-6) was decreased significantly and the release of anti-inflammatory cytokines (IL-10) were elevated significantly. Moreover, DKK-SP1 could significantly decrease the NaV1.8 current in acutelyisolated rat DRG neurons. In the acetic acid-writhing and ION-CCI model, DKK-SP2 displayed significant analgesic activity by inhibiting the expression of the NaV1.7 channel. Moreover, DKK-SP2 could significantly inhibit the NaV1.7 current in the hNaV1.7-CHO cells.

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2021 – L-carnitine suppresses transient receptor potential vanilloid type 1 activity and myofibroblast transdifferentiation in human corneal keratocytes
Port-a-Patch Publication in Lab Invest (2021) Authors: Turan E., Valtink M., Reinach P.S., Skupin A., Luo H., Brockmann T., Salem M.H.O.B., Pleyer U., Mergler S.

Corneal stromal wound healing is a well-balanced process promoted by overlapping phases including keratocyte proliferation, inflammatory-related events, and tissue remodeling. L-carnitine as a natural antioxidant has shown potential to reduce stromal fibrosis, yet the underlying pathway is still unknown. Since transient receptor potential vanilloid 1 (TRPV1) is a potential drug target for improving the outcome of inflammatory/fibrogenic wound healing, we investigated if L-carnitine can mediate inhibition of the fibrotic response through suppression of TRPV1 activation in human corneal keratocytes (HCK). We determined TRPV1-induced intracellular calcium transients using fluorescence calcium imaging, channel currents by planar patch-clamping, and cell migration by scratch assay for wound healing. The potential L-carnitine effect on TRPV1-induced myofibroblast transdifferentiation was evaluated by immunocytochemical detection of alpha smooth muscle actin. RT-PCR analysis confirmed TRPV1 mRNA expression in HCK. L-carnitine (1 mmol/l) inhibited either capsaicin (CAP) (10 µmol/l), hypertonic stress (450 mOsmol/l), or thermal increase (>43 °C) induced Ca2+ transients and corresponding increases in TRPV1-induced inward and outward whole-cell currents. This was accompanied by suppression of injury-induced increases in myofibroblast transdifferentiation and cell migration. In conclusion, L-carnitine contributes to inhibit stromal scarring through suppressing an injury-induced intrinsic TRPV1 activity that is linked with induction of myofibroblast transdifferentiation in HCK cells.

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2021 – Regulatory effects of non-steroidal anti-inflammatory drugs on cardiac ion channels NaV1.5 and KV11.1
Port-a-Patch Publication in Chemico-Biological Interactions (2021) Authors: Xu Y., Li W., Han Y., Liu H., Zhang S., Yan J., Sun J., Liu Y., Zhang J., Zhao M.

Non-steroidal Anti-inflammatory Drugs (NSAIDs) are widely used because of their excellent anti-inflammatory and analgesic effects. However, NSAIDs could cause certain cardiac side effects, such as myocardial infarction, heart failure, atrial fibrillation, arrhythmia and sudden cardiac death. Therefore, meloxicam, nimesulide, piroxicam, and diclofenac were selected and the whole cell patch clamp technique was used to investigate the electrophysiological regulatory effects of them on the sodium channel hNaV1.5 and potassium channel hKV11.1, which were closely associated to the biotoxicity of cardiac, and to explore the potential cardiac risk mechanism. The results showed that the four NSAIDs could inhibit the peak currents of hNaV1.5 and hKV11.1. Furthermore, the four NSAIDs could affect both the activation and inactivation processes of hNaV1.5 with I–V curves left-shifted to hyperpolarized direction in activation phase. These data indicate that the inhibition effects of NaV1.5 and KV11.1 by meloxicam, nimesulide, piroxicam, and diclofenac might contribute to their potential cardiac risk. These findings provide a basis for the discovery of other potential cardiac risk targets for NSAIDs.

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2021 – Gating the channel pore of ionotropic glutamate receptors with bacterial substrate binding proteins
Port-a-Patch prePublication in bioRxiv (2021) Authors: Bernhard M., Laube B.

Tetrameric ionotropic glutamate receptors (iGluRs) mediate excitatory neurotransmission in the mammalian central nervous system and are involved in learning, memory formation, and pathological processes. Based on structural and sequence similarities of the ligand-binding and channel domains of iGluR subunits to bacterial binding proteins and potassium channels, iGluRs are thought to have originally arisen from their fusion. Here we report the functional coupling of the bacterial ectoine binding protein EhuB to the channel pore-forming transmembrane domains of the bacterial GluR0 receptor by stabilization of dimeric binding domains. Insertion of a disulfide bridge in the dimer interface abolished desensitization of the channel current analogous to mammalian iGluRs. These results demonstrate the functional compatibility of bacterial binding proteins to the gate of the channel pore of an iGluR. Moreover, our results highlight the modular structure and crucial role of binding domain dimerization in the functional evolution of iGluRs.

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2021 – Glucosylsphingosine evokes pruritus via activation of 5-HT2A receptor and TRPV4 in sensory neurons
Port-a-Patch Publication in British Journal of Pharmacology (2021) Authors: Sanjel B., Kim B-H., Song M-H., Carstens E., Shim W-S.

Background and Purpose: Glucosylsphingosine (GS), an endogenous sphingolipid, is highly accumulated in the epidermis of patients with atopic dermatitis (AD) due to abnormal ceramide metabolism. More importantly, GS can evoke scratching behaviors. However, the precise molecular mechanism by which GS induces pruritus has been elusive. Thus, the present study aimed to elucidate the molecular signaling pathway of GS, especially at the peripheral sensory neuronal levels.Experimental Approach: Calcium imaging was used to investigate the responses of HEK293T cells or mouse dorsal root ganglion (DRG) neurons to application of GS. Scratching behavior tests were also performed with wild-type and Trpv4 knockout mice.Key Results: GS activated DRG neurons in a manner involving both the 5-HT2A receptor and TRPV4. Furthermore, GS-induced responses were significantly suppressed by various inhibitors, including ketanserin (5-HT2A receptor antagonist), YM254890 (Gαq/11 inhibitor), gallein (Gβγ complex inhibitor), U73122 (phospholipase C inhibitor), bisindolylmaleimide I (PKC inhibitor), and HC067047 (TRPV4 antagonist). Moreover, DRG neurons from Trpv4 knockout mice exhibited significantly reduced responses to GS. Additionally, GS-evoked scratching behaviors were greatly decreased by pretreatment with inhibitors of either 5-HT2A receptor or TRPV4. As expected, GS-evoked scratching behavior was also significantly decreased in Trpv4 knockout mice.Conclusion and Implications: Overall, the present study provides evidence for a novel molecular signaling pathway for GS-evoked pruritus, which utilizes both 5-HT2A receptor and TRPV4 in mouse sensory neurons. Considering the high accumulation of GS in the epidermis of patients with AD, GS could be another pruritogen in patients with AD.

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2021 – An advanced automated patch clamp protocol design to investigate drug – ion channel binding dynamics
Port-a-Patch Publication in Front. Pharmacol. (2021) Authors:  Lukacs P., Pesti K., Földi M.C., Zboray K.,  Toth A.V., Papp G, Mike A.

Standard high throughput screening projects using automated patch-clamp instruments often fail to grasp essential details of the mechanism of action, such as binding/unbinding dynamics and modulation of gating. In this study, we aim to demonstrate that depth of analysis can be combined with acceptable throughput on such instruments. Using the microfluidics-based automated patch clamp, IonFlux Mercury, we developed a method for a rapid assessment of the mechanism of action of sodium channel inhibitors, including their state-dependent association and dissociation kinetics. The method is based on a complex voltage protocol, which is repeated at 1 Hz. Using this time resolution we could monitor the onset and offset of both channel block and modulation of gating upon drug perfusion and washout. Our results show that the onset and the offset of drug effects are complex processes, involving several steps, which may occur on different time scales. We could identify distinct sub-processes on the millisecond time scale, as well as on the second time scale. Automated analysis of the results allows collection of detailed information regarding the mechanism of action of individual compounds, which may help the assessment of therapeutic potential for hyperexcitability-related disorders, such as epilepsies, pain syndromes, neuromuscular disorders, or neurodegenerative diseases. 

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2021 – An antibiotic-resistance conferring mutation in a neisserial porin: Structure, ion flux, and ampicillin binding
Port-a-Patch Pre-Print Publication in Biochimica et Biophysica Acta (BBA) - Biomembranes (2021) Authors: Bartsch A., Ives C.M., Kattner C., Pein F., Diehn M., Tanabe M., Munk A., Zachariae U., Steinem C., Llabrés S.

Gram-negative bacteria cause the majority of highly drug-resistant bacterial infections. To cross the outer membrane of the complex Gram-negative cell envelope, antibiotics permeate through porins, trimeric channel proteins that enable the exchange of small polar molecules. Mutations in porins contribute to the development of drug-resistant phenotypes. In this work, we show that a single point mutation in the porin PorB from Neisseria meningitidis, the causative agent of bacterial meningitis, can strongly affect the binding and permeation of beta-lactam antibiotics. Using X-ray crystallography, high-resolution electrophysiology, atomistic biomolecular simulation, and liposome swelling experiments, we demonstrate differences in drug binding affinity, ion selectivity and drug permeability of PorB. Our work further reveals distinct interactions between the transversal electric field in the porin eyelet and the zwitterionic drugs, which manifest themselves under applied electric fields in electrophysiology and are altered by the mutation. These observations may apply more broadly to drug-porin interactions in other channels. Our results improve the molecular understanding of porin-based drug-resistance in Gram-negative bacteria.

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2021 – A glutamate concentration-biased allosteric modulator potentiates NMDA-induced ion influx in neurons
Port-a-Patch Publication in PRP Pharmacology Research & Perspectives (2021) Authors: Costa B. M., Kwapisz L. C., Mehrkens B., Bledsoe D. N., Vacca B. N., Johnston T. V., Razzaq R., Manickam D., Klein B. G.

Precisely controlled synaptic glutamate concentration is essential for the normal function of the N-methyl D-aspartate (NMDA) receptors. Atypical fluctuations in synaptic glutamate homeostasis lead to aberrant NMDA receptor activity that results in the pathogenesis of neurological and psychiatric disorders. Therefore, glutamate concentration-dependent NMDA receptor modulators would be clinically useful agents with fewer on-target adverse effects. In the present study, we have characterized a novel compound (CNS4) that potentiates NMDA receptor currents based on glutamate concentration. This compound alters glutamate potency and exhibits no voltage-dependent effect. Patch-clamp electrophysiology recordings confirmed agonist concentration-dependent changes in maximum inducible currents. Dynamic Ca2+ and Na+ imaging assays using rat brain cortical, striatal and cerebellar neurons revealed CNS4 potentiated ion influx through native NMDA receptor activity. Overall, CNS4 is novel in chemical structure, mechanism of action and agonist concentration-biased allosteric modulatory effect. This compound or its future analogs will serve as useful candidates to develop drug-like compounds for the treatment of treatment-resistant schizophrenia and major depression disorders associated with hypoglutamatergic neurotransmission.

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2021 – Abnormal podocyte TRPML1 channel activity and exosome release in mice with podocyte-specific Asah1 gene deletion
Port-a-Patch Publication in BBA - Molecular and Cell Biology of Lipids (2021) Authors: Li G., Huang D., Bhat O.M., Poklis J.L., Zhang A., Zou Y., Kidd J., Gehr T.W.B., Li P-L.

Podocytopathy and associated nephrotic syndrome (NS) have been reported in a knockout mouse strain (Asah1fl/fl/PodoCre) with a podocyte-specific deletion of α subunit (the main catalytic subunit) of acid ceramidase (Ac). However, the pathogenesis of podocytopathy of these mice remains unknown. The present study tested whether exosome release from podocytes is enhanced due to Asah1 gene knockout, which may serve as a pathogenic mechanism switching on podocytopathy and associated NS in Asah1fl/fl/PodoCre mice. We first demonstrated the remarkable elevation of urinary exosome excretion in Asah1fl/fl/PodoCre mice compared with WT/WT mice, which was accompanied by significant Annexin-II (an exosome marker) accumulation in glomeruli of Asah1fl/fl/PodoCre mice, as detected by immunohistochemistry. In cell studies, we also confirmed that Asah1 gene knockout enhanced exosome release in the primary cultures of podocyte isolated from Asah1fl/fl/PodoCre mice compared to WT/WT mice. In the podocytes from Asah1fl/fl/PodoCre mice, the interactions of lysosome and multivesicular body (MVB) were demonstrated to be decreased in comparison with those from their control littermates, suggesting reduced MVB degradation that may lead to increase in exosome release. Given the critical role of transient receptor potential mucolipin 1 (TRPML1) channel in Ca2+-dependent lysosome trafficking and consequent lysosome-MVB interaction, we tested whether lysosomal Ca2+ release through TRPML1 channels is inhibited in the podocytes of Asah1fl/fl/PodoCre mice. By GCaMP3 Ca2+ imaging, it was found that lysosomal Ca2+ release through TRPML1 channels was substantially suppressed in podocytes with Asah1 gene deletion. As an Ac product, sphingosine was found to rescue TRPML1 channel activity and thereby recover lysosome-MVB interaction and reduce exosome release of podocytes from Asah1fl/fl/PodoCre mice. Combination of N, N-dimethylsphingosine (DMS), a potent sphingosine kinase inhibitor, and sphingosine significantly inhibited urinary exosome excretion of Asah1fl/fl/PodoCre mice. Moreover, rescue of Aash1 gene expression in podocytes of Asah1fl/fl/PodoCre mice showed normal ceramide metabolism and exosome secretion. Based on these results, we conclude that the normal expression of Ac importantly contributes to the control of TRPML1 channel activity, lysosome-MVB interaction, and consequent exosome release from podocytes. Asah1 gene defect inhibits TRPML1 channel activity and thereby enhances exosome release, which may contribute to the development of podocytopathy and associated NS.

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2020 – Trp, a conserved aromatic residue crucial to the interaction of a scorpion peptide with sodium channels
Port-a-Patch Publication found in The Journal of Biochemistry (2020) Authors: Xu Y., Sun J., Yu Y., Kong X., Meng X., Liu Y., Cui Y., Su Y., Zhao M., Zhang J. 

Antitumor-analgesic peptide (AGAP), one scorpion toxin purified from Buthus martensii Karsch, was known as its analgesic and antitumor activities. Trp38, a conserved aromatic residue of AGAP, might play important roles in its interaction with sodium channels. In this study, a mutant W38F was generated and effects of W38F were examined on hNaV1.4, hNaV1.5, and hNaV1.7 by using whole cell patch clamp, which were closely associated to the biotoxicity of skeletal and cardiac muscles, and pain signaling. The data showed that W38F decreased the inhibition effects of peak currents of hNaV1.7, hNaV1.4, and hNaV1.5 compared with AGAP, notably, W38F reduced the analgesic activity compared with AGAP. The results suggested that Trp38 be a crucial amino acid involved in the interaction with these three sodium channels. The decreased analgesic activity of W38F might result from its much less inhibition of hNaV1.7. These findings provided more information about the relationship between structure and function of AGAP and may facilitate the modification of other scorpion toxins with pharmacological effects.

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2020 – Wireless Charging Electrochemiluminescence System for Ionic Channel Manipulation in Living Cells
Port-a-Patch Publication in ACS Applied Materials & Interfaces (2020) Authors: Peng K., Liu S., Lv F., Fu X., Hussain S., Zhao H., Liu L., Wang S.

Optogenetics holds great potential for precisely altering living cell behavior with the aid of light because of its high temporospatial resolution. However, the light-dependent manner severely limits its applications in deep tissues, particularly to those in the visible region. Here, we propose a wireless charging electrochemiluminescence (ECL) system, featured with long-time delayed luminescence, to remotely activate the light-gated ion channel (channelrhodopsin-2, ChR2) on the living cell membrane, followed by the intracellular influx of Ca2+ ions. Upon wireless charging ECL illumination, the influx of Ca2+ into the living cells triggers strong ion indicator fluorescence, suggesting the successful remote control on ChR2. As such, the wireless charging ECL strategy exhibits great potential to wireless control of optogenetics in deep tissues by implanting a device in vivo.

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2020 – Mechanism of non-blocking inhibition of sodium channels revealed by conformation-selective photolabeling
Port-a-Patch pre-Publication in BioRxiv (2020) Authors: Földi M.C., Pesti K., Zboray K., Hegedűs T., Málnási-Csizmadia A., Lukács P., Mike A.

Sodium channel inhibitor drugs can exert their effect by either blocking, or modulating the channel. The extent of modulation versus channel block is crucial regarding the therapeutic potential of drug candidates. Modulation can be selective for pathological hyperactivity, while channel block affects vital physiological function as much as pathological activity. Previous results indicated that riluzole, a drug with neuroprotective and antiepileptic effects, may have a unique mechanism of action, where modulation is predominant, and channel block is negligible. We studied the effects of riluzole on rNaV1.4 channels expressed in HEK cells. We observed that inhibition by riluzole disappeared and reappeared at a rate that could not be explained by association/dissociation dynamics. In order to verify the mechanism of non-blocking modulation, we synchronized photolabeling with the voltage clamp protocol of patch-clamp experiments. Using this method, we could bind a photoreactive riluzole analog covalently to specific conformations of the channel. Photolabeling was ineffective at resting conformation, but effective at inactivated conformation, as judged from persisting modulated gating after removal of unbound photoactive drug from the solution. Mutation of the key residue of the local anesthetic binding site (F1579A) did not fully prevent ligand binding and inhibition, however, it eliminated most of the modulation caused by ligand binding. Our results indicate that riluzole binds with highest affinity to the local anesthetic binding site, which transmits inhibition by the unique non-blocking modulation mechanism. Our results also suggest the existence of one or more additional binding sites, with lower affinity, and different inhibition mechanism.

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2020 – New natural agonists of the transient receptor potential Ankyrin 1 (TRPA1) channel
Port-a-Patch Publication in Nature Scientific Reports (2020) Authors: Legrand C., Merlini J.M., de Senarclens‑Bezençon C., Michlig S.

The transient receptor potential (TRP) channels family are cationic channels involved in various physiological processes as pain, inflammation, metabolism, swallowing function, gut motility, thermoregulation or adipogenesis. In the oral CaVity, TRP channels are involved in chemesthesis, the sensory chemical transduction of spicy ingredients. Among them, TRPA1 is activated by natural molecules producing pungent, tingling or irritating sensations during their consumption. TRPA1 can be activated by different chemicals found in plants or spices such as the electrophiles isothiocyanates, thiosulfinates or unsaturated aldehydes. TRPA1 has been as well associated to various physiological mechanisms like gut motility, inflammation or pain. Cinnamaldehyde, its well known potent agonist from cinnamon, is reported to impact metabolism and exert anti-obesity and anti-hyperglycemic effects. Recently, a structurally similar molecule to cinnamaldehyde, cuminaldehyde was shown to possess anti-obesity and anti-hyperglycemic effect as well. We hypothesized that both cinnamaldehyde and cuminaldehyde might exert this metabolic effects through TRPA1 activation and evaluated the impact of cuminaldehyde on TRPA1. The results presented here show that cuminaldehyde activates TRPA1 as well. Additionally, a new natural agonist of TRPA1, tiglic aldehyde, was identified and p-anisaldehyde confirmed.

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2020 – Homocysteine-induced electrical remodeling via the mediation of IP 3 R1/NaV1.5 signaling pathway
Port-a-Patch Publication in American Journal of Translational Research (2020) Authors: Han L., Wu A., Li Q., Xia Z., Wu Y., Hong K., Xia Z., Li J.

Inositol-1,4,5-triphosphate-receptor 1 (IP3R1), a Ca2+ channel in the sarcoplasmic reticulum membrane, is an effective regulator of Ca2+ release involved in the pathology of most cardiovascular diseases. Our study aim to investigate the underlying mechanism by which IP3R1 signaling mediates the process of homocysteine (Hcy)-induced Ca2+ accumulation via interaction with sodium current (NaV1.5) in atrium. We utilized whole-cell patch-clamp analysis and flow cytometry to detect the abnormal electrical activity in mouse atrial myocytes (MACs) obtained from C57B6 mice fed with high-Hcy diet. The results represented not only an increase in protein levels of NaV1.5 and IP3R1, but also an enhanced intracellular levels of Ca2+, and prolonged action potential duration (APD). However, the inhibition of IP3R1 or NaV1.5 gene could both attenuate Ca2+ accumulation in MACs triggered by Hcy, as well as abnormal electrical activity. In addition, Hcy increased the interaction between IP3R1 and NaV1.5. These data suggest that Hcy induced Ca2+ accumulation is mediated by the IP3R1/NaV1.5 signaling pathway, accompanied with the influx of Na+ and Ca2+, which act as triggers for electrical remodeling.

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2020 – Interferon-γ and high glucose-induced opening of Cx43 hemichannels causes endothelial cell dysfunction and damage
Port-a-Patch Publication in Biochimica et Biophysica Acta (BBA) - Molecular Cell Research (2020) Authors: Sáez J.C., Contreras-Duarte S., Labra V.C., Santibañez C.A., Mellado L.A., Inostroza C.A., Alvear T.F., Retamal M.A., Velarde V., Orellana J.A..

Both IFN-γ or high glucose have been linked to systemic inflammatory imbalance with serious repercussions not only for endothelial function but also for the formation of the atherosclerotic plaque. Although the uncontrolled opening of connexin hemichannels underpins the progression of various diseases, whether they are implicated in endothelial cell dysfunction and damage evoked by IFN-γ plus high glucose remains to be fully elucidated. In this study, by using live cell imaging and biochemical approaches, we demonstrate that IFN-γ plus high glucose augment endothelial connexin43 hemichannel activity, resulting in the increase of ATP release, ATP-mediated Ca2+ dynamics and production of nitric oxide and superoxide anion, as well as impaired insulin-mediated uptake and intercellular diffusion of glucose and cell survival. Based on our results, we propose that connexin 43 hemichannel inhibition could serve as a new approach for tackling the activation of detrimental signaling resulting in endothelial cell dysfunction and death caused by inflammatory mediators during atherosclerosis secondary to diabetes mellitus.

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2020 – Chronic hepatitis C virus infection impairs insulin secretion by regulation of p38δ MAPK-dependent exocytosis in pancreatic β-cells
Port-a-Patch Publication in Clinical Science (2020) Authors: Chen J., Wang F., Zhou Y., Jiang J., Ksimu S., Zhang X., Li J.Z., Niu J., Wang Q.

Chronic hepatitis C virus (HCV) infection has a close association with type 2 diabetes mellitus. Although the mechanisms of insulin resistance in chronic hepatitis C (CHC) patients have been extensively studied, little attention has been given to the role of β-cell function in HCV-associated diabetes. Here, we analysed β-cell function in CHC patients and HCV-infected mouse model and found in addition to insulin resistance, impaired pancreatic β-cell function occurred in CHC patients and HCV-infected C/OTg mice, not only in diabetic individuals but also in individuals with impaired fasting glucose levels. Both first-phase and second-phase insulin secretion were impaired, at least partially due to the reduction of exocytosis of secretory insulin-containing granules following HCV infection. Up-regulated p38δ in HCV-infected β-cells resulted in inactivation of protein kinase D (PKD), which was responsible for impaired insulin secretory capacity of β-cells. Thus, impaired insulin secretion due to HCV infection in β-cells contributes to HCV-associated type 2 diabetes. These findings provided a new inspiration for the important prognostic and therapeutic implications in the management of CHC patients with impaired fasting glucose.

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2020 – Fenamates Inhibit Human Sodium Channel NaV1.2 and Protect Glutamate-Induced Injury in SH-SY5Y Cells
Port-a-Patch Publication in Cellular and Molecular Neurobiology (2020) Authors: Sun J.F., Zhao M.Y., Xu Y.J., Su Y., Kong X.H., Wang Z.Y.

Voltage-gated sodium channels are crucial mediators of neuronal damage in ischemic and excitotoxicity disease models. Fenamates have been reported to have anti-inflammatory properties following a decrease in prostaglandin synthesis. Several researches showed that fenamates appear to be ion channel modulators and potential neuroprotectants. In this study, the neuroprotective effects of tolfenamic acid, flufenamic acid, and mefenamic acid were tested by glutamate-induced injury in SH-SY5Y cells. Following this, fenamates' effects were examined on both the expression level and the function of hNaV1.1 and hNaV1.2, which were closely associated with neuroprotection, using Western blot and patch clamp. Finally, the effect of fenamates on the expression of apoptosis-related proteins in SH-SY5Y cells was examined. The results showed that both flufenamic acid and mefenamic acid exhibited neuroprotective effects against glutamate-induced injury in SH-SY5Y cells. They inhibited peak currents of both hNaV1.1 and hNaV1.2. However, fenamates exhibited decreased inhibitory effects on hNaV1.1 when compared to hNaV1.2. Correspondingly, the inhibitory effect of fenamates was found to be consistent with the level of neuroprotective effects in vitro. Fenamates inhibited glutamate-induced apoptosis through the modulation of the Bcl-2/Bax-dependent cell death pathways. Taken together, NaV1.2 might play a part in fenamates' neuroprotection mechanism. NaV1.2 and NMDAR might take part in the neuroprotection mechanism of the fenamates. The fenamates inhibited glutamate-induced apoptosis through modulation of the Bcl-2/Bax-dependent cell death pathways.

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2020 – Ascorbate-induced oxidative stress mediates TRP channel activation and cytotoxicity in human etoposide-sensitive and -resistant retinoblastoma cells
Port a Patch Publication in Laboratory Investigation (2020) Authors: Oronowicz J., Reinhard J., Reinach P.S., Ludwiczak S., Luo H., Salem M.H.O.B., Kraemer M.M., Biebermann H., Kakkassery V., Mergler S.

There are indications that pharmacological doses of ascorbate (Asc) used as an adjuvant improve the chemotherapeutic management of cancer. This favorable outcome stems from its cytotoxic effects due to prooxidative mechanisms. Since regulation of intracellular Ca2+ levels contributes to the maintenance of cell viability, we hypothesized that one of the effects of Asc includes disrupting regulation of intracellular Ca2+ homeostasis. Accordingly, we determined if Asc induced intracellular Ca2+ influx through activation of pertussis sensitive Gi/o-coupled GPCR which in turn activated transient receptor potential (TRP) channels in both etoposide-resistant and -sensitive retinoblastoma (WERI-Rb1) tumor cells. Ca2+ imaging, whole-cell patch-clamping, and quantitative real-time PCR (qRT-PCR) were performed in parallel with measurements of RB cell survival using Trypan Blue cell dye exclusion. TRPM7 gene expression levels were similar in both cell lines whereas TRPV1, TRPM2, TRPA1, TRPC5, TRPV4, and TRPM8 gene expression levels were downregulated in the etoposide-resistant WERI-Rb1 cells. In the presence of extracellular Ca2+, 1 mM Asc induced larger intracellular Ca2+ transients in the etoposide-resistant WERI-Rb1 than in their etoposide-sensitive counterpart. With either 100 µM CPZ, 500 µM La3+, 10 mM NAC, or 100 µM 2-APB, these Ca2+ transients were markedly diminished. These inhibitors also had corresponding inhibitory effects on Asc-induced rises in whole-cell currents. Pertussis toxin (PTX) preincubation blocked rises in Ca2+ influx. Microscopic analyses showed that after 4 days of exposure to 1 mM Asc cell viability fell by nearly 100% in both RB cell lines. Taken together, one of the effects underlying oxidative mediated Asc-induced WERI-Rb1 cytotoxicity stems from its promotion of Gi/o coupled GPCR mediated increases in intracellular Ca2+ influx through TRP channels. Therefore, designing drugs targeting TRP channel modulation may be a viable approach to increase the efficacy of chemotherapeutic treatment of RB. Furthermore, Asc may be indicated as a possible supportive agent in anti-cancer therapies.

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2020 – Bioluminescence Methodology for Ion Channel Studies
Port-a-Patch Chapter in Patch Clamp Electrophysiology (2020) Authors: Wadsworth P.A., Singh A.K., Nguyen N., Stephan C., Laezza F.

As key players in cell function, ion channels are important targets for drug discovery and therapeutic development against a wide range of health conditions. Thus, developing assays to reconstitute ion channel macromolecular complexes in physiological conditions and screen for chemical modifiers of protein–protein interactions within these complexes is timely in drug discovery campaigns. For most ion channels, expressing their pore-forming subunit in heterologous mammalian cells has now become a routine procedure. However, reconstituting protein-channel complexes in physiological environments is still challenging, limiting our ability to identify tools and probes based on allosteric mechanisms, which could lead to more targeted and precise modulation of the channel function. Here, we describe the assay development steps to stably reconstitute the interaction between voltage-gated Na+ (NaV) channel NaV1.6 and its accessory protein, fibroblast growth factor 14 (FGF14) using the split-luciferase complementation assay (LCA), followed by assay miniaturization and optimization in 384-well plates for in-cell high-throughput screening (HTS) against protein-channel interactions. This optimized LCA can subsequently be used for rapid estimation of hit potency and efficacy via dose-dependency studies, enabling ranking of hits prior to more labor-intensive validation studies. Lastly, we introduce the methodology for rapid functional hit validation studies using semi-automated planar patch-clamp electrophysiology. Our robust, in-cell HTS platform can be adapted to any suitable ion channel complex to explore regulatory pathways of cellular signaling using kinase inhibitors, as well as to screen small molecules for probe development and drug repurposing toward new targets/areas of medicine. Overall, the flexibility of this assay allows users to broadly explore therapeutic options for channelopathy-associated diseases at a fast pace, enabling rapid hypothesis generation in early phase drug discovery campaigns and narrowing down targets prior to more labor-intensive in vivo studies.

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2020 – Addressing hERG activity while maintaining favorable potency, selectivity and pharmacokinetic properties of PPARδ modulators
Port-a Patch Publication in Bioorganic & Medicinal Chemistry Letters (2020) Authors: Lagu B., Senaiar R.S., Kluge A.F., Mallesh B., Ramakrishna M., Bhat R., Patane M.A.

One of the most commonly used strategies to reduce hERG (human ether-a-go-go) activity in the drug candidates is introduction of a carboxylic acid group. During the optimization of PPARδ modulators, some of the compounds containing a carboxylic acid were found to inhibit the hERG channel in a patch clamp assay. By modifying the basicity of the imidazole core, potent and selective PPARδ modulators that do not inhibit hERG channel were identified. Some of the modulators have excellent pharmacokinetic profiles in mice.

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2020 – Arterial Medial Calcification through Enhanced small Extracellular Vesicle Release in Smooth Muscle-Specific Asah1 Gene Knockout Mice
Port-a-Patch Publication in Nature Scientific Reports (2020) Authors: Bhat O.M., Li G., Yuan X., Huang D., Gulbins E., Kukreja R.C., Li P-L.

Arterial medial calcification (AMC) involves an increased small extracellular vesicle (sEV) secretion and apatite calcium precipitation in the arterial wall. The mechanisms mediating AMC remain poorly understood. In the present study, smooth muscle-specific acid ceramidase (Ac) gene knockout mice (Asah1fl/fl/SMCre) were used to demonstrate the role of lysosomal ceramide signaling pathway in AMC. Asah1fl/fl/SMCre mice were found to have more severe AMC in both aorta and coronary arteries compared to their littermates (Asah1fl/fl/SMwt and WT/WT mice) after receiving a high dose vitamin D. These mice also had pronounced upregulation of osteopontin and RUNX2 (osteogenic markers), CD63, AnX2 (sEV markers) and ALP expression (mineralization marker) in the arterial media. In cultured coronary arterial smooth muscle cells (CASMCs) from Asah1fl/fl/SMCre mice, high dose of Pi led to a significantly increased calcium deposition, phenotypic change and sEV secretion compared to WT CASMCs, which was associated with reduced lysosome-multivesicular body (MVB) interaction. Also, GW4869, sEV release inhibitor decreased sEV secretion and calcification in these cells. Lysosomal transient receptor potential mucolipin 1 (TRPML1) channels regulating lysosome interaction with MVBs were found remarkably inhibited in Asah1fl/fl/SMCre CASMCs as shown by GCaMP3 Ca2+ imaging and Port-a-Patch patch clamping of lysosomes. Lysosomal Ac in SMCs controls sEV release by regulating lysosomal TRPML1 channel activity and lysosome-MVB interaction, which importantly contributes to phenotypic transition and AMC.

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2019 – Postpartum hormones oxytocin and prolactin cause pro-arrhythmic prolongation of cardiac repolarization in long QT syndrome type 2
Port-a Patch Publication in EP Europace (2019) Authors: Bodi I., Sorge J., Castiglione A., Glatz S.M., Wuelfers E.M., Franke G., Perez-Feliz S., Koren G., Zehender M., Bugger H., Seemann G., Brunner M., Bode C., Odening K.E.

AimsWomen with long QT syndrome 2 (LQT2) have a particularly high postpartal risk for lethal arrhythmias. We aimed at investigating whether oxytocin and prolactin contribute to this risk by affecting repolarization.Methods and resultsIn female transgenic LQT2 rabbits (HERG-G628S, loss of IKr), hormone effects on QT/action potential duration (APD) were assessed (0.2–200 ng/L). Hormone effects (200 ng/L) on ion currents and cellular APD were determined in transfected cells and LQT2 cardiomyocytes. Hormone effects on ion channels were assessed with qPCR and western blot. Experimental data were incorporated into in silico models to determine the pro-arrhythmic potential. Oxytocin prolonged QTc and steepened QT/RR-slope in vivo and prolonged ex vivo APD75 in LQT2 hearts. Prolactin prolonged APD75 at high concentrations. As underlying mechanisms, we identified an oxytocin- and prolactin-induced acute reduction of IKs-tail and IKs-steady (−25.5%, oxytocin; −13.3%, prolactin, P  0.05) in CHO-cells and LQT2-cardiomyocytes. IKr currents were not altered. This oxytocin-/prolactin-induced IKs reduction caused APD90 prolongation (+11.9%/+13%, P  0.05) in the context of reduced/absent IKr in LQT2 cardiomyocytes. Hormones had no effect on IK1 and ICa,L in cardiomyocytes. Protein and mRNA levels of CACNA1C/CaV1.2 and RyR2 were enhanced by oxytocin and prolactin. Incorporating these hormone effects into computational models resulted in reduced repolarization reserve and increased propensity to pro-arrhythmic permanent depolarization, lack of capture and early afterdepolarizations formation.ConclusionsPostpartum hormones oxytocin and prolactin prolong QT/APD in LQT2 by reducing IKs and by increasing CaV1.2 and RyR2 expression/transcription, thereby contributing to the increased postpartal arrhythmic risk in LQT2.

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2019 – Screening by changes in stereotypical behavior during cell motility
Port-a Patch Publication in Scientific Reports (2019) Authors: Tweedy L., Witzel P., Heinrich D., Insall R.H., Endres R.G.

Stereotyped behaviors are series of postures that show very little variability between repeats. They have been used to classify the dynamics of individuals, groups and species without reference to the lower-level mechanisms that drive them. Stereotypes are easily identified in animals due to strong constraints on the number, shape, and relative positions of anatomical features, such as limbs, that may be used as landmarks for posture identification. In contrast, the identification of stereotypes in single cells poses a significant challenge as the cell lacks these landmark features, and finding constraints on cell shape is a non-trivial task. Here, we use the maximum caliber variational method to build a minimal model of cell behavior during migration. Without reference to biochemical details, we are able to make behavioral predictions over timescales of minutes using only changes in cell shape over timescales of seconds. We use drug treatment and genetics to demonstrate that maximum caliber descriptors can discriminate between healthy and aberrant migration, thereby showing potential applications for maximum caliber methods in automated disease screening, for example in the identification of behaviors associated with cancer metastasis.

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2019 – NMDA Receptor-Mediated Signaling Pathways Enhance Radiation Resistance, Survival and Migration in Glioblastoma Cells-A Potential Target for Adjuvant Radiotherapy
Port-a Patch Publication in Cancers (2019) Authors: Müller-Längle A., Lutz H., Hehlgans S., Rödel F., Rau K., Laube B.

Glioblastoma is one of the most aggressive malignant brain tumors, with a survival time less than 15 months and characterized by a high radioresistance and the property of infiltrating the brain. Recent data indicate that the malignancy of glioblastomas depends on glutamatergic signaling via ionotropic glutamate receptors. In this study we revealed functional expression of Ca2+-permeable NMDARs in three glioblastoma cell lines. Therefore, we investigated the impact of this receptor on cell survival, migration and DNA double-strand break (DSB) repair in the presence of both, glutamate and NMDAR antagonists, and after clinically relevant doses of ionizing radiation. Our results indicate that treatment with NMDAR antagonists slowed the growth and migration of glutamate-releasing LN229 cells, suggesting that activation of NMDARs facilitate tumor expansion. Furthermore, we found that DSB-repair upon radiation was more effective in the presence of glutamate. In contrast, antagonizing the NMDAR or the Ca2+-dependent transcription factor CREB impaired DSB-repair similarly and resulted in a radiosensitizing effect in LN229 and U-87MG cells, indicating a common link between NMDAR signaling and CREB activity in glioblastoma. Since the FDA-approved NMDAR antagonists memantine and ifenprodil showed differential radiosensitizing effects, these compounds may constitute novel optimizations for therapeutic interventions in glioblastoma..

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2019 – NPPB prevents postoperative peritoneal adhesion formation by blocking volume-activated Cl− current
Port-a Patch Publication in Naunyn-Schmiedeberg's Archives of Pharmacology (2019) Authors: Zhong, J., Qin Z., Yu H., Liu X., Li C., Shi J., Mao J., Xu B.

5-Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) is a non-specific chloride channel blocker. Peritoneal adhesion is an inevitable complication of abdominal surgery and remains an important clinical problem, leading to chronic pain, intestinal obstruction, and female infertility. The aim of this study is to observe the effects of NPPB on peritoneal adhesions and uncover the underlying mechanism. The formation of postoperative peritoneal adhesions was induced by mechanical injury to the peritoneum of rats. MTT assay and wound-healing assay were used to evaluate proliferation and migration of primary cultured adhesion fibroblasts (AFB) respectively. Whole-cell chloride currents were measured using a fully automated patch-clamp workstation. Cell volume changes were monitored by light microscopy and video imaging. Our results demonstrated that NPPB could significantly prevent the formation of peritoneal adhesion in rats and inhibit the proliferation of AFB in a concentration-dependent manner. NPPB also reduced the migration of AFB cells with an IC50 of 53.09 μM. A 47% hypotonic solution successfully activated the ICl,vol in AFB cells. The current could be blocked by extracellular treatment with NPPB. Moreover, 100 μM NPPB almost completely eliminated the capacity of regulatory volume decrease (RVD) in these cells. These data indicate that NPPB could prevent the formation of postoperative peritoneal adhesions. The possible mechanism may be through the inhibition of the proliferation and migration of AFB cells by modulating ICl,vol and cell volume. These results suggest a potential clinical use of NPPB for preventing the formation of peritoneal adhesions.

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2019 – Mitragynine, an euphoric compound inhibits hERG1a/1b channel current and upregulates the complexation of hERG1a-Hsp90 in HEK293-hERG1a/1b cells
Port-a Patch Publication in Nature Scientific Reports (2019) Authors: Tay Y.L., Amanah A., Adenan M.I., Wahab H.A., Tan M.L.

Mitragyna speciosa Korth (M. speciosa) has been widely used as a recreational product, however, there are growing concerns on the abuse potentials and toxicity of the plant. Several poisoning and fatal cases involving kratom and mitragynine have been reported but the underlying causes remain unclear. The human ether-a-go-go-related gene 1 (hERG1) encodes the pore-forming subunit underlying cardiac rapidly delayed rectifier potassium current (IKr). Pharmacological blockade of the IKr can cause acquired long QT syndrome, leading to lethal cardiac arrhythmias. This study aims to elucidate the mechanisms of mitragynine-induced inhibition on hERG1a/1b current. Electrophysiology experiments were carried out using Port-a-Patch system. Quantitative RT-PCR, Western blot analysis, immunofluorescence and co-immunoprecipitation methods were used to determine the effects of mitragynine on hERG1a/1b expression and hERG1-cytosolic chaperones interaction. Mitragynine was found to inhibit the IKr current with an IC50 value of 332.70 nM. It causes a significant reduction of the fully-glycosylated (fg) hERG1a protein expression but upregulates both core-glycosylated (cg) expression and hERG1a-Hsp90 complexes, suggesting possible impaired hERG1a trafficking. In conclusion, mitragynine inhibits hERG1a/1b current through direct channel blockade at lower concentration, but at higher concentration, it upregulates the complexation of hERG1a-Hsp90 which may be inhibitory towards channel trafficking.

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2019 – Modulation of the potassium channel KcsA by anionic phospholipids: Role of arginines at the non-annular lipid binding sites
Port-a Patch Publication in BBA - Biomembranes (2019) Authors: Poveda J.A., Giudici A.M., Renart M.L., Millet O., Morales A., González-Ros J.M., Oakes V., Furini S., Domene C

The role of arginines R64 and R89 at non-annular lipid binding sites of KcsA, on the modulation of channel activity by anionic lipids has been investigated. In wild-type (WT) KcsA reconstituted into asolectin lipid membranes, addition of phosphatidic acid (PA) drastically reduces inactivation in macroscopic current recordings. Consistent to this, PA increases current amplitude, mean open time and open probability at the single channel level. Moreover, kinetic analysis reveals that addition of PA causes longer open channel lifetimes and decreased closing rate constants. Effects akin to those of PA on WT-KcsA are observed when R64 and/or R89 are mutated to alanine, regardless of the added anionic lipids. We interpret these results as a consequence of interactions between the arginines and the anionic PA bound to the non-annular sites. NMR data shows indeed that at least R64 is involved in binding PA. Moreover, molecular dynamics (MD) simulations predict that R64, R89 and surrounding residues such as T61, mediate persistent binding of PA to the non-annular sites. Channel inactivation depends on interactions within the inactivation triad (E71-D80-W67) behind the selectivity filter. Therefore, it is expected that such interactions are affected when PA binds the arginines at the non-annular sites. In support of this, MD simulations reveal that PA binding prevents interaction between R89 and D80, which seems critical to the effectiveness of the inactivation triad. This mechanism depends on the stability of the bound lipid, favoring anionic headgroups such as that of PA, which thrive on the positive charge of the arginines.

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2019 – Inhibition of Polyamine Biosynthesis Reverses Ca2+ Channel Remodeling in Colon Cancer Cells
Port-a-Patch Publication in Cancers (2019) Authors: Gutiérrez L.G., Hernández-Morales M., Núñez L., Villalobos C.

Store-operated Ca2+ entry (SOCE) is the most important Ca2+ entry pathway in non-excitable cells. Colorectal cancer (CRC) shows decreased Ca2+ store content and enhanced SOCE that correlate with cancer hallmarks and are associated to remodeling of store-operated channels (SOCs). Normal colonic cells display small, Ca2+-selective currents driven by Orai1 channels. In contrast, CRC cells display larger, non-selective currents driven by Orai1 and transient receptor potential canonical type 1 channels (TRPC1). Difluoromethylornithine (DFMO), a suicide inhibitor of ornithine decarboxylase (ODC), the limiting step in polyamine biosynthesis, strongly prevents CRC, particularly when combined with sulindac. We asked whether DFMO may reverse SOC remodeling in CRC. We found that CRC cells overexpress ODC and treatment with DFMO decreases cancer hallmarks including enhanced cell proliferation and apoptosis resistance. Consistently, DFMO enhances Ca2+ store content and decreases SOCE in CRC cells. Moreover, DFMO abolish selectively the TRPC1-dependent component of SOCs characteristic of CRC cells and this effect is reversed by the polyamine putrescine. Combination of DFMO and sulindac inhibit both SOC components and abolish SOCE in CRC cells. Finally, DFMO treatment inhibits expression of TRPC1 and stromal interaction protein 1 (STIM1) in CRC cells. These results suggest that polyamines contribute to Ca2+ channel remodeling in CRC, and DFMO may prevent CRC by reversing channel remodeling

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2019 – KCa1.1 and KV1.3 channels regulate the interactions between fibroblast-like synoviocytes and T lymphocytes during rheumatoid arthritis
Port-a-Patch Publication in Arthritis Research & Therapy (2019) Authors: Tanner M.R., Pennington M.W., Chauhan S.S., Laragione T., Gulko P.S., Beeton C.

Background: Fibroblast-like synoviocytes (FLS) and CCR7- effector memory T (TEM) cells are two of the major cell types implicated in the progression of rheumatoid arthritis (RA). In particular, FLS become highly invasive, whereas TEM cells proliferate and secrete proinflammatory cytokines, during RA. FLS and T cells may also interact and influence each other's phenotypes. Inhibition of the pathogenic phenotypes of both FLS and TEM cells can be accomplished by selectively blocking the predominant potassium channels that they upregulate during RA: KCa1.1 (BK, Slo1, MaxiK, KCNMA1) upregulated by FLS and KV1.3 (KCNA3) upregulated by activated TEM cells. In this study, we investigated the roles of KCa1.1 and KV1.3 in regulating the interactions between FLS and TEM cells and determined if combination therapies of KCa1.1- and KV1.3-selective blockers are more efficacious than monotherapies in ameliorating disease in rat models of RA.
Methods: We used in vitro functional assays to assess the effects of selective KCa1.1 and KV1.3 channel inhibitors on the interactions of FLS isolated from rats with collagen-induced arthritis (CIA) with syngeneic TEM cells. We also used flow cytometric analyses to determine the effects of KCa1.1 blockers on the expression of proteins used for antigen presentation on CIA-FLS. Finally, we used the CIA and pristane-induced arthritis models to determine the efficacy of combinatorial therapies of KCa1.1 and KV1.3 blockers in reducing disease severity compared with monotherapies.
Results: We show that the interactions of FLS from rats with CIA and of rat TEM cells are regulated by KCa1.1 and KV1.3. Inhibiting KCa1.1 on FLS reduces the ability of FLS to stimulate TEM cell proliferation and migration, and inhibiting KV1.3 on TEM cells reduces TEM cells' ability to enhance FLS expression of KCa1.1 and major histocompatibility complex class II protein, as well as stimulates their invasion. Furthermore, we show that combination therapies of selective KCa1.1 and KV1.3 blockers are more efficacious than monotherapies at reducing signs of disease in two rat models of RA.
Conclusions: Our results demonstrate the importance of KCa1.1 and KV1.3 in regulating FLS and TEM cells during RA, as well as the value of combined therapies targeting both of these cell types to treat RA.

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2019 – Fenamates inhibit human sodium channel NaV1.7 and NaV1.8
Port-a-Patch Publication in Neuroscience Letters (2019) Authors: Sun, J-F., Xu, Y-J., Kong, X-H., Su, Y., Wang, Z-Y.

Fenamates are N-substituted anthranilic acid derivatives, clinically used as nonsteroidal anti-inflammatory drugs (NSAIDs) in fever, pain and inflammation treatments. Previous studies have shown that they are also modulators of diverse ion channels, exhibiting either activation or inhibitory effects. However, the effects of fenamates on sodium channel subtypes are still unknown. In this study, fenamates, including mefenamic acid, flufenamic acid and tolfenamic acid, were examined by whole-cell patch clamp techniques on the sodium channels hNaV1.7 and hNaV1.8, which are closely associated with pain. The results showed that the mefenamic acid, flufenamic acid, and tolfenamic acid inhibited the peak currents of hNaV1.7 and hNaV1.8 in CHO cells stably expressing hNaV1.7 and hNaV1.8. However, much lighter inhibition effects of hNaV1.8 were registered in the experimental system. Furthermore, the mefenamic acid, flufenamic acid and tolfenamic acid significantly affected the inactivation processes of hNaV1.7 and hNaV1.8 with I-V curves left-shifted to hyperpolarized direction. These data indicate that the inhibition effects of NaV1.7 and NaV1.8 by mefenamic acid, flufenamic acid and tolfenamic acid might contribute to their analgesic activity in addition to their inhibition of cyclooxygenase. These findings provide a basis for further studies in the discovery of other potential targets for NSAIDs.

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2019 – High-resolution experimental and computational electrophysiology reveals weak β-lactam binding events in the porin PorB
Port-a-Patch Publication in Nature Scientific Reports (2019) Authors: Bartsch A., Llabrés S., Pein F., Kattner C., Schön M., Diehn M., Tanabe M., Munk A., Zachariae U., Steinem C.

The permeation of most antibiotics through the outer membrane of Gram-negative bacteria occurs through porin channels. To design drugs with increased activity against Gram-negative bacteria in the face of the antibiotic resistance crisis, the strict constraints on the physicochemical properties of the permeants imposed by these channels must be better understood. Here we show that a combination of high-resolution electrophysiology, new noise-filtering analysis protocols and atomistic biomolecular simulations reveals weak binding events between the beta-lactam antibiotic ampicillin and the porin PorB from the pathogenic bacterium Neisseria meningitidis. In particular, an asymmetry often seen in the electrophysiological characteristics of ligand-bound channels is utilised to characterise the binding site and molecular interactions in detail, based on the principles of electro-osmotic flow through the channel. Our results provide a rationale for the determinants that govern the binding and permeation of zwitterionic antibiotics in anion-selective porin channels.

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2019 – Breaching the Barrier: Quantifying Antibiotic Permeability across Gram-Negative Bacterial Membranes
Port-a Patch Publication in Journal of Molecular Biology (2019) Authors: Cama J., MaeHenney A., Winterhalter M.

The double membrane cell envelope of Gram negative bacteria is a sophisticated barrier that facilitates the uptake of nutrients and protects the organism from toxic compounds. An antibiotic molecule must find its way through the negatively charged lipopolysaccharide layer on the outer surface, pass through either a porin or the hydrophobic layer of the outer membrane, then traverse the hydrophilic peptidoglycan layer only to find another hydrophobic lipid bilayer before it finally enters the cytoplasm, where it typically finds its target. This complex uptake pathway with very different physico-chemical properties is one reason that Gram-negatives are intrinsically protected against multiple classes of antibiotic-like molecules, and is likely the main reason that in vitro target based screening programmes have failed to deliver novel antibiotics for these organisms. Due to the lack of general methods available for quantifying the flux of drugs into the cell, little is known about permeation rates, transport pathways and accumulation at the target sites for particular molecules. Here we summarise the current tools available for measuring antibiotic uptake across the different compartments of Gram-negative bacteria.

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2019 – Control of Lysosomal TRPML1 Channel Activity and Exosome Release by Acid Ceramidase in Mouse Podocytes
Port-a Patch Publication in American Journal of Physiology Cell Physiology (2019) Authors: Li G., Huang D., Hong J., Bhat O.M., Yuan X., Li P.L.

The transient receptor potential mucolipin 1 (TRPML1) channel has been reported to mediate lysosomal Ca2+ release that is involved in Ca2+-dependent lysosome trafficking and autophagic flux. However, this regulatory mechanism of lysosomal TRPML1 channel activity in podocytes remains poorly understood. In the present study, we tested whether TRPML1 channel in podocytes mediates lysosome trafficking which is essential for multivesicular body (MVB) degradation by lysosomes. We first demonstrated the abundant expression of TRPML1 channel in podocytes. By GCaMP3 Ca2+ imaging, we characterized the lysosomal specificity of TRPML1 channel-mediated Ca2+ release in podocytes. Given the important role of acid ceramidase (AC) in lysosome function and podocyte injury, we tested whether AC regulates this TRPML1 channel-mediated Ca2+ release and consequent lysosome-dependent MVB degradation in podocytes. Pharmacologically, it was found that TRPML1 channel activity was remarkably attenuated by AC inhibitor, carmofur. Sphingosine, as an AC product, was demonstrated to induce TRPML1-mediated Ca2+ release, which was inhibited by a TRPML1 blocker, verapamil. Using a Port-a-Patch planar patch-clamp system, we found that AC-associated sphingolipids, sphingomyelin, ceramide, and sphingosine had different effects on TRPML1 channel activity in podocytes. Functionally, the inhibition of AC or blockade of TRPML1 channels was found to suppress the interaction of lysosomes and MVBs, leading to increased exosome release from podocytes. These results suggest that AC is critical for TRPML1 channel-mediated Ca2+ release, which controls lysosome-MVB interaction and exosome release in podocytes.

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2019 – Abolishing cAMP sensitivity in HCN2 pacemaker channels induces generalized seizures
Port-a Patch and Internal Perfusion Publication in JCI Insight (2019) Authors: Hammelmann, V., Stieglitz, M.S., Hülle, H., Le Meur, K., Kass, J., Brümmer, M., Gruner, C., Rötzer, R.D., Fenske, S., Hartmann, J., Zott, B., Lüthi, A., Spahn, S., Moser, M,, Isbrandt, D., Ludwig, A., Konnerth, A., Wahl-Schott, C., Biel M.

Hyperpolarization-activated cyclic nucleotide–gated (HCN) channels are dually gated channels that are operated by voltage and by neurotransmitters via the cAMP system. cAMP-dependent HCN regulation has been proposed to play a key role in regulating circuit behavior in the thalamus. By analyzing a knockin mouse model (HCN2EA), in which binding of cAMP to HCN2 was abolished by 2 amino acid exchanges (R591E, T592A), we found that cAMP gating of HCN2 is essential for regulating the transition between the burst and tonic modes of firing in thalamic dorsal-lateral geniculate (dLGN) and ventrobasal (VB) nuclei. HCN2EA mice display impaired visual learning, generalized seizures of thalamic origin, and altered NREM sleep properties. VB-specific deletion of HCN2, but not of HCN4, also induced these generalized seizures of the absence type, corroborating a key role of HCN2 in this particular nucleus for controlling consciousness. Together, our data define distinct pathological phenotypes resulting from the loss of cAMP-mediated gating of a neuronal HCN channel.

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2019 – Action of MK‐7264 (Gefapixant) at human P2X3 and P2X2/3 receptors and in vivo efficacy in models of sensitisation
Port-a-Patch Publication in British Journal of Pharmacology (2019) Authors: Richards D., Gever J.R., Ford A.P., Fountain S.J.

Background & Purpose:The P2X3 receptor is an ATP‐gated ion channel expressed by sensory afferent neurons, and is as a target to treat chronic sensitisation conditions. The first‐in‐class, selective P2X3 and P2X2/3 receptor antagonist, the diaminopyrimidine MK‐7264 (Gefapixant), has progressed to Phase III trials for refractory or unexplained chronic cough. We have used patch‐clamp to elucidate the pharmacology and kinetics of MK‐7264 and rat models of hypersensitivity and hyperalgesia to test efficacy in these conditions.Experimental Approach:Whole‐cell patch‐clamp of 1321N1 cells expressing human P2X3 and P2X2/3 receptors was used to determine mode of MK‐7264 action, potency and kinetics. The analgesic efficacy was assessed using paw withdrawal threshold and limb weight distribution in rat models of inflammatory, osteoarthritic and neuropathic sensitisation.Key Results:MK‐7264 is a reversible allosteric antagonist at human P2X3 and P2X2/3 receptors with IC50 values of 153 and 220nM, respectively. Experiments with the slowly desensitising P2X2/3 heteromer revealed concentration and state‐dependency to wash‐on, with faster rates and greater inhibition when applied before agonist compared to during agonist application. Wash‐on rate (τ value) for MK‐7264 at maximal concentrations was 19s and 146s when applied before and during agonist application, respectively. In vivo, MK‐7264 (30 mg/kg) displayed efficacy comparable to naproxen (20 mg/kg) in inflammatory and osteoarthritic sensitisation models, and gabapentin (100 mg/kg) in neuropathic sensitisation models, increasing paw withdrawal threshold and decreasing weight bearing discomfort.Conclusions and Implications:MK‐7264 is a reversible and selective P2X3 and P2X2/3 antagonist, exerting allosteric antagonism via preferential activity at closed channels. Efficacy in rat models supports clinical investigation of chronic sensitisation conditions.

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2018 – TRPM8 Activation via 3-Iodothyronamine Blunts VEGF-Induced Transactivation of TRPV1 in Human Uveal Melanoma Cells
Port-a-Patch Publication in Frontiers in Pharmacology (2018) Authors: Walcher, L., Budde, C., Böhm, A., Reinach, P.S., Dhandapani, P., Ljubojevic, N., Schweiger, M.W., von der Waydbrink, H., Reimers, I., Köhrle, J., Mergler, S.

In human uveal melanoma (UM), tumor enlargement is associated with increases in aqueous humor vascular endothelial growth factor-A (VEGF-A) content that induce neovascularization. 3-Iodothyronamine (3-T1AM), an endogenous thyroid hormone metabolite, activates TRP melastatin 8 (TRPM8), which blunts TRP vanilloid 1 (TRPV1) activation by capsaicin (CAP) in human corneal, conjunctival epithelial cells, and stromal cells. We compare here the effects of TRPM8 activation on VEGF-induced transactivation of TRPV1 in an UM cell line (92.1) with those in normal primary porcine melanocytes (PM) since TRPM8 is upregulated in melanoma. Fluorescence Ca2+-imaging and planar patch-clamping characterized functional channel activities. CAP (20 μM) induced Ca2+ transients and increased whole-cell currents in both the UM cell line and PM whereas TRPM8 agonists, 100 μM menthol and 20 μM icilin, blunted such responses in the UM cells. VEGF (10 ng/ml) elicited Ca2+ transients and augmented whole-cell currents, which were blocked by capsazepine (CPZ; 20 μM) but not by a highly selective TRPM8 blocker, AMTB (20 μM). The VEGF-induced current increases were not augmented by CAP. Both 3-T1AM (1 μM) and menthol (100 μM) increased the whole-cell currents, whereas 20 μM AMTB blocked them. 3-T1AM exposure suppressed both VEGF-induced Ca2+ transients and increases in underlying whole-cell currents. Taken together, functional TRPM8 upregulation in UM 92.1 cells suggests that TRPM8 is a potential drug target for suppressing VEGF induced increases in neovascularization and UM tumor growth since TRPM8 activation blocked VEGF transactivation of TRPV1.

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2018 – Vascular Endothelial Growth Factor (VEGF) Induced Downstream Responses to Transient Receptor Potential Vanilloid 1 (TRPV1) and 3-Iodothyronamine (3-T1AM) in Human Corneal Keratocytes
Port-a-Patch Publication in Frontiers in Pharmacology (2018) Authors: Türker, E., Garreis, F., Khajavi, N., Reinach, P.S., Joshi, P., Brockmann, T., Lucius, A., Ljubojevic, N., Turan, E., Cooper, D., Schick, F., Reinholz, R., Pleyer, U., Köhrle, J., Mergler, S.

This study was undertaken to determine if crosstalk among the transient receptor potential (TRP) melastatin 8 (TRPM8), TRP vanilloid 1 (TRPV1), and vascular endothelial growth factor (VEGF) receptor triad modulates VEGF-induced Ca2+ signaling in human corneal keratocytes. Using RT-PCR, qPCR and immunohistochemistry, we determined TRPV1 and TRPM8 gene and protein coexpression in a human corneal keratocyte cell line (HCK) and human corneal cross sections. Fluorescence Ca2+ imaging using both a photomultiplier and a single cell digital imaging system as well as planar patch-clamping measured relative intracellular Ca2+ levels and underlying whole-cell currents. The TRPV1 agonist capsaicin increased both intracellular Ca2+ levels and whole-cell currents, while the antagonist capsazepine (CPZ) inhibited them. VEGF-induced Ca2+ transients and rises in whole-cell currents were suppressed by CPZ, whereas a selective TRPM8 antagonist, AMTB, increased VEGF signaling. In contrast, an endogenous thyroid hormone-derived metabolite 3-Iodothyronamine (3-T1AM) suppressed increases in the VEGF-induced current. The TRPM8 agonist menthol increased the currents, while AMTB suppressed this response. The VEGF-induced increases in Ca2+ influx and their underlying ionic currents stem from crosstalk between VEGFR and TRPV1, which can be impeded by 3-T1AM-induced TRPM8 activation. Such suppression in turn blocks VEGF-induced TRPV1 activation. Therefore, crosstalk between TRPM8 and TRPV1 inhibits VEGFR-induced activation of TRPV1.

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2018 – Temperature-Induced Modulation of Voltage-Gated Ion Channels in Human Lung Cancer Cell Line A549 Using Automated Patch Clamp Technology
Port-a-Patch book chapter in World Congress on Medical Physics and Biomedical Engineering 2018. Authors: Langthaler S., Bergmoser K., Lassnig A., Baumgartner C.

In cancer cells specific ion channels exhibit altered channel expression, which can drive malignant and metastatic cell behavior. Hence, therapeutic strategies modulating ion channels prove to be promising in cancer therapeutics. Alterations in temperature, even small deviations from normothermia, may cause changes in electrophysiological processes, since activation and conductivity of various ion channels are temperature-dependent. In this pilot study, we focused on a basic understanding of the effects of temperature-alterations on voltage-gated ion channels of A549 cells using an automated patch-clamp system. The measurements were carried out in whole-cell voltage-clamped configuration applying test pulses between −60 and +60 mV. For positive voltages the ion-current curves showed an instantaneously increased conductance, followed by a slow current increase provoked by later activating voltage-gated ion channels, indicating the time-delayed response of additional channels. To investigate the temperature-dependent electrophysiological behavior, six cells (passages 7–10, n = 34) were examined at room temperature and normal body temperature. Compared to normal body temperature, reduced temperatures revealed a higher whole-cell current at negative voltages (63.4% (±18.5%), −60 mV) and lower currents (52.6% (±27.3%), +60 mV) at positive voltages, indicating a hypothermia-induced modulation of voltage-gated channels in the lung cancer cell line A549.

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2018 – Thymol Induces Conidial Apoptosis in Aspergillus flavus via Stimulating K+ Eruption
Port-a-Patch Publication in Journal of Agricultural and Food Chemistry (2018) Authors: Hu L.-B., Ban F.-F., Li H.-B., Qian P.-P., Shen Q.-S., Yan-Zhao Y., Mo H.-Z., Zhou X.

Aspergillus flavus is a notorious foodborne fungus, posing a significant risk to humans in the form of hepatocellular carcinoma or aspergillosis. Thymol, as a food preservative, could efficiently kill conidia of A. flavus. However, the underlying mechanisms by which thymol kills A. flavus are not completely understood. With specific fluorescent dyes, we detected several apoptotic hallmarks, including chromatin condensation, phosphatidylserine externalization, DNA damage, mitochondrial depolarization, and caspase 9 activation in conidia exposed to 200 μg/mL of thymol, indicating that thymol induced a caspase-dependent conidial apoptosis in A. flavus. Chemical–protein interactome (CPI) and autodock analyses showed that KCNAB, homologue to the β-subunit of the voltage-gated potassium channel (KV) and aldo-keto reductase, was the potential target of thymol. Following studies demonstrated that thymol could activate the aldo-keto reductase activity of KCNAB in vitro and stimulate a transient K+ efflux in conidia, as determined using a Port-a-Patch. Blocking K+ eruption by 4-aminopyridine (a universal inhibitor of KV) could significantly alleviate thymol-mediated conidial apoptosis, indicating that activation of KV was responsible for the apoptosis. Taken together, our results revealed a K+ efflux-mediated apoptotic pathway in A. flavus, which greatly contributed to the development of an alternative strategy to control this pathogen.

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2018 – Structure of the mechanosensitive OSCA channels
Port-a-Patch Suction Control Pro Publication in Nature Structural & Molecular Biology (2018) Authors: Zhang M., Wang D., Kang Y., Wu J.-X., Yao F., Pan C., Yan Z., Song C., Chen L.

Mechanosensitive ion channels convert mechanical stimuli into a flow of ions. These channels are widely distributed from bacteria to higher plants and humans, and are involved in many crucial physiological processes. Here we show that two members of the OSCA protein family in Arabidopsis thaliana, namely AtOSCA1.1 and AtOSCA3.1, belong to a new class of mechanosensitive ion channels. We solve the structure of the AtOSCA1.1 channel at 3.5-Å resolution and AtOSCA3.1 at 4.8-Å resolution by cryo-electron microscopy. OSCA channels are symmetric dimers that are mediated by cytosolic inter-subunit interactions. Strikingly, they have structural similarity to the mammalian TMEM16 family proteins. Our structural analysis accompanied with electrophysiological studies identifies the ion permeation pathway within each subunit and suggests a conformational change model for activation.

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2018 – Targeting KCa1.1 channels with a scorpion venom peptide for the therapy of rat models of rheumatoid arthritis
Port-a-Patch Publication in The Journal of Pharmacology and Experimental Therapeutics (2018) Authors: Tanner M.R., Pennington R.W., Chamberlain B.H., Huq R., Gehrmann E.J., Laragione, T., Gulko P.S., Beeton C.

Fibroblast-like synoviocytes (FLS) are a key cell-type involved in rheumatoid arthritis (RA) progression. We previously identified the KCa1.1 potassium channel (Maxi-K, BK, Slo 1, KCNMA1) as a regulator of FLS and that KCa1.1 inhibition reduces disease severity in RA animal models. However, systemic KCa1.1 block causes multiple side effects and in this study, we aimed to determine whether the KCa1.1 β1-3-specific venom peptide blocker iberiotoxin (IbTX) reduces disease severity in animal models of RA without inducing major side effects. We used immunohistochemistry to identify IbTX-sensitive KCa1.1 subunits in joints of rats with a model of RA. Patch clamp and functional assays were used to determine if IbTX can regulate FLS through targeting KCa1.1. We then tested the efficacy of IbTX in ameliorating disease in two rat models of RA. Finally, we determined if IbTX causes side-effects including incontinence or tremors in rats, compared to those treated with the small molecule KCa1.1 blocker paxilline. IbTX-sensitive subunits of KCa1.1 are expressed by FLS in joints of rats with experimental arthritis. IbTX inhibits KCa1.1 channels expressed by FLS from patients with RA and by FLS from rat models of RA and reduces FLS invasiveness. IbTX significantly reduces disease severity in two rat models of RA. Unlike paxilline, IbTX does not induce tremors or incontinence in rats. Overall, IbTX inhibits KCa1.1 channels on FLS and treats rat models of RA without inducing side effects associated with non-specific KCa1.1 blockade and could become the basis for the development of a new treatment for RA.

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2018 – Powering the ABC multidrug exporter LmrA: How nucleotides embrace the ion-motive force
Port-a-Patch Publication in Science Advances (2018) Authors: Agboh K., Lau C.H.F., Khoo Y.S.K., Singh H., Raturi S., Nair A.V., Howard J., Chiapello M., Feret R., Deery M.J., Murakami S., van Veen H.W.

LmrA is a bacterial ATP-binding cassette (ABC) multidrug exporter that uses metabolic energy to transport ions, cytotoxic drugs, and lipids. Voltage clamping in a Port-a-Patch was used to monitor electrical currents associated with the transport of monovalent cationic HEPES+ by single-LmrA transporters and ensembles of transporters. In these experiments, one proton and one chloride ion are effluxed together with each HEPES+ ion out of the inner compartment, whereas two sodium ions are transported into this compartment. Consequently, the sodium-motive force (interior negative and low) can drive this electrogenic ion exchange mechanism in cells under physiological conditions. The same mechanism is also relevant for the efflux of monovalent cationic ethidium, a typical multidrug transporter substrate. Studies in the presence of Mg-ATP (adenosine 5′-triphosphate) show that ion-coupled HEPES+ transport is associated with ATP-bound LmrA, whereas ion-coupled ethidium transport requires ATP binding and hydrolysis. HEPES+ is highly soluble in a water-based environment, whereas ethidium has a strong preference for residence in the water-repelling plasma membrane. We conclude that the mechanism of the ABC transporter LmrA is fundamentally related to that of an ion antiporter that uses extra steps (ATP binding and hydrolysis) to retrieve and transport membrane-soluble substrates from the phospholipid bilayer.

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2018 – Role of the Fourth Transmembrane Segment in TRAAK Channel Mechanosensitivity
Port-a-Patch Suction Control Pro pre-Publication in bioRxiv (2018) Authors: Zhang M., Yao F., Pan C., Yan Z.

Mechanosensitive ion channels such as Piezo, TRAAK, TRPs and OSCA are important transmembrane proteins that are involved in many physiological processes such as touch, hearing and blood pressure regulation. Unlike ligand-gated channels or voltage-gated ion channels, which have a canonical ligand-binding domain or voltage-sensing domain, the mechanosensitive domain and related gating mechanism remain elusive. TRAAK channels are mechanosensitive channels that convert a physical mechanical force into a flow of potassium ions. The structures of TRAAK channels have been solved, however, the functional roles of the structural domains associated with channel mechanosensitivity remains unclear. Here, we generated a series of chimeric mutations between TRAAK and a non-mechanosensitive silent TWIK-1 K2P channel. We found that the selectivity filter region functions as the major gate of outward rectification and found that lower part of fourth transmembrane domain (M4) is necessary for TRAAK channel mechanosensitivity. We further demonstrated that upper part of M4 can modulate the mechanosensitivity of TRAAK channel. Furthermore, we found that hydrophilic substitutions of W262 and F121 facing each other, and hydrophobic substitutions of Q258 and G124, which are above and below W262 and F121, respectively, greatly increase mechanosensitivity, which suggests that dynamic interactions in the upper part of M4 and PH1 domain are involved in TRAAK channel mechanosensitivity. Interestingly, these gain-of-function mutations are sensitive to cell-poking stimuli, indicating that cell-poking stimuli generate a low membrane mechanical force that opens TRAAK channels. Our results thus showed that fourth transmembrane domain of TRAAK is critical for the gating of TRAAK by mechanical force and suggested that multiple dynamic interactions in the upper part of M4 and PH1 domain are involved in this process.

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2018 – Interaction of Gravity with Cellular Compounds
Port-a-Patch book chapter in Gravitational Biology II (2018) Authors: Hanke W., Kohn F.P.M., Neef M. Hampp R. 

During evolution, the majority of organisms have developed specific sensors for gravity, the only constant environmental cue on earth. Nevertheless, a variety of gravity effects on molecular, cellular, and physiological level has also been reported in single-cell organisms and cell types of plants and animals which do not seem to possess specific sensors. We have found that the cellular membrane, common to all cells, itself is interacting with gravity by changing its fluidity. Thus, it delivers a basic mechanism for gravity perception for all existing cells and living systems. In the following, we discuss the physical principles and the consequences of our findings for membrane-bound processes, for life on earth, and for manned space travel. In addition, a first model is proposed, how a sensor system for gravity based on membrane thermodynamics could be structured.

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2018 – Non-blocking modulation contributes to sodium channel inhibition by a covalently attached photoreactive riluzole analog
Port-a-Patch Publication in Nature Scientific Reports (2018) Authors: Lukacs P., Mátyás F.C., Valánszki L., Casanova E., Biri-Kovács B., Nyitray L., Málnási-Csizmadia A., Mike A.

Sodium channel inhibitor drugs decrease pathological hyperactivity in various diseases including pain syndromes, myotonia, arrhythmias, nerve injuries and epilepsies. Inhibiting pathological but not physiological activity, however, is a major challenge in drug development. Sodium channel inhibitors exert their efects by a dual action: they obstruct ion fow (“block”), and they alter the energetics of channel opening and closing (“modulation”). Ideal drugs would be modulators without blocking effect, because modulation is inherently activity-dependent, therefore selective for pathological hyperactivity. Can block and modulation be separated? It has been difficult to tell, because the effect of modulation is obscured by conformation-dependent association/dissociation of the drug. To eliminate dynamic association/dissociation, we used a photoreactive riluzole analog which could be covalently bound to the channel; and found, unexpectedly, that drug-bound channels could still conduct ions, although with modulated gating. The finding that non-blocking modulation is possible, may open a novel avenue for drug development because non-blocking modulators could be more specifc in treating hyperactivity-linked diseases.

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2018 – Enhanced Cardiomyocyte NLRP3 Inflammasome Signaling Promotes Atrial Fibrillation
Port-a-Patch Publication in Circulation (2018) Authors: Yao C., Veleva T., Scott L., Cao S., Li L., Chen G., Jeyabal P., Pan X., Alsina K.M., Abu-Taha I., Ghezelbash S., Reynolds C.L., Shen Y.H., LeMaire S.A., Schmitz W., Müller F.U., El-Armouche A., Eissa N.T., Beeton C., Nattel S. , Wehrens X.H.T., Dobrev D., Li N.

Background:Atrial fibrillation (AF) is frequently associated with enhanced inflammatory response. The “NACHT, LRR and PYD domain containing protein 3” (NLRP3)-inflammasome mediates caspase-1 activation and interleukin-1β release in immune cells, but is not known to play a role in cardiomyocytes (CMs). Here, we assessed the role of CM NLRP3-inflammasome in AF.Methods:NLRP3-inflammasome activation was assessed by immunoblot in atrial whole-tissue lysates and CMs from patients with paroxysmal (pAF) or long-standing persistent (chronic) AF (cAF). To determine whether CM-specific activation of NLPR3 is sufficient to promote AF, a CM-specific knock-in mouse model expressing constitutively active NLRP3 (CM-KI) wasestablished. In vivo electrophysiology was used to assess atrial arrhythmia vulnerability. To evaluate the mechanism of AF, electrical activation pattern, Ca2+ spark frequency (CaSF), atrialeffective refractory period (AERP), and morphology of atria were evaluated in CM-KI mice and WT littermates.Results:NLRP3-inflammasome activity was increased in atrial CMs of pAF and cAF patients. CM-KI mice developed spontaneous premature atrial contractions and inducible AF, which wasattenuated by a specific NLRP3-inflammasome inhibitor, MCC950. CM-KI mice exhibited ectopic activity, abnormal sarcoplasmic-reticulum Ca2+-release, AERP shortening and atrialhypertrophy. Adeno-associated virus subtype-9 mediated CM-specific knockdown of Nlrp3 suppressed AF development in CM-KI mice. Finally, genetic inhibition of Nlrp3 prevented AFdevelopment in CREM transgenic mice, a well-characterized mouse model of spontaneous AF.Conclusions:Our study establishes a novel pathophysiological role for CM NLRP3-inflammasome signaling with a mechanistic link to the pathogenesis of AF, and establishes inhibition of NLRP3 as a potential novel AF-therapy approach.

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2018 – Inhibition of pannexin-1 channel activity by adiponectin in podocytes: Role of acid ceramidase activation
Port-a-Patch Publication in BBA - Molecular and Cell Biology of Lipids (2018) Authors: Li G., Zhang Q., Hong J., Ritter J.K., Li P.-L.

The pannexin-1 (Panx1) channel has been reported to mediate the release of ATP that is involved in local tissue inflammation, obesity, and many chronic degenerative diseases. It remains unknown whether Panx1 is present in podocytes and whether this channel in podocytes mediates ATP release leading to glomerular inflammation or fibrosis. To answer these questions, we first characterized the expression of Panx channels in podocytes. Among the three known pannexins, Panx1 was the most enriched in podocytes, either cultured or native in mouse glomeruli. Using a Port-a-Patch planar patch-clamp system, we recorded a large voltage-gated outward current through podocyte membrane under the Cs+in/Na+out gradient. Substitution of gluconate or aspartate for chloride in the bath solution blocked voltage-gated outward currents and shifted the reversal potential of Panx1 currents to the right, indicating the anion permeability of this channel. Pharmacologically, the recorded voltage-gated outward currents were substantially attenuated by specific Panx1 channel inhibitors. Given the anti-inflammatory and intracellular ATP restorative effects of adiponectin, we tested whether this adipokine inhibits Panx1 channel activity to block ATP release. Adiponectin blocked Panx1 channel activity in podocytes. Mechanistically, inhibition of acid ceramidase (AC) remarkably enhanced Panx1 channel activity under control conditions and prevented the inhibition of Panx1 channel by adiponectin. Correspondingly, intracellular addition of AC products, sphingosine or sphingosine-1-phosphate (S1P), blocked Panx1 channel activity, while elevation of intracellular ceramide had no effect on Panx1 channel activity. These results suggest that adiponectin inhibits Panx1 channel activity in podocytes through activation of AC and associated elevation of intracellular S1P.

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2018 – An allosteric mechanism of inactivation in the calcium-dependent chloride channel BEST1
Port-a-Patch Publication in Journal of General Physiology (2018) Authors: Vaisey G., Long S.B.

Bestrophin proteins are calcium (Ca2+)-activated chloride channels. Mutations in bestrophin 1 (BEST1) cause macular degenerative disorders. Whole-cell recordings show that ionic currents through BEST1 run down over time, but it is unclear whether this behavior is intrinsic to the channel or the result of cellular factors. Here, using planar lipid bilayer recordings of purified BEST1, we show that current rundown is an inherent property of the channel that can now be characterized as inactivation. Inactivation depends on the cytosolic concentration of Ca2+, such that higher concentrations stimulate inactivation. We identify a C-terminal inactivation peptide that is necessary for inactivation and dynamically interacts with a receptor site on the channel. Alterations of the peptide or its receptor dramatically reduce inactivation. Unlike inactivation peptides of voltage-gated channels that bind within the ion pore, the receptor for the inactivation peptide is on the cytosolic surface of the channel and separated from the pore. Biochemical, structural, and electrophysiological analyses indicate that binding of the peptide to its receptor promotes inactivation, whereas dissociation prevents it. Using additional mutational studies we find that the “neck” constriction of the pore, which we have previously shown to act as the Ca2+-dependent activation gate, also functions as the inactivation gate. Our results indicate that unlike a ball-and-chain inactivation mechanism involving physical occlusion of the pore, inactivation in BEST1 occurs through an allosteric mechanism wherein binding of a peptide to a surface-exposed receptor controls a structurally distant gate.

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2018 – Electrophysiological experiments in microgravity: lessons learned and future challenges
Port-a-Patch Publication in Microgravity (2018) Authors: Wuest S.L., Gantenbein B., Ille F.,Egli M.

Advances in electrophysiological experiments have led to the discovery of mechanosensitive ion channels (MSCs) and the identification of the physiological function of specific MSCs. They are believed to play important roles in mechanosensitive pathways by allowing for cells to sense their mechanical environment. However, the physiological function of many MSCs has not been conclusively identified. Therefore, experiments have been developed that expose cells to various mechanical loads, such as shear flow, membrane indentation, osmotic challenges and hydrostatic pressure. In line with these experiments, mechanical unloading, as experienced in microgravity, represents an interesting alternative condition, since exposure to microgravity leads to a series of physiological adaption processes. As outlined in this review, electrophysiological experiments performed in microgravity have shown an influence of gravity on biological functions depending on ion channels at all hierarchical levels, from the cellular level to organs. In this context, calcium signaling represents an interesting cellular pathway, as it involves the direct action of calcium-permeable ion channels, and specific gravitatic cells have linked graviperception to this pathway. Multiple key proteins in the graviperception pathways have been identified. However, measurements on vertebrae cells have revealed controversial results. In conclusion, electrophysiological experiments in microgravity have shown that ion-channel-dependent physiological processes are altered in mechanically unloaded conditions. Future experiments may provide a better understanding of the underlying mechanisms.

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2017 – Synthetic Channel Specifically Inserts into the Lipid Bilayer of Gram-Positive Bacteria but not that of Mammalian Erythrocytes
Port-a-Patch Publication in Angewandte Chemie (2017) Authors: Zhang M., Zhu P.P., Xin P., Si W., Li Z.T., Hou J.L.

A series of tubular molecules with different lengths have been synthesized by attaching Trp-incorporated peptides to the pillar[5]arene backbone. The tubular molecules are able to insert into the lipid bilayer to form unimolecular transmembrane channels. One of the channels has been revealed to specifically insert into the bilayer of the Gram-positive bacteria. In contrast, this channel cannot insert into the membranes of the mammalian rat erythrocytes even at the high concentration of 100 μm. It was further demonstrated that, as a result of this high membrane selectivity, the channel exhibits efficient antimicrobial activity for the Gram-positive bacteria and very low hemolytic toxicity for mammalian erythrocytes.

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2018 – 3-Iodothyronamine Activates a Set of Membrane Proteins in Murine Hypothalamic Cell Lines
Port-a-Patch Publication in Frontiers in Endocrinology (2018) Authors: Bräunig J., Mergler S., Jyrch S., Hoefig C.S., Rosowski M., Mittag J., Biebermann H., Khajavi N.

3-Iodothyronamine (3-T1AM) is an endogenous thyroid hormone metabolite. The profound pharmacological effects of 3-T1AM on energy metabolism and thermal homeostasis have raised interest to elucidate its signaling properties in tissues that pertain to metabolic regulation and thermogenesis. Previous studies identified G protein-coupled receptors (GPCRs) and transient receptor potential channels (TRPs) as targets of 3-T1AM in different cell types. These two superfamilies of membrane proteins are largely expressed in tissue which influences energy balance and metabolism. As the first indication that 3-T1AM virtually modulates the function of the neurons in hypothalamus, we observed that intraperitoneal administration of 50 mg/kg bodyweight of 3-T1AM significantly increased the c-FOS activation in the paraventricular nucleus (PVN) of C57BL/6 mice. To elucidate the underlying mechanism behind this 3-T1AM-induced signalosome, we used three different murine hypothalamic cell lines, which are all known to express PVN markers, GT1-7, mHypoE-N39 (N39) and mHypoE-N41 (N41). Various aminergic GPCRs, which are the known targets of 3-T1AM, as well as numerous members of TRP channel superfamily, are expressed in these cell lines. Effects of 3-T1AM on activation of GPCRs were tested for the two major signaling pathways, the action of Gαs/adenylyl cyclase and TRPM. Here, we demonstrated that this thyroid hormone metabolite has no significant effect on Gi/o signaling and only a minor effect on the Gαs/adenylyl cyclase pathway, despite the expression of known GPCR targets of 3-T1AM. Next, to test for other potential mechanisms involved in 3-T1AM-induced c-FOS activation in PVN, we evaluated the effect of 3-T1AM on the intracellular Ca2+ concentration and whole-cell currents. The fluorescence-optic measurements showed a significant increase of intracellular Ca2+ concentration in the three cell lines in the presence of 10 μM 3-T1AM. Furthermore, this thyroid hormone metabolite led to an increase of whole-cell currents in N41 cells. Interestingly, the TRPM8 selective inhibitor (10 μM AMTB) reduced the 3-T1AM stimulatory effects on cytosolic Ca2+ and whole-cell currents. Our results suggest that the profound pharmacological effects of 3-T1AM on selected brain nuclei of murine hypothalamus, which are known to be involved in energy metabolism and thermoregulation, might be partially attributable to TRP channel activation in hypothalamic cells.

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2017 – Peptidomic analysis of the venom of the solitary bee Xylocopa appendiculata circumvolans
Port-a-Patch Publication in Journal of Venomous Animals and Toxins including Tropical Diseases (2017) Authors: Kazuma K., Ando K., Nihei K., Wang X., Rangel M., Franzolin M.R., Mori-Yasumoto K., Sekita S., Kadowaki M., Satake M., Konno K.

Background: Among the hymenopteran insect venoms, those from social wasps and bees – such as honeybee, hornets and paper wasps – have been well documented. Their venoms are composed of a number of peptides and proteins and used for defending their nests and themselves from predators. In contrast, the venoms of solitary wasps and bees have not been the object of further research. In case of solitary bees, only major peptide components in a few venoms have been addressed. Therefore, the aim of the present study was to explore the peptide component profile of the venom from the solitary bee Xylocopa appendiculata circumvolans by peptidomic analysis with using LC-MS. Methods: A reverse-phase HPLC connected to ESI-OrbiTrap MS was used for LC-MS. On-line mass fingerprinting was made from TIC, and data-dependent tandem mass spectrometry gave MSMS spectra. A major peptide component was isolated by reverse-phase HPLC by conventional way, and its sequence was determined by Edman degradation, which was finally corroborated by solid phase synthesis. Using the synthetic specimen, biological activities (antimicrobial activity, mast cell devaluation, hemolysis, leishmanicidal activity) and pore formation in artificial lipid bilayer were evaluated. Results: On-line mass fingerprinting revealed that the crude venom contained 124 components. MS/MS analysis gave 75 full sequences of the peptide components. Most of these are related to the major and novel peptide, xylopin. Its sequence, GFVALLKKLPLILKHLH-NH2, has characteristic features of linear cationic α-helical peptides; rich in hydrophobic and basic amino acids with no disulfide bond, and accordingly, it can be predicted to adopt an amphipathic α-helix secondary structure. In biological evaluation, xylopin exhibited broad-spectrum antimicrobial activity, and moderate mast cell degranulation and leishmanicidal activities, but showed virtually no hemolytic activity. Additionally, the peptide was able to incorporate pores in artificial lipid bilayers of azolectin, confirming the mechanism of the cytolytic activity by pore formation in biological membranes. Conclusions: LC-ESI-MS and MS/MS analysis of the crude venom extract from a solitary bee Xylocopa appendiculata circumvolans revealed that the component profile of this venom mostly consisted of small peptides. The major peptide components, xylopin and xylopinin, were purified and characterized in a conventional manner. Their chemical and biological characteristics, belonging to linear cationic α-helical peptides, are similar to the known solitary bee venom peptides, melectin and osmin. Pore formation in artificial lipid bilayers was demonstrated for the first time with a solitary bee peptide.

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2017 – Prolonged immunomodulation in inflammatory arthritis using the selective KV1.3 channel blocker HsTX1[R14A] and its PEGylated analog
Port-a-Patch Publication in Clinical Immunology (2017) Authors: Tanner M.R., Tajhya R.B., Huq R., Gehrmann E.J., Rodarte K.E., Atik M.A., Norton R.S., Pennington R.W., Beeton C.

Effector memory T lymphocytes (TEM cells) that lack expression of CCR7 are major drivers of inflammation in a number of autoimmune diseases, including multiple sclerosis and rheumatoid arthritis. The KV1.3 potassium channel is a key regulator of CCR7− TEM cell activation. Blocking KV1.3 inhibits TEM cell activation and attenuates inflammation in autoimmunity, and as such, KV1.3 has emerged as a promising target for the treatment of TEM cell-mediated autoimmune diseases. The scorpion venom-derived peptide HsTX1 and its analog HsTX1[R14A] are potent KV1.3 blockers and HsTX1[R14A] is selective for KV1.3 over closely-related KV1 channels. PEGylation of HsTX1[R14A] to create a KV1.3 blocker with a long circulating half-life reduced its affinity but not its selectivity for KV1.3, dramatically reduced its adsorption to inert surfaces, and enhanced its circulating half-life in rats. PEG-HsTX1[R14A] is equipotent to HsTX1[R14A] in preferential inhibition of human and rat CCR7− TEM cell proliferation, leaving CCR7+ naïve and central memory T cells able to proliferate. It reduced inflammation in an active delayed-type hypersensitivity model and in the pristane-induced arthritis (PIA) model of rheumatoid arthritis (RA). Importantly, a single subcutaneous dose of PEG-HsTX1[R14A] reduced inflammation in PIA for a longer period of time than the non-PEGylated HsTX1[R14A]. Together, these data indicate that HsTX1[R14A] and PEG-HsTX1[R14A] are effective in a model of RA and are therefore potential therapeutics for TEM cell-mediated autoimmune diseases. PEG-HsTX1[R14A] has the additional advantages of reduced non-specific adsorption to inert surfaces and enhanced circulating half-life.

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2017 – Cytotoxic effect of zinc oxide nanoparticles on murine photoreceptor cells via potassium channel block and Na+/K+‐ATPase inhibition
Port-a-Patch Publication in Cell Proliferation (2017) Authors: Chen C. Bu W., Ding H., Li Q., Wang D., Bi H., Guo D.

Objective: Zinc oxide (ZnO) nanoparticles can exhibit toxicity towards organisms and oxidative stress is often hypothesized to be one of the most important factors. Nevertheless, the detailed mechanism of toxicity‐induced by ZnO nanoparticles has not been completely addressed. The present study aimed to investigate the toxic effects of ZnO nanoparticles on the expression and activity of Na+/K+‐ATPase and on potassium channel block. Materials and methods: In the present study, we explored the cytotoxic effect of ZnO nanoparticles on murine photoreceptor cells using lactate dehydrogenase (LDH) release assay, reactive oxygen species (ROS) determination, mitochondrial membrane potential (Δφm) measurement, delayed rectifier potassium current recordings and Na+/K+‐ATPase expression and activity monitoring. Results: The results indicated that ZnO nanoparticles could increase the LDH release in medium, aggravate the ROS level within cells, collapse the Δφm, block the delayed rectifier potassium current, and attenuate the expressions of Na+/K+‐ATPase at both mRNA and protein levels and its activity, and thus exert cytotoxic effects on murine photoreceptor cells, finally damaging target cells. Conclusion: Our findings will facilitate the understanding of the mechanism involved in ZnO nanoparticle‐induced cytotoxicity in murine photoreceptor cells via potassium channel block and Na+/K+‐ATPase inhibition.

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2017 – Mitochondria sustain store-operated currents in colon cancer cells but not in normal colonic cells: reversal by non-steroidal anti-inflammatory drugs
Port-a-Patch Publication in Oncotarget (2017) Authors: Hernández-Morales M., Sobradillo D., Valero R.A., Muñoz E., Ubierna D., Moyer M.P., Núñez L., Villalobos C.

Tumor cells undergo a critical remodeling of intracellular Ca2+ homeostasis that contribute to important cancer hallmarks. Store-operated Ca2+ entry (SOCE), a Ca2+ entry pathway modulated by mitochondria, is dramatically enhanced in colon cancer cells. In addition, most cancer cells display the Warburg effect, a metabolic switch from mitochondrial metabolism to glycolysis that provides survival advantages. Accordingly, we investigated mitochondria control of store-operated currents (SOCs) in two cell lines previously selected for representing human normal colonic cells and colon cancer cells. We found that, in normal cells, mitochondria are important for SOCs activity but they are unable to prevent current inactivation. In contrast, in colon cancer cells, mitochondria are dispensable for SOCs activation but are able to prevent the slow, Ca2+-dependent inactivation of SOCs. This effect is associated to increased ability of tumor cell mitochondria to take up Ca2+ due to increased mitochondrial potential (ΔΨ) linked to the Warburg effect. Consistently with this view, selected non-steroidal anti-inflammatory drugs (NSAIDs) depolarize mitochondria, inhibit mitochondrial Ca2+ uptake and promote SOC inactivation, leading to inhibition of both SOCE and cancer cell proliferation. Thus, mitochondria sustain store-operated currents in colon cancer cells but not in normal colonic cells and this effect is counteracted by selected NSAIDs providing a mechanism for cancer chemoprevention.

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2017 – A mutant of the Buthus martensii Karsch antitumor-analgesic peptide exhibits reduced inhibition to hNaV1.4 and hNaV1.5 channels while retaining analgesic activity
Port-a-Patch Publication in Journal of Biological Chemistry (2017) Authors: Xu Y., Meng X., Hou X., Sun J., Kong X., Sun Y., Liu Z., Ma Y., Niu Y., Song Y., Cui Y., Zhao M., Zhang J.

Scorpion toxins can kill other animals by inducing paralysis and arrhythmia, which limits the potential applications of these agents in the clinical management of diseases. Antitumor-analgesic peptide (AGAP), purified from Buthus martensii Karsch, has been proved to possess analgesic and antitumor activities. Trp38, a conserved aromatic residue of AGAP, might play an important role in mediating AGAP activities according to the sequence and homology-modeling analyses. Therefore, an AGAP mutant, W38G, was generated, and effects of both AGAP and the mutant W38G were examined by whole-cell patch clamp techniques on the sodium channels hNaV1.4 and hNaV1.5, which were closely associated with the biotoxicity of skeletal and cardiac muscles, respectively. The data showed that both W38G and AGAP inhibited the peak currents of hNaV1.4 and hNaV1.5; however, W38G induced a much weaker inhibition of both channels than AGAP. Accordingly, W38G exhibited much less toxic effect on both skeletal and cardiac muscles than AGAP in vivo. The analgesic activity of W38G and AGAP were verified in vivo as well, and W38G retained analgesic activity similar to AGAP. Inhibition to both NaV1.7 and NaV1.8 was involved in the analgesic mechanism of AGAP and W38G. These findings indicated that Trp38 was a key amino acid involved in the biotoxicity of AGAP, and the AGAP mutant W38G might be a safer alternative for clinical application because it retains the analgesic efficacy with less toxicity to skeletal and cardiac muscles.

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2017 – Buserelin alleviates chloride transport defect in human cystic fibrosis nasal epithelial cells
Port-a-Patch Publication in PLoS ONE (2017) Authors: Calvez M.L., Benz N., Huguet F., Saint-Pierre A., Rouillé E., Coraux C., Férec C., Kerbiriou M., Trouvé P.

Cystic fibrosis (CF) is the most common autosomal recessive disease in Caucasians caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) chloride (Cl-) channel regulated by protein kinases, phosphatases, divalent cations and by protein-protein interactions. Among protein-protein interactions, we previously showed that Annexin A5 (AnxA5) binds to CFTR and is involved in the channel localization within membranes and in its Cl- channel function. The deletion of phenylalanine at position 508 (F508del) is the most common mutation in CF which leads to an altered protein (F508del-CFTR) folding with a nascent protein retained within the ER and is quickly degraded. We previously showed that AnxA5 binds to F508del-CFTR and that its increased expression due to a Gonadoliberin (GnRH) augments Cl- efflux in cells expressing F508del-CFTR. The aim of the present work was to use the GnRH analog buserelin which is already used in medicine. Human nasal epithelial cells from controls and CF patients (F508del/F508del) were treated with buserelin and we show here that the treatment alleviates Cl- channel defects in CF cells. Using proteomics we highlighted some proteins explaining this result. Finally, we propose that buserelin is a potential new pharmaceutical compound that can be used in CF and that bronchus can be targeted since we show here that they express GnRH-R.

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2016 – Upregulation of Transient Receptor Potential Vanilloid Type-1 Channel Activity and Ca2+ Influx Dysfunction in Human Pterygial Cells
Port-a-Patch Publication in Investigative Opthalmology & Visual Science (2016) Authors: Garreis F., Schröder A., Reinach P.S., Zoll S., Khajavi N., Dhandapani P., Lucius A., Pleyer U., Paulsen F., Mergler S.

Purpose:The heat-sensitive transient receptor potential vanilloid type-1 (TRPV1) channel (i.e., capsaicin [CAP] receptor) is upregulated in numerous cancers. This study determined if this response occurs in fresh and cultured hyperplastic human pterygial epithelial tissues.Methods:Reverse transcriptase PCR and quantitative real-time PCR, along with immunohistochemistry and Western blotting, characterized TRPV1 expression patterns in pterygial and healthy conjunctival tissue, primary and immortalized pterygial cells (hPtEC), and primary and immortalized conjunctival epithelial cells (HCjEC). Imaging of Ca2+ and planar whole-cell patch-clamping evaluated TRP channel activity. An MTS assay measured cell metabolic activity and a cell growth assay monitored proliferation.Results:Capsaicin (20 μM) and elevating bath temperature above 43°C activated Ca2+ transients more in hPtEC than HCjEC. Capsaicin induced corresponding changes in inward currents that were inhibited by 20 μM capsazepine (CPZ). Vascular endothelial growth factor (VEGF) also increased Ca2+-influx and induced corresponding inward currents more in hPtEC than in HCjEC, whereas CPZ (20 μM), BCTC (20 μM), or La3+ (500 μM) reduced these responses, respectively. Whereas epidermal growth factor (EGF) increased proliferation more in hPtEC than in HCjEC, VEGF had no effect on this response. Capsazepine suppressed hPtEC proliferation induced by EGF and VEGF, whereas it was cytotoxic to HCjEC.Conclusions:Mitogenic responses to EGF and VEGF are mediated through TRPV1 transactivation. Only in hPtEC do the increases in proliferation induced by EGF exceed those in HCjEC. Therefore, TRPV1 is a potential drug target whose clinical relevance in treating pterygium warrants further assessment.

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2016 – Voltage Dependence of Conformational Dynamics and Subconducting States of VDAC-1
Port-a-Patch Publication in Biophysical Journal (2016) Authors: Briones R., Weichbrodt C., Paltrinieri L., Mey I., Villinger S., Giller K., Lange A., Zweckstetter M., Griesinger C., Becker S., Steinem C., de Groot B.L.

The voltage-dependent anion channel 1 (VDAC-1) is an important protein of the outer mitochondrial membrane that transports energy metabolites and is involved in apoptosis. The available structures of VDAC proteins show a wide β-stranded barrel pore, with its N-terminal α-helix (N-α) bound to its interior. Electrophysiology experiments revealed that voltage, its polarity, and membrane composition modulate VDAC currents. Experiments with VDAC-1 mutants identified amino acids that regulate the gating process. However, the mechanisms for how these factors regulate VDAC-1, and which changes they trigger in the channel, are still unknown. In this study, molecular dynamics simulations and single-channel experiments of VDAC-1 show agreement for the current-voltage relationships of an “open” channel and they also show several subconducting transient states that are more cation selective in the simulations. We observed voltage-dependent asymmetric distortions of the VDAC-1 barrel and the displacement of particular charged amino acids. We constructed conformational models of the protein voltage response and the pore changes that consistently explain the protein conformations observed at opposite voltage polarities, either in phosphatidylethanolamine or phosphatidylcholine membranes. The submicrosecond VDAC-1 voltage response shows intrinsic structural changes that explain the role of key gating amino acids and support some of the current gating hypotheses. These voltage-dependent protein changes include asymmetric barrel distortion, its interaction with the membrane, and significant displacement of N-α amino acids.

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2016 – Distinct regions that control ion selectivity and calcium-dependent activation in the bestrophin ion channel
Port-a-Patch Publication in Proceedings of the National Academy of Sciences of the United States of America (2016) Authors: Vaisey G., Miller A.N., Long S.B.

Abstract: Cytoplasmic calcium (Ca2+) activates the bestrophin anion channel, allowing chloride ions to flow down their electrochemical gradient. Mutations in bestrophin 1 (BEST1) cause macular degenerative disorders. Previously, we determined an X-ray structure of chicken BEST1 that revealed the architecture of the channel. Here, we present electrophysiological studies of purified wild-type and mutant BEST1 channels and an X-ray structure of a Ca2+-independent mutant. From these experiments, we identify regions of BEST1 responsible for Ca2+ activation and ion selectivity. A “Ca2+ clasp” within the channel’s intracellular region acts as a sensor of cytoplasmic Ca2+. Alanine substitutions within a hydrophobic “neck” of the pore, which widen it, cause the channel to be constitutively active, irrespective of Ca2+. We conclude that the primary function of the neck is as a “gate” that controls chloride permeation in a Ca2+-dependent manner. In contrast to what others have proposed, we find that the neck is not a major contributor to the channel’s ion selectivity. We find that mutation of a cytosolic “aperture” of the pore does not perturb the Ca2+ dependence of the channel or its preference for anions over cations, but its mutation dramatically alters relative permeabilities among anions. The data suggest that the aperture functions as a size-selective filter that permits the passage of small entities such as partially dehydrated chloride ions while excluding larger molecules such as amino acids. Thus, unlike ion channels that have a single “selectivity filter,” in bestrophin, distinct regions of the pore govern anion-vs.-cation selectivity and the relative permeabilities among anions.Significance:BEST1 is a Ca2+-activated chloride channel found in a variety of cell types that allows chloride to traverse the plasma membrane. Mutations in BEST1 can cause macular degeneration. The mechanisms for anion selectivity and Ca2+-dependent activation of BEST1 are unknown. Here, we show that a hydrophobic “neck” region of the channel’s pore does not play a major role in ion selectivity but acts as an effective gate, responding to Ca2+ binding at a cytosolic sensor. Mutation of a cytosolic “aperture” dramatically affects relative permeabilities among anions. These insights help rationalize how disease-causing mutations in BEST1 affect channel behavior and contribute to a broader understanding of ion channel gating and selectivity mechanisms.

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2016 – Reversible control of current across lipid membranes by local heating
Port-a-Patch Publication in Scientific Reports (2016) Authors: Urban P., Kirchner S.R., Mühlbauer C., Lohmüller T., Feldmann J.

Lipid membranes are almost impermeable for charged molecules and ions that can pass the membrane barrier only with the help of specialized transport proteins. Here, we report how temperature manipulation at the nanoscale can be employed to reversibly control the electrical resistance and the amount of current that flows through a bilayer membrane with pA resolution. For this experiment, heating is achieved by irradiating gold nanoparticles that are attached to the bilayer membrane with laser light at their plasmon resonance frequency. We found that controlling the temperature on the nanoscale renders it possible to reproducibly regulate the current across a phospholipid membrane and the membrane of living cells in absence of any ion channels.

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2015 – The Functional Property Changes of Muscular NaV1.4 and Cardiac NaV1.5 Induced by Scorpion Toxin BmK AGP-SYPU1 Mutants Y42F and Y5F
Port-a-Patch Publication in Biochemistry (2015) Authors: Meng x., Xu Y., Zhao M., Wang F., Ma Y., Jin Y., Liu Y., Song Y., Zhang J.

Scorpion toxins are invaluable therapeutic leads and pharmacological tools which influence the voltage-gated sodium channels. However, the details were still unclear about the structure–function relationship of scorpion toxins on VGSC subtypes. In the previous study, we reported one α-type scorpion toxin Bmk AGP-SYPU1 and its two mutants (Y5F and Y42F) which had been demonstrated to ease pain in mice acetic acid writhing test. However, the function of Bmk AGP-SYPU1 on VGSCs is still unknown. In this study, we examined the effects of BmK AGP-SYPU1 and its two mutants (Y5F and Y42F) on hNaV1.4 and hNaV1.5 heterologously expressed CHO cell lines by using Na+-specialized fluorescent dye and whole-cell patch clamp. The data showed that BmK AGP-SYPU1 displayed as an activator of hNaV1.4 and hNaV1.5, which might indeed contribute to its biotoxicity to muscular and cardiac system and exhibited the functional properties of both the α-type and β-type scorpion toxin. Notably, Y5F mutant exhibited lower activatory effects on hNaV1.4 and hNaV1.5 compared with BmK AGP-SYPU1. Y42F was an enhanced activator and confirmed that the conserved Tyr42 was the key amino acid involved in bioactivity or biotoxicity. These data provided a deep insight into the structure–function relationship of BmK AGP-SYPU1, which may be the guidance for engineering α-toxin with high selectivity on VGSC subtypes.

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2016 – Defined Medium Conditions for the Induction and Expansion of Human Pluripotent Stem Cell-Derived Retinal Pigment Epithelium
Port-a-Patch Publication in Stem Cell Reviews and Reports (2016) Authors: Lidgerwood G.E., Lim S.Y., Crombie D.E., Ali R., Gill K.P., Hernández D., Kie J., Conquest A., Waugh H.S., Wong R.C., Liang H.H., Hewitt A.W., Davidson K.C., Pébay A.

We demonstrate that a combination of Noggin, Dickkopf-1, Insulin Growth Factor 1 and basic Fibroblast Growth Factor, promotes the differentiation of human pluripotent stem cells into retinal pigment epithelium (RPE) cells. We describe an efficient one-step approach that allows the generation of RPE cells from both human embryonic stem cells and human induced pluripotent stem cells within 40–60 days without the need for manual excision, floating aggregates or imbedded cysts. Compared to methods that rely on spontaneous differentiation, our protocol results in faster differentiation into RPE cells. This pro-retinal culture medium promotes the growth of functional RPE cells that exhibit key characteristics of the RPE including pigmentation, polygonal morphology, expression of mature RPE markers, electrophysiological membrane potential and the ability to phagocytose photoreceptor outer segments. This protocol can be adapted for feeder, feeder-free and serum-free conditions. This method thereby provides a rapid and simplified production of RPE cells for downstream applications such as disease modelling and drug screening.

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2015 – K2P channel gating mechanisms revealed by structures of TREK-2 and a complex with Prozac
Port-a-Patch Publication in Science (2015) Authors: Dong Y.Y., Pike A.C., Mackenzie A., McClenaghan C., Aryal P., Dong L., Quigley A., Grieben M., Goubin S., Mukhopadhyay S., Ruda G.F., Clausen M.V., Cao L., Brennan P.E., Burgess-Brown N.A., Sansom M.S., Tucker S.J., Carpenter E.P.

TREK-2 (KCNK10/K2P10), a two-pore domain potassium (K2P) channel, is gated by multiple stimuli such as stretch, fatty acids, and pH and by several drugs. However, the mechanisms that control channel gating are unclear. Here we present crystal structures of the human TREK-2 channel (up to 3.4 angstrom resolution) in two conformations and in complex with norfluoxetine, the active metabolite of fluoxetine (Prozac) and a state-dependent blocker of TREK channels. Norfluoxetine binds within intramembrane fenestrations found in only one of these two conformations. Channel activation by arachidonic acid and mechanical stretch involves conversion between these states through movement of the pore-lining helices. These results provide an explanation for TREK channel mechanosensitivity, regulation by diverse stimuli, and possible off-target effects of the serotonin reuptake inhibitor Prozac

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2015 – The application of the Escherichia coli giant spheroplast for drug screening with automated planar patch clamp system
Port-a-Patch Publication in Biotechnology Reports (2015) Authors: Kikuchi K., Sugiura M., Nishizawa-Harada C., Kimura T. 

KV2.1, the voltage-gated ion channel, is ubiquitously expressed in variety of tissues and dysfunction of this ion channel is responsible for multiple diseases. Electrophysiological properties of ion channels are so far characterized with eukaryotic cells using the manual patch clamp which requires skilful operators and expensive equipments. In this research, we created a simple and sensitive drug screen method using bacterial giant spheroplasts and the automated patch clamp which does not require special skills. We expressed a eukaryotic voltage-gated ion channel KV2.1 in Escherichia coli using prokaryotic codon, and prepared giant spheroplasts large enough for the patch clamp. Human KV2.1 currents were successfully recorded from giant spheroplasts with the automated system, and KV2.1-expressed E. coli spheroplasts could steadily reacted to the dose–response assay with TEA and 4-AP. Collectively, our results indicate for the first time that the bacterial giant spheroplast can be applied for practical pharmaceutical assay using the automated patch clamp.

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2014 – L-Carnitine Reduces in Human Conjunctival Epithelial Cells Hypertonic- Induced Shrinkage through Interacting with TRPV1 Channels
Port-a-Patch Publication in Cellular Physiology and Biochemistry (2014) Authors: Khajavi N., Reinach P.S., Skrzypski M., LudeA., Mergler S.

Background/Aims: Ocular surface health depends on conjunctival epithelial (HCjE) layer integrity since it protects against pathogenic infiltration and contributes to tissue hydration maintenance. As the same increases in tear film hyperosmolarity described in dry eye disease can increase corneal epithelial transient receptor potential vanilloid type-1 (TRPV1) channel activity, we evaluated its involvement in mediating an osmoprotective effect by L-carnitine against such stress. Methods: Using siRNA gene silencing, Ca2+ imaging, planar patch-clamping and relative cell volume measurements, we determined if the protective effects of this osmolyte stem from its interaction with TRPV1. Results: TRPV1 activation by capsaicin (CAP) and an increase in osmolarity to ≈ 450 mOsM both induced increases in Ca2+ levels. In contrast, blocking TRPV1 activation with capsazepine (CPZ) fully reversed this response. Similarly, L-carnitine (1 mM) also reduced underlying whole-cell currents. In calcein-AM loaded cells, hypertonic-induced relative cell volume shrinkage was fully blocked during exposure to L-carnitine. On the other hand, in TRPV1 gene-silenced cells, this protective effect by L-carnitine was obviated.Conclusion:The described L-carnitine osmoprotective effect is elicited through suppression of hypertonic-induced TRPV1 activation leading to increases in L-carnitine uptake through a described Na+-dependent L-carnitine transporter.

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2014 – Ultra-stable glass microcraters for on-chip patch clamping
Port-a-Patch Publication in Joyal Society of Chemistry Advances (2014) Authors: Stava E., Shin H.C.,Yu M., Bhat A., Resto P.J., Seshadri A., Williams J.C., Blick R.H.

We report on a single-step fabrication procedure of borosilicate glass micropores surrounded by a smooth microcrater. By inserting a thin air-gap between a borosilicate glass substrate and a reflective layer, we achieve dual-sided laser ablation of the device. The resultant crater provides a smoother, curved surface onto which cells settle during planar patch clamping. Gigaohm seals, which are more easily achievable on these devices as compared to conventional micropores, are achieved by patch clamping human embryonic kidney (HEK 293) cells. Further, the microcraters show enhanced mechanical stability of the planar patch clamped cells during perfusion. We integrate polydimethylsiloxane microfluidic devices with the microcraters and use passive pumping to perfuse the cells. We find that passive pumping increases the pressure within the device by 1.85 Pa. However, due to the enhanced stability of the microcrater, fluidic shearing reduces the seal resistance by only 6.8 MΩ on average, which is less than one percent of the gigaohm seal resistance.

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2014 – Improvement of Chloride Transport Defect by Gonadotropin-Releasing Hormone (GnRH) in Cystic Fibrosis Epithelial Cells
Port-a-Patch Publication in PLoS One (2014) Authors: Benz N., Le Hir S., Norez C., Kerbiriou M., Calvez M.-L., Becq F., Trouvé P. , Férec C.

Cystic fibrosis (CF), the most common autosomal recessive disease in Caucasians, is due to mutations in the CFTR gene. F508del, the most frequent mutation in patients, impairs CFTR protein folding and biosynthesis. The F508del-CFTR protein is retained in the endoplasmic reticulum (ER) and its traffic to the plasma membrane is altered. Nevertheless, if it reaches the cell surface, it exhibits a Cl− channel function despite a short half-life. Pharmacological treatments may target the F508del-CFTR defect directly by binding to the mutant protein or indirectly by altering cellular proteostasis, and promote its plasma membrane targeting and stability. We previously showed that annexine A5 (AnxA5) directly binds to F508del-CFTR and, when overexpressed, promotes its membrane stability, leading to the restoration of some Cl− channel function in cells. Because Gonadotropin-Releasing Hormone (GnRH) increases AnxA5 expression in some cells, we tested it in CF cells. We showed that human epithelial cells express GnRH-receptors (GnRH-R) and that GnRH induces an AnxA5 overexpression and an increased Cl− channel function in F508del-CFTR cells, due to an increased stability of the protein in the membranes. Beside the numerous physiological implications of the GnRH-R expression in epithelial cells, we propose that a topical use of GnRH is a potential treatment in CF.

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2014 – KCNMA1 Encoded Cardiac BK Channels Afford Protection against Ischemia-Reperfusion Injury
Port-a-Patch Publication in PLoS One (2014) Authors: Soltysinska E., Bentzen B.H., Barthmes M., Hattel H., Thrush A.B., Harper M.E., Qvortrup K., Larsen F.J., Schiffer T.A., Losa-Reyna J., Straubinger J., Kniess A., Thomsen M.B., Brüggemann A., Fenske S., Biel M., Ruth P., Wahl-Schott C., Boushel R.C., Olesen S.P., Lukowski R.

Mitochondrial potassium channels have been implicated in myocardial protection mediated through pre-/postconditioning. Compounds that open the Ca2+- and voltage-activated potassium channel of big-conductance (BK) have a pre-conditioning-like effect on survival of cardiomyocytes after ischemia/reperfusion injury. Recently, mitochondrial BK channels (mitoBKs) in cardiomyocytes were implicated as infarct-limiting factors that derive directly from the KCNMA1 gene encoding for canonical BKs usually present at the plasma membrane of cells. However, some studies challenged these cardio-protective roles of mitoBKs. Herein, we present electrophysiological evidence for paxilline- and NS11021-sensitive BK-mediated currents of 190 pS conductance in mitoplasts from wild-type but not BK−/− cardiomyocytes. Transmission electron microscopy of BK−/− ventricular muscles fibres showed normal ultra-structures and matrix dimension, but oxidative phosphorylation capacities at normoxia and upon re-oxygenation after anoxia were significantly attenuated in BK−/− permeabilized cardiomyocytes. In the absence of BK, post-anoxic reactive oxygen species (ROS) production from cardiomyocyte mitochondria was elevated indicating that mitoBK fine-tune the oxidative state at hypoxia and re-oxygenation. Because ROS and the capacity of the myocardium for oxidative metabolism are important determinants of cellular survival, we tested BK−/− hearts for their response in an ex-vivo model of ischemia/reperfusion (I/R) injury. Infarct areas, coronary flow and heart rates were not different between wild-type and BK−/− hearts upon I/R injury in the absence of ischemic pre-conditioning (IP), but differed upon IP. While the area of infarction comprised 28±3% of the area at risk in wild-type, it was increased to 58±5% in BK−/− hearts suggesting that BK mediates the beneficial effects of IP. These findings suggest that cardiac BK channels are important for proper oxidative energy supply of cardiomyocytes at normoxia and upon re-oxygenation after prolonged anoxia and that IP might indeed favor survival of the myocardium upon I/R injury in a BK-dependent mode stemming from both mitochondrial post-anoxic ROS modulation and non-mitochondrial localizations.

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2014 – Crystal structure of a human GABAA receptor
Port-a-Patch Publication in Nature (2014) Authors: Miller P.S., Aricescu A.R.

Type-A γ-aminobutyric acid receptors (GABAARs) are the principal mediators of rapid inhibitory synaptic transmission in the human brain. A decline in GABAAR signalling triggers hyperactive neurological disorders such as insomnia, anxiety and epilepsy. Here we present the first three-dimensional structure of a GABAAR, the human β3 homopentamer, at 3 Å resolution. This structure reveals architectural elements unique to eukaryotic Cys-loop receptors, explains the mechanistic consequences of multiple human disease mutations and shows an unexpected structural role for a conserved N-linked glycan. The receptor was crystallized bound to a previously unknown agonist, benzamidine, opening a new avenue for the rational design of GABAAR modulators. The channel region forms a closed gate at the base of the pore, representative of a desensitized state. These results offer new insights into the signalling mechanisms of pentameric ligand-gated ion channels and enhance current understanding of GABAergic neurotransmission.

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2014 – High susceptibility to fatty liver disease in two-pore channel 2-deficient mice
Port-a-Patch Publication in Nature Communications (2014) Authors: Grimm C., Holdt L.M., Chen C.C., Hassan S., Müller C., Jörs S., Cuny H., Kissing S., Schröder B., Butz B., Northoff B., Castonguay J., Luber C.A., Moser M., Spahn S., Lüllmann-Rauch R., Fendel C., Klugbauer N., Griesbeck O., Haas A., Mann M., Bracher F., Teupser D., Saftig P., Biel M., Wahl-Schott C.

Endolysosomal organelles play a key role in trafficking, breakdown and receptor-mediated recycling of different macromolecules such as low-density lipoprotein (LDL)-cholesterol, epithelial growth factor (EGF) or transferrin. Here we examine the role of two-pore channel (TPC) 2, an endolysosomal cation channel, in these processes. Embryonic mouse fibroblasts and hepatocytes lacking TPC2 display a profound impairment of LDL-cholesterol and EGF/EGF-receptor trafficking. Mechanistically, both defects can be attributed to a dysfunction of the endolysosomal degradation pathway most likely on the level of late endosome to lysosome fusion. Importantly, endolysosomal acidification or lysosomal enzyme function are normal in TPC2-deficient cells. TPC2-deficient mice are highly susceptible to hepatic cholesterol overload and liver damage consistent with non-alcoholic fatty liver hepatitis. These findings indicate reduced metabolic reserve of hepatic cholesterol handling. Our results suggest that TPC2 plays a crucial role in trafficking in the endolysosomal degradation pathway and, thus, is potentially involved in the homoeostatic control of many macromolecules and cell metabolites.

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2014 – A small molecule restores function to TRPML1 mutant isoforms responsible for mucolipidosis type IV
Port-a-Patch Publication in Nature Communications (2014) Authors: Chen C.C., Keller M., Hess M., Schiffmann R., Urban N., Wolfgardt A., Schaefer M., Bracher F., Biel M., Wahl-Schott C., Grimm C.

Mucolipidosis type IV (MLIV) is an autosomal recessive lysosomal storage disorder often characterized by severe neurodevelopmental abnormalities and neuro-retinal degeneration. Mutations in the TRPML1 gene are causative for MLIV. We used lead optimization strategies to identify—and MLIV patient fibroblasts to test—small-molecule activators for their potential to restore TRPML1 mutant channel function. Using the whole-lysosome planar patch-clamp technique, we found that activation of MLIV mutant isoforms by the endogenous ligand PI(3,5)P2 is strongly reduced, while activity can be increased using synthetic ligands. We also found that the F465L mutation renders TRPML1 pH insensitive, while F408Δ impacts synthetic ligand binding. Trafficking defects and accumulation of zinc in lysosomes of MLIV mutant fibroblasts can be rescued by the small molecule treatment. Collectively, our data demonstrate that small molecules can be used to restore channel function and rescue disease associated abnormalities in patient cells expressing specific MLIV point mutations.

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2014 – Calcium regulation by temperature-sensitive transient receptor potential channels in human uveal melanoma cells
Port-a-Patch Publication in Cellular Signalling (2014) Authors: Mergler S., Derckx R., Reinach P.S., Garreis F., Böhm A., Schmelzer L., Skosyrski S., Ramesh N., Abdelmessih S., Polat O.K., Khajavi N., Riechardt A.I.

Uveal melanoma (UM) is both the most common and fatal intraocular cancer among adults worldwide. As with all types of neoplasia, changes in Ca2+ channel regulation can contribute to the onset and progression of this pathological condition. Transient receptor potential channels (TRPs) and cannabinoid receptor type 1 (CB1) are two different types of Ca2+ permeation pathways that can be dysregulated during neoplasia. We determined in malignant human UM and healthy uvea and four different UM cell lines whether there is gene and functional expression of TRP subtypes and CB1 since they could serve as drug targets to either prevent or inhibit initiation and progression of UM. RT-PCR, Ca2+ transients, immunohistochemistry and planar patch-clamp analysis probed for their gene expression and functional activity, respectively. In UM cells, TRPV1 and TRPM8 gene expression was identified. Capsaicin (CAP), menthol or icilin induced Ca2+ transients as well as changes in ion current behavior characteristic of TRPV1 and TRPM8 expression. Such effects were blocked with either La3+, capsazepine (CPZ) or BCTC. TRPA1 and CB1 are highly expressed in human uvea, but TRPA1 is not expressed in all UM cell lines. In UM cells, the CB1 agonist, WIN 55,212-2, induced Ca2+ transients, which were suppressed by La3+ and CPZ whereas CAP-induced Ca2+ transients could also be suppressed by CB1 activation. Identification of functional TRPV1, TRPM8, TRPA1 and CB1 expression in these tissues may provide novel drug targets for treatment of this aggressive neoplastic disease.

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2013 – Monoacylglycerols Activate TRPV1 – A Link between Phospholipase C and TRPV1
Port-a-Patch Publication in PLoS One (2013) Authors: Zygmunt P.M., Ermund A., Movahed P., Andersson D.A., Simonsen C., Jönsson B.A., Blomgren A., Birnir B., Bevan S., Eschalier A., Mallet C., Gomis A., Högestätt E.D.

Phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate generates diacylglycerol, inositol 1,4,5-trisphosphate and protons, all of which can regulate TRPV1 activity via different mechanisms. Here we explored the possibility that the diacylglycerol metabolites 2-arachidonoylglycerol and 1-arachidonoylglycerol, and not metabolites of these monoacylglycerols, activate TRPV1 and contribute to this signaling cascade. 2-Arachidonoylglycerol and 1-arachidonoylglycerol activated native TRPV1 on vascular sensory nerve fibers and heterologously expressed TRPV1 in whole cells and inside-out membrane patches. The monoacylglycerol lipase inhibitors methylarachidonoyl-fluorophosphonate and JZL184 prevented the metabolism of deuterium-labeled 2-arachidonoylglycerol and deuterium-labeled 1-arachidonoylglycerol in arterial homogenates, and enhanced TRPV1-mediated vasodilator responses to both monoacylglycerols. In mesenteric arteries from TRPV1 knock-out mice, vasodilator responses to 2-arachidonoylglycerol were minor. Bradykinin and adenosine triphosphate, ligands of phospholipase C-coupled membrane receptors, increased the content of 2-arachidonoylglycerol in dorsal root ganglia. In HEK293 cells expressing the phospholipase C-coupled histamine H1 receptor, exposure to histamine stimulated the formation of 2-AG, and this effect was augmented in the presence of JZL184. These effects were prevented by the diacylglycerol lipase inhibitor tetrahydrolipstatin. Histamine induced large whole cell currents in HEK293 cells co-expressing TRPV1 and the histamine H1 receptor, and the TRPV1 antagonist capsazepine abolished these currents. JZL184 increased the histamine-induced currents and tetrahydrolipstatin prevented this effect. The calcineurin inhibitor ciclosporin and the endogenous “entourage” compound palmitoylethanolamide potentiated the vasodilator response to 2-arachidonoylglycerol, disclosing TRPV1 activation of this monoacylglycerol at nanomolar concentrations. Furthermore, intracerebroventricular injection of JZL184 produced TRPV1-dependent antinociception in the mouse formalin test. Our results show that intact 2-arachidonoylglycerol and 1-arachidonoylglycerol are endogenous TRPV1 activators, contributing to phospholipase C-dependent TRPV1 channel activation and TRPV1-mediated antinociceptive signaling in the brain.

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2014 – A potent and KV1.3-selective analogue of the scorpion toxin HsTX1 as a potential therapeutic for autoimmune diseases
Port-a-Patch Publication in Nature Scientific Reports (2014) Authors: Rashid M.H., Huq R., Tanner M.R., Chhabra S., Khoo K.K., Estrada R., Dhawan V., Chauhan S., Pennington M.W., Beeton C., Kuyucak S., Norton R.S.

HsTX1 toxin, from the scorpion Heterometrus spinnifer, is a 34-residue, C-terminally amidated peptide cross-linked by four disulfide bridges. Here we describe new HsTX1 analogues with an Ala, Phe, Val or Abu substitution at position 14. Complexes of HsTX1 with the voltage-gated potassium channels KV1.3 and KV1.1 were created using docking and molecular dynamics simulations, then umbrella sampling simulations were performed to construct the potential of mean force (PMF) of the ligand and calculate the corresponding binding free energy for the most stable configuration. The PMF method predicted that the R14A mutation in HsTX1 would yield a > 2 kcal/mol gain for the KV1.3/KV1.1 selectivity free energy relative to the wild-type peptide. Functional assays confirmed the predicted selectivity gain for HsTX1[R14A] and HsTX1[R14Abu], with an affinity for KV1.3 in the low picomolar range and a selectivity of more than 2,000-fold for KV1.3 over KV1.1. This remarkable potency and selectivity for KV1.3, which is significantly up-regulated in activated effector memory cells in humans, suggest that these analogues represent valuable leads in the development of therapeutics for autoimmune diseases.

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2013 – Differential Effects of the β‐Adrenoceptor Blockers Carvedilol and Metoprolol on SQT1‐ and SQT2‐Mutant Channels
Port-a-Patch Publication in Journal of Cardiovascular Electrophysiology (2013) Authors: Bodi I., Franke G., Pantulu N.D., Wu K., Perez-Feliz S., Bode C., Zehender M., Zur Hausen A., Brunner M., Odening K.

Background: N588K-KCNH2 and V307L-KCNQ1 mutations lead to a gain-of-function of IKr and IKs thus causing short-QT syndromes (SQT1, SQT2). Combined pharmacotherapies using K+-channel-blockers and β-blockers are effective in SQTS. Since β-blockers can block IKr and IKs, we aimed at determining carvedilol's and metoprolol's electrophysiological effects on N588K-KCNH2 and V307L-KCNQ1 channels. Methods Wild-type (WT)-KCNH2, WT-KCNQ1 and mutant N588K-KCNH2 and V307L-KCNQ1 channels were expressed in CHO-K1 or HEK-293T cells and IKs and IKr were recorded at baseline and during β-blocker exposure. Results Carvedilol (10 μM) reduced IKs tail in WT- and V307L-KCNQ1 by 36.5 ± 5% and 18.6 ± 9% (P 0.05). IC50 values were 16.3 μM (WT) and 46.1 μM (V307L), indicating a 2.8-fold decrease in carvedilol's IKs-blocking potency in V307L-KCNQ1. Carvedilol's (1 μM) inhibition of the IKr tail was attenuated in N588K-KCNH2 (4.5 ± 3% vs 50.3 ± 4%, WT, P 0.001) with IC50 values of 2.8 μM (WT) and 25.4 μM (N588K). Carvedilol's IKr end-pulse inhibition, however, was increased in N588K-KCNH2 (10 μM, 60.7 ± 6% vs 36.5 ± 5%, WT, P 0.01).Metoprolol (100 μM) reduced IKr end-pulse by 0.23 ± 3% (WT) and 74.1 ± 7% (N588K, P 0.05), IKr tail by 32.9 ± 10% (WT) and 68.8 ± 7% (N588K, P 0.05), and reduced IKs end-pulse by 18.3 ± 5% (WT) and 57.1 ± 11% (V307L, P 0.05) and IKs tail by 3.3 ± 1% (WT) and 45.1 ± 13 % (V307L, P 0.05), indicating an increased sensitivity to metoprolol in SQT mutated channels. Conclusions N588K-KCNH2 and V307L-KCNQ1 mutations decrease carvedilol's inhibition of the IKs or IKr tail but increase carvedilol's IKr end-pulse inhibition and metoprolol's inhibition of tail and end-pulse currents. These different effects on SQT1 and SQT2 mutated channels should be considered when using β-blocker therapy in SQTS patients.

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2013 – Functional significance of thermosensitive transient receptor potential melastatin channel 8 (TRPM8) expression in immortalized human corneal endothelial cells
Port-a-Patch Publication in Experimental Eye Research (2013) Authors: Mergler S., Mertens C., Valtink M., Reinach P.S., Castelo Székely V., Slavi N., Abdelmessih S., Türker E., Fels G., Pleyer U.

Human corneal endothelial cells (HCEC) maintain appropriate tissue hydration and transparency by eliciting net ion transport coupled to fluid egress from the stroma into the anterior chamber. Such activity offsets tissue swelling caused by stromal imbibition of fluid. As corneal endothelial (HCE) transport function is modulated by temperature changes, we probed for thermosensitive transient receptor potential melastatin 8 (TRPM8) functional activity in immortalized human corneal endothelial cells (HCEC-12) and freshly isolated human corneal endothelial cells (HCEC) as a control. This channel is either activated upon lowering to 28 °C or by menthol, eucalyptol and icilin. RT-PCR and quantitative real-time PCR (qPCR) verified TRPM8 gene expression. Ca2+ transients induced by either menthol (500 μmol/l), eucalyptol (3 mmol/l), or icilin (2–60 μmol/l) were identified using cell fluorescence imaging. The TRP channel blocker lanthanum III chloride (La3+, 100 μmol/l) as well as the TRPM8 blockers BCTC (10 μmol/l) and capsazepine (CPZ, 10 μmol/l) suppressed icilin-induced Ca2+ increases. In and outward currents induced by application of menthol (500 μmol/l) or icilin (50 μmol/l) were detected using the planar patch-clamp technique. A thermal transition from room temperature to ≈ 18 °C led to Ca2+ increases that were inhibited by a TRPM8 blocker BCTC (10 μmol/l). Other thermosensitive TRP pathways whose heterogeneous Ca2+ response patterns are suggestive of other Ca2+ handling pathways were also detected upon strong cooling (≈10 °C). Taken together, functional TRPM8 expression in HCEC-12 and freshly dissociated HCEC suggests that HCE function can adapt to thermal variations through activation of this channel subtype.

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2013 – A Potent and Selective Peptide Blocker of the KV1.3 Channel: Prediction from Free-Energy Simulations and Experimental Confirmation
Port-a-Patch Publication in PLoS ONE (2013) Authors: Rashid M.H., Heinzelmann G., Huq R., Tajhya R.B., Chang S.C., Chhabra S., Pennington M.W., Beeton C., Norton R.S., Kuyucak S.

The voltage-gated potassium channel KV1.3 is a well-established target for treatment of autoimmune diseases. ShK peptide from a sea anemone is one of the most potent blockers of KV1.3 but its application as a therapeutic agent for autoimmune diseases is limited by its lack of selectivity against other KV channels, in particular KV1.1. Accurate models of KV1.x-ShK complexes suggest that specific charge mutations on ShK could considerably enhance its specificity for KV1.3. Here we evaluate the K18A mutation on ShK, and calculate the change in binding free energy associated with this mutation using the path-independent free energy perturbation and thermodynamic integration methods, with a novel implementation that avoids convergence problems. To check the accuracy of the results, the binding free energy differences were also determined from path-dependent potential of mean force calculations. The two methods yield consistent results for the K18A mutation in ShK and predict a 2 kcal/mol gain in KV1.3/KV1.1 selectivity free energy relative to wild-type peptide. Functional assays confirm the predicted selectivity gain for ShK[K18A] and suggest that it will be a valuable lead in the development of therapeutics for autoimmune diseases.

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2013 – Crystal structure and functional mechanism of a human antimicrobial membrane channel
Port-a-Patch Publication in Proceedings of the National Academy of Sciences of the United States of America (2013) Authors: Songa C., Weichbrodt C., Salnikov E.S., Dynowski M.,Forsberg B.O., Bechinger B., Steinem C., de Groot B.L., Zachariae U. and Zeth K.

Multicellular organisms fight bacterial and fungal infections by producing peptide-derived broad-spectrum antibiotics. These host-defense peptides compromise the integrity of microbial cell membranes and thus evade pathways by which bacteria develop rapid antibiotic resistance. Although more than 1,700 host-defense peptides have been identified, the structural and mechanistic basis of their action remains speculative. This impedes the desired rational development of these agents into next-generation antibiotics. We present the X-ray crystal structure as well as solid-state NMR spectroscopy, electrophysiology, and MD simulations of human dermcidin in membranes that reveal the antibiotic mechanism of this major human antimicrobial, found to suppress Staphylococcus aureus growth on the epidermal surface. Dermcidin forms an architecture of high-conductance transmembrane channels, composed of zinc-connected trimers of antiparallel helix pairs. Molecular dynamics simulations elucidate the unusual membrane permeation pathway for ions and show adjustment of the pore to various membranes. Our study unravels the comprehensive mechanism for the membrane-disruptive action of this mammalian host-defense peptide at atomistic level. The results may form a foundation for the structure-based design of peptide antibiotics.

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2012 – Selective modification of the N-Terminal structure of polytheonamide B significantly changes its cytotoxicity and activity as an ion channel
Port-a-Patch Publication in ChemMedChem (2012) Authors: Shinohara N., Itoh H., Matsuoka S., Inoue M.

Chemical point mutation: Polytheonamide B is a naturally occurring polypeptide containing 48 amino acids. It both displays potent cytotoxicity and acts as a monovalent cation channel in vitro. Chemoselective methods to modify the 44th, N-, and C-terminal residues of the natural product have been developed, and evaluation of the resultant derivatives suggests that the intrinsic activities of the peptide can only be altered by switching its N-terminal substitution.

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2012 – Thermo-sensitive transient receptor potential vanilloid channel-1 regulates intracellular calcium and triggers chromogranin A secretion in pancreatic neuroendocrine BON-1 tumor cells
Port-a-Patch Publication in Cellular Signalling (2012) Authors: Mergler S., Skrzypski M., Sassek M., Pietrzak P., Pucci C., Wiedenmann B., Strowski M.Z.

Transient receptor potential channels (TRPs) regulate tumor growth via calcium-dependent mechanisms. The (thermosensitive) capsaicin receptor TRPV1 is overexpressed in numerous highly aggressive cancers. TRPV1 has potent antiproliferative activity and is therefore potentially applicable in targeted therapy of malignancies. Recently, we characterized TRPM8 functions in pancreatic neuroendocrine tumors (NETs), however, the role of TRPV1 is unknown. Here, we studied the expression and the role of TRPV1 in regulating intracellular Ca2+ and chromogranin A (CgA) secretion in pancreatic NET BON-1 cell line and in primary NET cells (prNET). TRPV1 expression was detected by RT-PCR, Western blot and immunofluorescence. Intracellular free Ca2+ ([Ca2+]i) was measured by fura-2; TRPV1 channel currents by the planar patch-clamp technique. Nonselective cation currents were analyzed by a color-coded plot method and CgA secretion by ELISA. Pancreatic BON-1 cells and NETs express TRPV1. Pharmacological blockade of TRPs by La3+ (100 μM) or by ruthenium-red (RuR) or by capsazepine (CPZ) (both at 10 μM) suppressed the capsaicin (CAP)- or heat-stimulated increase of [Ca2+]i in NET cells. CAP (20 μM) also increased nonselective cation channel currents in BON-1 cells. Furthermore, CAP (10 μM) stimulated CgA secretion, which was inhibited by CPZ or by RuR (both 10 μM). La3+ potently reduced both stimulated and the basal CgA secretion. Our study shows for the first time that TRPV1 is expressed in pancreatic NETs. Activation of TRPV1 translates into changes of intracellular Ca2+, a known mechanism triggering the secretion of CgA. The clinical relevance of TRPV1 activation in NETs requires further investigations.

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2012 – Isolation, characterization and total regioselective synthesis of the novel μO-conotoxin MfVIA from Conus magnificus that targets voltage-gated sodium channels
Port-a-Patch Publication in Biochemical Pharmacology (2012) Authors: Vetter I., Dekan Z., Knapp O., Adams D.J., Alewood P.F., Lewis R.J.

The μO-conotoxins are notable for their unique selectivity for NaV1.8 over other sodium channel isoforms, making them attractive drug leads for the treatment of neuropathic pain. We describe the discovery of a novel μO-conotoxin, MfVIA, from the venom of Conus magnificus using high-throughput screening approaches. MfVIA was found to be a hydrophobic 32-residue peptide (amino acid sequence RDCQEKWEYCIVPILGFVYCCPGLICGPFVCV) with highest sequence homology to μO-conotoxin MrVIB. To overcome the synthetic challenges posed by μO-conotoxins due to their hydrophobic nature and difficult folding, we developed a novel regioselective approach for the synthesis of μO-conotoxins. Performing selective oxidative deprotections of the cysteine side-chain protecting groups of the fully protected peptide allowed manipulations in organic solvents with no chromatography required between steps. Using this approach, we obtained correctly folded MfVIA with increased synthetic yields. Biological activity of MfVIA was assessed using membrane potential-sensitive dyes and electrophysiological recording techniques. MfVIA preferentially inhibits NaV1.8 (IC50 95.9 ± 74.3 nM) and NaV1.4 (IC50 81 ± 16 nM), with significantly lower affinity for other NaV subtypes (IC50 431–6203 nM; NaV1.5 > 1.6 ∼ 1.7 ∼ 1.3 ∼ 1.1 ∼ 1.2). This improved approach to μO-conotoxin synthesis will facilitate the optimization of μO-conotoxins as novel analgesic molecules to improve pain management.

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2012 – Regulation of Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) Channel Activity by cCMP
Port-a-Patch Publication in Journal of Biological Chemistry (2012) Authors: Zong X., Krause S., Chen C.C., Krüger J., Gruner C., Cao-Ehlker X., Fenske S., Wahl-Schott C., Biel M.

Activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels is facilitated in vivo by direct binding of the second messenger cAMP. This process plays a fundamental role in the fine-tuning of HCN channel activity and is critical for the modulation of cardiac and neuronal rhythmicity. Here, we identify the pyrimidine cyclic nucleotide cCMP as another regulator of HCN channels. We demonstrate that cCMP shifts the activation curves of two members of the HCN channel family, HCN2 and HCN4, to more depolarized voltages. Moreover, cCMP speeds up activation and slows down deactivation kinetics of these channels. The two other members of the HCN channel family, HCN1 and HCN3, are not sensitive to cCMP. The modulatory effect of cCMP is reversible and requires the presence of a functional cyclic nucleotide-binding domain. We determined an EC50 value of ∼30 μm for cCMP compared with 1 μm for cAMP. Notably, cCMP is a partial agonist of HCN channels, displaying an efficacy of ∼0.6. cCMP increases the frequency of pacemaker potentials from isolated sinoatrial pacemaker cells in the presence of endogenous cAMP concentrations. Electrophysiological recordings indicated that this increase is caused by a depolarizing shift in the activation curve of the native HCN current, which in turn leads to an enhancement of the slope of the diastolic depolarization of sinoatrial node cells. In conclusion, our findings establish cCMP as a gating regulator of HCN channels and indicate that this cyclic nucleotide has to be considered in HCN channel-regulated processes.

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2012 – Insulin-secreting INS-1E cells express functional TRPV1 channels
Port-a-Patch Publication in Islets (2012) Authors: Fågelskiöld A.J., Kannisto K., Boström A., Hadrovic B., Farre C., Eweida M., Wester K., Islam MD.S.

We have studied whether functional TRPV1 channels exist in the INS-1E cells, a cell type used as a model for β-cells, and in primary β-cells from rat and human. The effects of the TRPV1 agonists capsaicin and AM404 on the intracellular free Ca2+ concentration ([Ca2+]i) in the INS-1E cells were studied by fura-2 based microfluorometry. Capsaicin increased [Ca2+]i in a concentration-dependent manner, and the [Ca2+]i increase was dependent on extracellular Ca2+. AM404 also increased [Ca2+]i in the INS-1E cells. Capsazepine, a specific antagonist of TRPV1, completely blocked the capsaicin- and AM404-induced [Ca2+]i increases. Capsaicin did not increase [Ca2+]i in the primary β-cells from rat and human. Whole cell patch clamp configuration was used to record currents across the plasma membrane in the INS-1E cells. Capsaicin elicited inward currents that were inhibited by capsazepine. Western blot analysis detected TRPV1 proteins in the INS-1E cells and the human islets. Immunohistochemistry was used to study the expression of TRPV1, but no TRPV1 protein immunoreactivity was detected in the human islet cells and the human insulinoma cells. We conclude that the INS-1E cells, but not the primary β-cells, express functional TRPV1 channels.

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2012 – Interleukin-1R3 mediates interleukin-1–induced potassium current increase through fast activation of Akt kinase
Port-a-Patch Publication in Proceedings of the National Academy of Sciences of the Unites States of America (2012) Authors: Qian J., Zhu L., Li Q., Belevych N., Chen Q., Zhao F., Herness S., Quan N.

Inflammatory cytokine interleukin-1 (IL-1) performs multiple functions in the central nervous system. The type 1 IL-1 receptor (IL-1R1) and the IL-1 receptor accessory protein (IL-1RAcP) form a functional IL-1 receptor complex that is thought to mediate most, if not all, IL-1–induced effects. Several recent studies, however, suggest the existence of a heretofore-unidentified receptor for IL-1. In this study, we report that the IL-1R1 gene contains an internal promoter that drives the transcription of a shortened IL-1R1 mRNA. This mRNA is the template for a unique IL-1R protein that is identical to IL-1R1 at the C terminus, but with a shorter extracellular domain at the N terminus. We have termed this molecule IL-1R3. The mRNA and protein for IL-1R3 are expressed in normal and two strains of commercially available IL-1R1 knockout mice. Western blot analysis shows IL-1R3 is preferentially expressed in neural tissues. Furthermore, IL-1β binds specifically to IL-1R3 when it is complexed with the newly discovered alternative IL-1 receptor accessory protein, IL-1RAcPb. Stimulation of neurons expressing both IL-1R3 and IL-1RAcPb with IL-1β causes fast activation of the Akt kinase, which leads to an increase in voltage-gated potassium current. These results demonstrate that IL-1R3/IL-1RAcPb complex mediates a unique subset of IL-1 activity that accounts for many previously unexplained IL-1 effects in the central nervous system.

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2012 – DNA unzipping and protein unfolding using nanopores
Port-a-Patch Publication in Methods of Molecular Biology (2012) Authors: Merstorf C., Cressiot B., Pastoriza-Gallego M., Oukhaled A.G., Bacri L., Gierak J., Pelta J., Auvray L., Mathé J.

We present here an overview on unfolding of biomolecular structures as DNA double strands or protein folds. After some theoretical considerations giving orders of magnitude about transport timescales through pores, forces involved in unzipping processes … we present our experiments on DNA unzipping or protein unfolding using a nanopore. We point out the difficulties that can be encountered during these experiments, such as the signal analysis problems, noise issues, or experimental limitations of such system.

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2012 – Effects of the Antitussive Drug Cloperastine on Ventricular Repolarization in Halothane-Anesthetized Guinea Pigs
Port-a-Patch Publication in Journal of Pharmacologigal Sciences (2012) Authors: Takahara A., Fujiwara K., Ohtsuki A., Oka T., Namekata I., Tanaka H.

Cloperastine is an antitussive drug, which can be received as an over-the-counter cold medicine. The chemical structure of cloperastine is quite similar to that of the antihistamine drug diphenhydramine, which is reported to inhibit hERG K+ channels and clinically induce long QT syndrome after overdose. To analyze its proarrhythmic potential, we compared effects of cloperastine and diphenhydramine on the hERG K+ channels expressed in HEK293 cells. We further assessed their effects on the halothane-anesthetized guinea-pig heart under the monitoring of monophasic action potential (MAP) of the ventricle. Cloperastine inhibited the hERG K+ currents in a concentration-dependent manner with an IC50 value of 0.027 μM, whose potency was 100 times greater than that of diphenhydramine (IC50; 2.7 μM). In the anesthetized guinea pigs, cloperastine at a therapeutic dose of 1 mg/kg prolonged the QT interval and MAP duration without affecting PR interval or QRS width. Diphenhydramine at a therapeutic dose of 10 mg/kg prolonged the QT interval and MAP duration together with increase in PR interval and QRS width. The present results suggest that cloperastine may be categorized as a QT-prolonging drug that possibly induces arrhythmia at overdoses like diphenhydramine does.

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2012 – Alternative splicing of the N-terminal cytosolic and transmembrane domains of P2X7 controls gating of the ion channel by ADP-ribosylation
Port-a-Patch Publication in PLoS ONE (2012) Authors: Schwarz N., Drouot L., Nicke A., Fliegert R., Boyer O., Guse A.H., Haag F., Adriouch S., Koch-Nolte F.

P2X7 is a homotrimeric ion channel with two transmembrane domains and a large extracellular ATP-binding domain. It plays a key role in the response of immune cells to danger signals released from cells at sites of inflammation. Gating of murine P2X7 can be induced by the soluble ligand ATP, as well as by NAD(+)-dependent ADP-ribosylation of arginine 125, a posttranslational protein modification catalyzed by the toxin-related ecto-enzymes ART2.1 and ART2.2. R125 is located at the edge of the ligand-binding crevice. Recently, an alternative splice variant of P2X7, designated P2X7(k), was discovered that differs from the previously described variant P2X7(a) in the N-terminal 42 amino acid residues composing the first cytosolic domain and most of the Tm1 domain. Here we compare the two splice variants of murine P2X7 with respect to their sensitivities to gating by ADP-ribosylation in transfected HEK cells. Our results show that the P2X7(k) variant is sensitive to activation by ADP-ribosylation whereas the P2X7(a) variant is insensitive, despite higher cell surface expression levels. Interestingly, a single point mutation (R276K) renders the P2X7(a) variant sensitive to activation by ADP-ribosylation. Residue 276 is located at the interface of neighboring subunits approximately halfway between the ADP-ribosylation site and the transmembrane domains. Moreover, we show that naive and regulatory T cells preferentially express the more sensitive P2X7(k) variant, while macrophages preferentially express the P2X7(a) variant. Our results indicate that differential splicing of alternative exons encoding the N-terminal cytosolic and transmembrane domains of P2X7 control the sensitivity of different immune cells to extracellular NAD(+) and ATP.

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2012 – Calcium regulation by thermo- and osmosensing transient receptor potential vanilloid channels (TRPVs) in human conjunctival epithelial cells
Port-a-Patch Publication in Histochemistry and Cell Biology (2012) Authors: Mergler S., Garreis F., Sahlmüller M., Lyras E.-M., Reinach P.S., Dwarakanath A., Paulsen F., Pleyer U.

Transient receptor potential vanilloid (TRPV) channels respond to polymodal stresses to induce pain, inflammation and tissue fibrosis. In this study, we probed for their functional expression in human conjunctival epithelial (HCjE) cells and ex vivo human conjunctivas. Notably, patients suffering from dry eye syndrome experience the same type of symptomology induced by TRPV channel activation in other ocular tissues. TRPV gene and protein expression were determined by RT-PCR and immunohistochemistry in HCjE cells and human conjunctivas (body donors). The planar patch-clamp technique was used to record nonselective cation channel currents. Ca2+ transients were monitored in fura-2 loaded cells. Cultivated HCjE cells and human conjunctiva express TRPV1, TRPV2, and TRPV4 mRNA. TRPV1 and TRPV4 localization was identified in human conjunctiva. Whereas the TRPV1 agonist capsaicin (CAP) (5–20 μM) -induced Ca2+ transients were blocked by capsazepine (CPZ) (10 μM), the TRPV4 activator 4α-PDD (10 μM) -induced Ca2+ increases were reduced by ruthenium-red (RuR) (20 μM). Different heating (40°C or >43°C) led to Ca2+ increases, which were also reduced by RuR. Hypotonic challenges of either 25 or 50% induced Ca2+ transients and nonselective cation channel currents. In conclusion, conjunctiva express TRPV1, TRPV2, and TRPV4 channels which may provide novel drug targets for dry eye therapeutics. Their usage may have fewer side effects than those currently encountered with less selective drugs.

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2011 – Physiology of the Human Corneal Endothelium – New Insights from Electrophysiological Investigations
Port-a-Patch Publication in Klinische Monatsblätter für Augenheilkunde (2011) Authors: Mergler S. and Pleyer U.

Currently, the identification of apoptotic or damaged human corneal endothelial (HCE) cells is limited to a morphological assessment and vital staining. Specific electrophysiological investigations may prospectively help to identify damaged HCE cells at an earlier stage. Besides calcium imaging, the so-called patch-clamp technique is an important test method enabling one to assay the effect of various substances on ion channels and receptors of the cell membrane. First electrophysiological pilot experiments with cultivated and freshly isolated HCE cells have revealed promising results. In this way, the expression of certain transient receptor potential channels (TRPs) could be demonstrated. However, the function of these channels is still not fully elucidated. In humans, TRPs play a crucial role in the sense of taste, pheromones, temperature and pain and are involved in osmolarity. This review summarises the current literature on the electrophysiology of the human corneal endothelium and deduces potential approaches to a sensitive vitality and function test under utilisation of the electrophysiological properties of HCE cells.

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2011 – Thermosensitive transient receptor potential channels in human corneal epithelial cells
Port-a-Patch Publication in Journal of Cellular Physiology (2011) Authors: Mergler S., Garreis F., Sahlmüller M., Reinach P.S., Paulsen F., and Pleyer U.

Thermosensitive transient receptor potential (TRP) proteins such as TRPV1–TRPV4 are all heat-activated non-selective cation channels that are modestly permeable to Ca2+. TRPV1, TRPV3, and TRPV4 functional expression were previously identified in human corneal epithelial cells (HCEC). However, the membrane currents were not described underlying their activation by either selective agonists or thermal variation. This study characterized the membrane currents and [Ca 2+]i transients induced by thermal and agonist TRPV1 and 4 stimulation. TRPV1 and 4 expressions were confirmed by RT-PCR and TRPV2 transcripts were also detected. In fura2-loaded HCEC, a TRPV1–3 selective agonist, 100 µM 2-aminoethoxydiphenyl borate (2-APB), induced intracellular Ca2+ transients and an increase in non-selective cation outward currents that were suppressed by ruthenium-red (RuR) (10–20 µM), a non-selective TRPV channel blocker. These changes were also elicited by rises in ambient temperature from 25 to over 40°C. RuR (5 µM) and a selective TRPV1 channel blocker capsazepine CPZ (10 µM) or another related blocker, lanthanum chloride (La3+) (100 µM) suppressed these temperature-induced Ca2+ increases. Planar patch-clamp technique was used to characterize the currents underlying Ca2+ transients. Increasing the temperature to over 40°C induced reversible rises in non-selective cation currents. Moreover, a hypotonic challenge (25%) increased non-selective cation currents confirming TRPV4 activity. We conclude that HCEC possess in addition to thermo-sensitive TRPV3 activity TRPV1, TRPV2, and TRPV4 activity. Their activation confers temperature sensitivity at the ocular surface, which may protect the cornea against such stress.

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2011 – Chemical and biological characterization of four new linear cationic a-helical peptides from the venoms of two solitary eumenine wasps
Port-a-Patch Publication in Toxicon (2011) Authors: Rangel M., dos Santos Cabrera M.P., Kazuma K., Ando K., Wang X., Kato M., Nihei K., HirataI.Y., Cross T.J., Garcia .N., Faquim-Mauro E.L., Franzolin M.R., Fuchino H., Mori-Yasumoto K., SekitaS., Kadowaki M., SatakeM., KonnoK.

Four novel peptides were isolated from the venoms of the solitary eumenine wasps Eumenes rubrofemoratus and Eumenes fraterculus. Their sequences were determined by MALDI-TOF/TOF (matrix assisted laser desorption/ionization time-of-flight mass spectrometry) analysis, Edman degradation and solid-phase synthesis. Two of them, eumenitin-R (LNLKGLIKKVASLLN) and eumenitin-F (LNLKGLFKKVASLLT), are highly homologous to eumenitin, an antimicrobial peptide from a solitary eumenine wasp, whereas the other two, EMP-ER (FDIMGLIKKVAGAL-NH2) and EMP-EF (FDVMGIIKKIAGAL-NH2), are similar to eumenine mastoparan-AF (EMP-AF), a mast cell degranulating peptide from a solitary eumenine wasp. These sequences have the characteristic features of linear cationic cytolytic peptides; rich in hydrophobic and basic amino acids with no disulfide bond, and accordingly, they can be predicted to adopt an amphipathic α-helix secondary structure. In fact, the CD (circular dichroism) spectra of these peptides showed significant α-helical conformation content in the presence of TFE (trifluoroethanol), SDS (sodium dodecylsulfate) and asolectin vesicles. In the biological evaluation, all the peptides exhibited a significant broad-spectrum antimicrobial activity, and moderate mast cell degranulation and leishmanicidal activities, but showed virtually no hemolytic activity.

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2011 – Functional Analysis of Synthetic Substructures of Polytheonamide B: A Transmembrane Channel-Forming Peptide
Port-a-Patch Publication in Angewandte Chemie International Edition (2011) Authors: Matsuoka S., Shinohara N., Takahashi T., Iida M., Inoue M.

Polytheonamide B, the largest nonribosomal linear peptide identified to date, is a transmembrane channel-forming peptide. Nine of its substructures have now been chemically synthesized. The membrane-disrupting and ion-channel-forming sequences as well as the cytotoxicity-enhancing sequence have been identified.

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2011 – Bacterial mechanosensitive channels as a paradigm for mechanosensory transduction
Port-a-Patch Publication in Cellular Physiology and Biochemistry (2011) Authors: Martinac B.

Research on bacterial mechanosensitive (MS) channels has since their discovery been at the forefront of the MS channel field due to extensive studies of the structure and function of MscL and MscS, two of the several different types of MS channels found in bacteria. Just a few years after these two MS channels were cloned their 3D structure was solved by X-ray crystallography. Today, the repertoire of multidisciplinary approaches used in experimental and theoretical studies following the cloning and crystallographic determination of the MscL and MscS structure has expanded by including electronparamagnetic resonance (EPR) and Förster resonance energy transfer (FRET) spectroscopy aided by computational modelling employing molecular dynamics as well as Brownian dynamics simulations, which significantly advanced the understanding of structural determinants of the gating and conduction properties of these two MS channels. These extensive multidisciplinary studies of MscL and MscS have greatly contributed to elucidation of the basic physical principles of MS channel gating by mechanical force. This review summarizes briefly the major experimental and conceptual advancements, which helped in establishing MscL and MscS as a major paradigm of mechanosensory transduction in living cells.

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2011 – Characterization of transient receptor potential vanilloid channel 4 (TRPV4) in human corneal endothelial cells
Port-a-Patch Publication in Experimental Eye Research (2011) Authors: Mergler S., Valtink M., Taetz K., Sahlmüller M., Fels G., Reinach P.S., Engelmann K., Pleyer U.

The transient receptor potential vanilloid 4 (TRPV4) is a Ca2+-and Mg2+ permeable cation channel that might be a cellular osmosensor since it is activated upon hypotonic cell swelling. TRPV4 is also thermosensitive and responds to moderate heat (from 24 to 27 °C) as well as to phorbol esters (4α-PDD) and several endogenous substances including arachidonic acid (AA), the endocannabinoids anandamide and 2-AG, and cytochrome P-450 metabolites of AA, such as epoxyeicosatrienoic acids. The resulting Ca2+ influx occurring in response to swelling induces regulatory volume decrease (RVD) behavior. As regulation of cell volume is essential for corneal endothelial function, we determined whether human corneal endothelial cells have functional TRPV4 channel activity. RT-PCR identified TRPV4 gene expression in the HCEC-12 cell line as well as two clonal daughter cell lines (HCEC-H9C1, HCEC-B4G12). [Ca2+]i transients were monitored in fura-2 loaded cells. Nonselective cation channel currents were recorded in the whole-cell mode of the planar patch-clamp technique. TRPV4 mRNA was found in HCEC-12 and the clonal daughter cell lines. TRPV4 channel agonists (4α-PDD and GSK1016790A; both 5 μmol/l) as well as moderate heat (40 °C) elicited [Ca2+]i transients. Hypotonicity increased [Ca2+]i and nonselective cation channel currents in HCEC-12 cells. There is functional TRPV4 expression in HCEC-12 and in its clonal daughter cell lines based on Ca2+ transients and underlying currents induced by known activators of this channel.

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2010 – Planar Patch Clamp Approach to Characterize Ionic Currents from Intact Lysosomes
Port-a-Patch Publication in Science Signaling (2010) Authors: Schieder M., Rötzer K., Brüggemann A., Biel M., Wahl-Schott C.

Since its launch in the early 1980s, the patch clamp method has been extensively used to study ion channels in the plasma membrane, but its application to the study of intracellular ion channels has been limited. Unlike the plasma membrane, intracellular membranes are usually not stable enough to withstand mechanical manipulation by glass electrodes during seal formation and rupturing of the membrane. To circumvent these problems, we developed a method involving the immobilization of isolated organelles on a solid matrix planar glass chip. This glass chip contains a microstructured hole that supports the formation of gigaseals and subsequent electrophysiological recordings despite the high fragility of intracellular membranes. Here, we report the experimental details of this method using lysosomes, which are the smallest cellular organelles, as a model system. We demonstrate that we can record endogenous ionic currents from wild-type lysosomes, as well as from lysosomes overexpressing ion channels, and expect that this method will provide electrophysiological access to a broad range of intracellular ion channels.

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2010 – TRPV channels mediate temperature-sensing in human corneal endothelial cells
Port-a-Patch Publication in Experimental Eye Research (2010) Authors: Mergler S., Valtink M., Coulson-Thomas V.J., Lindemann D., Reinach P.S., Engelmann K., Pleyer U.

The physiology and transparency of the cornea are dependent on corneal endothelial function. The role of temperature sensitive ion channels in maintaining such activity is unknown. This study was undertaken to probe for the functional expression of such pathways in human corneal endothelial cells (HCEC). We used HCEC-12, an immortalized population derived from whole corneal endothelium, and two morphologically distinct clonal cell lines derived from HCEC-12 (HCEC-H9C1, HCEC-B4G12) to probe for gene expression and function of transient receptor potential (TRP) channels of the vanilloid (V) isoform subfamily (i.e. TRPV1–3) in these cell types. Expression of TRPV isotypes 1, 2 and 3 were detected by RT-PCR. Protein expression of TRPV1 in situ was confirmed by immunostaining of corneoscleral remnants after keratoplasty. TRPV1–3 functional activity was evident based on capsaicin-induced Ca2+ transients and induction of these responses through rises in ambient temperature from 25 °C to over 40 °C. The currents underlying Ca2+ transients were characterized with a novel high throughput patch-clamp system. The TRPV1 selective agonist, capsaicin (CAP) (10–20 μM) increased non-selective cation whole-cell currents resulting in calcium increases that were fully blocked by either the TRPV1 antagonist capsazepine (CPZ) or removal of extracellular calcium. Similarly, heating from room temperature to over 40 °C increased the same currents resulting in calcium increases that were significantly reduced by the TRP channel blockers lanthanum chloride (La3+) (100 μM) and ruthenium-red (RuR) (10 μM), respectively. Moreover, application of the TRPV channel opener 2-aminoethoxydiphenyl borate (2-APB) (400 μM) led to a reversible increase in intracellular Ca2+ indicating putative TRPV1–3 channel activity. Taken together, TRPV activity modulation by temperature underlies essential homeostatic mechanisms contributing to the support of corneal endothelial function under different ambient conditions.

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2009 – T-type channel blocking properties and antiabsence activity of two imidazo[1,2-b]pyridazine derivatives structurally related to indomethacin
Port-a-Patch Publication in Neuropharmacology (2009) Authors: Rimoli M.G., Russo E., Cataldi M., Citraro R., Ambrosino P., Melisi D., Curcio A., De Lucia S., Patrignani P., De Sarro G., Abignente E.

It is presently unclear whether the antiseizure effects exerted by NSAIDs are totally dependent on COX inhibition or not. To clarify this point we investigated whether 7-methyl-2-phenylimidazo[1,2-b]pyridazine-3-carboxylic acid (DM1) and 6-methoxy-2-phenylimidazo[1,2-b]pyridazine-3-carboxylic acid (DM2), two imidazo[1,2-b]pyridazines structurally related to indomethacin (IDM) but ineffective in blocking COXs, retain IDM antiabsence activity. When administered by intraperitoneal injection in WAG/Rij rats, a rat strain which spontaneously develops SWDs, both DM1 and DM2 dose-dependently suppressed the occurrence of these seizures. Importantly, these compounds were both more potent in suppressing SWD occurrence than IDM. As T-type channel blockade is considered a mechanism of action common to many antiabsence drugs we explored by whole cell patch clamp electrophysiology in stably transfected HEK-293 the effect of DM1 and DM2 on CaV3.1 channels, the T-type channel subtype preferentially expressed in ventrobasal thalamic nuclei. Both these compounds dose-dependently suppressed the currents elicited by membrane depolarization in these cells. A similar T-type blocking effect was also observed when the cells were exposed to IDM. In conclusion, DM1 and DM2 whilst inactive on COXs, are potent antiabsence drugs. This suggests that compounds with structural features typical of NSAIDs may exert antiepileptic activity independently from COX inhibition and possibly by a direct interaction with T-type voltage-dependent Ca2+ channels.

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2010 – Characterization of two pore channel 2 (TPCN2) -mediated Ca2+ currents in isolated lysosomes
Port-a-Patch Publication in The Journal of Biological Chemistry (2010) Authors: Schieder M., Rötzer K., Brüggemann A., Biel M., Wahl-Schott C.A.

Two-pore channels (TPCNs) have been proposed to form lysosomal Ca2+ release channels that are activated by nicotinic acid adenine dinucleotide phosphate. Here, we employ a glass chip-based method to record for the first time nicotinic acid adenine dinucleotide phosphate -dependent currents through a two-pore channel (TPCN2) from intact lysosomes. We show that TPCN2 is a highly selective Ca2+ channel that is regulated by intralysosomal pH. Using site-directed mutagenesis, we identify an amino acid residue in the putative pore region that is crucial for conferring high Ca2+ selectivity. Our glass chip-based method will provide electrophysiological access not only to lysosomal TPCN channels but also to a broad range of other intracellular ion channels.

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2008 – TRPA1 is a major oxidant sensor in murine airway sensory neurons
Port-a-Patch Publication in The Journal of Clinical Investigation (2008) Authors: Bessac B.F., Sivula M., von Hehn C.A., Escalera J., Cohn L., Jordt S.E.

Sensory neurons in the airways are finely tuned to respond to reactive chemicals threatening airway function and integrity. Nasal trigeminal nerve endings are particularly sensitive to oxidants formed in polluted air and during oxidative stress as well as to chlorine, which is frequently released in industrial and domestic accidents. Oxidant activation of airway neurons induces respiratory depression, nasal obstruction, sneezing, cough, and pain. While normally protective, chemosensory airway reflexes can provoke severe complications in patients affected by inflammatory airway conditions like rhinitis and asthma. Here, we showed that both hypochlorite, the oxidizing mediator of chlorine, and hydrogen peroxide, a reactive oxygen species, activated Ca2+ influx and membrane currents in an oxidant-sensitive subpopulation of chemosensory neurons. These responses were absent in neurons from mice lacking TRPA1, an ion channel of the transient receptor potential (TRP) gene family. TRPA1 channels were strongly activated by hypochlorite and hydrogen peroxide in primary sensory neurons and heterologous cells. In tests of respiratory function, Trpa1–/– mice displayed profound deficiencies in hypochlorite- and hydrogen peroxide–induced respiratory depression as well as decreased oxidant-induced pain behavior. Our results indicate that TRPA1 is an oxidant sensor in sensory neurons, initiating neuronal excitation and subsequent physiological responses in vitro and in vivo.

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2009 – Transient receptor potential ankyrin 1 antagonists block the noxious effects of toxic industrial isocyanates and tear gases
Port-a-Patch Publication in FASEB (2009) Authors: Bessac B.F., Sivula M., von Hehn C.A., Caceres A.I., Escalera, J, Jordt S.

The release of methyl isocyanate in Bhopal, India, caused the worst industrial accident in history. Exposures to industrial isocyanates induce lacrimation, pain, airway irritation, and edema. Similar responses are elicited by chemicals used as tear gases. Despite frequent exposures, the biological targets of isocyanates and tear gases in vivo have not been identified, precluding the development of effective countermeasures. We use Ca2+ imaging and electrophysiology to show that the noxious effects of isocyanates and those of all major tear gas agents are caused by activation of Ca2+ influx and membrane currents in mustard oil-sensitive sensory neurons. These responses are mediated by transient receptor potential ankyrin 1 (TRPA1), an ion channel serving as a detector for reactive chemicals. In mice, genetic ablation or pharmacological inhibition of TRPA1 dramatically reduces isocyanate- and tear gas-induced nocifensive behavior after both ocular and cutaneous exposures. We conclude that isocyanates and tear gas agents target the same neuronal receptor, TRPA1. Treatment with TRPA1 antagonists may prevent and alleviate chemical irritation of the eyes, skin, and airways and reduce the adverse health effects of exposures to a wide range of toxic noxious chemicals.

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2008 – Planar patch-clamp force microscopy on living cells
Port-a-Patch Publication in Ultramicroscopy (2008) Authors: Pamir E., George M., Fertig N., Benoit M.

Here we report a new combination of the patch-clamp technique with the atomic force microscope (AFM). A planar patch-clamp chip microstructured from borosilicate glass was used as a support for mechanical probing of living cells. The setup not only allows for immobilizing even a non-adherent cell for measurements of its mechanical properties, but also for simultaneously measuring the electrophysiological properties of a single cell. As a proof of principle experiment we measured the voltage-induced membrane movement of HEK293 and Jurkat cells in the whole-cell voltage clamp configuration. The results of these measurements are in good agreement with previous studies. By using the planar patch-clamp chip for immobilization, the AFM not only can image non-adhering cells, but also gets easily access to an electrophysiologically controlled cellular probe at low vibrational noise.

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2008 – Synthesis and biological evaluation of chalcones as inhibitors of the voltage-gated potassium channel KV1.3
Port-a-Patch Publication in Bioorganic & Medicinal Chemistry Letters (2008) Authors: Cianci J., Baell J.B., Flynn B.L., Gable R.W., Mould J.A., Paul D., Harvey A.J.

Chalcone derivatives of the natural product khellinone were synthesised and screened for bioactivity against the voltage-gated potassium channel KV1.3. X-ray crystallography was employed to investigate relationships between the structure and function of a selection of the reported chalcones.

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2008 – High-throughput electrophysiology: an emerging paradigm for ion-channel screening and physiology
Port-a-Patch Publication in Nature Reviews Drug Discovery (2008) Authors: Dunlop J., Bowlby M., Peri R., Vasilyev D., Arias R.

Ion channels represent highly attractive targets for drug discovery and are implicated in a diverse range of disorders, in particular in the central nervous and cardiovascular systems. Moreover, assessment of cardiac ion-channel activity of new chemical entities is now an integral component of drug discovery programmes to assess potential for cardiovascular side effects. Despite their attractiveness as drug discovery targets ion channels remain an under-exploited target class, which is in large part due to the labour-intensive and low-throughput nature of patch-clamp electrophysiology. This Review provides an update on the current state-of-the-art for the various automated electrophysiology platforms that are now available and critically evaluates their impact in terms of ion-channel screening, lead optimization and the assessment of cardiac ion-channel safety liability.

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2008 – Planar patch clamp: Advances in electrophysiology
Port-a-Patch book chapter in "Potassium Channels" (2008) Authors: Brüggemann A., Farre C., Haarmann C., Haythornthwaite A., Kreir M., Stoelzle S., George M., Fertig N.

Ion channels have gained increased interest as therapeutic targets over recent years, since a growing number of human and animal diseases have been attributed to defects in ion channel function. Potassium channels are the largest and most diverse family of ion channels. Pharmaceutical agents such as Glibenclamide, an inhibitor of KATP channel activity which promotes insulin release, have been successfully sold on the market for many years. So far, only a small group of the known ion channels have been addressed as potential drug targets. The functional testing of drugs on these ion channels has always been the bottleneck in the development of these types of pharmaceutical compounds. New generations of automated patch clamp screening platforms allow a higher throughput for drug testing and widen this bottleneck. Due to their planar chip design not only is a higher throughput achieved, but new applications have also become possible. One of the advantages of planar patch clamp is the possibility of perfusing the intracellular side of the membrane during a patch clamp experiment in the whole-cell configuration. Furthermore, the extracellular membrane remains accessible for compound application during the experiment. Internal perfusion can be used not only for patch clamp experiments with cell membranes, but also for those with artificial lipid bilayers. In this chapter we describe how internal perfusion can be applied to potassium channels expressed in Jurkat cells, and to Gramicidin channels reconstituted in a lipid bilayer.

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2005 – The Port-a-Patch: The smallest patch clamp set up for high quality electrophysiology
Port-a-Patch Publication in HEKA Impulse (2005) Authors: Brüggemann A., George M., Klau M., Beckler M., Steindl J., Behrends J., Fertig N.

In evaluating ion channel function, patch clamping, provides the highest information content but every electrophysiologist knows the draw back of the size and complexity of a patch clamp rig. We present here patch clamp recordings in the whole cell con- figuration performed with planar patch clamp chips, which are micro- structured from borosilicate glass substrate. The chips are used in the Port-a-Patch, a simplified and miniaturized patch clamp setup that enables automated patch clamp experiments on a single cell. The PatchMaker software performs the experiment by executing user-determined protocols for cell positioning and in communication with the PULSE software also protocols for electrical stimulation and current readout. In various electro- physiological experiments, the high quality of recordings and the versatility of the perfusion of the recorded cells are demonstrated.

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2007 – Electrophysiological assessment of HERG blockade: Comparative study using automated and conventional patch clamp systems
Port-a-Patch Publication in Toxicological Letters (2007) Authors: Yook Y., Kim J., Cho H., Moon H., Kwak B.
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2004 – Ion channel drug discovery and research: The automated Nano-Patch-Clamp technology
Port-a-Patch Publication in Current Drug Discovery Technologies (2004) Authors: Brüggemann A., George M., Klau M., Beckler M., Steindl J., Behrends J.C., Fertig N.

Unlike the genomics revolution, which was largely enabled by a single technological advance (high throughput sequencing), rapid advancement in proteomics will require a broader effort to increase the throughput of a number of key tools for functional analysis of different types of proteins. In the case of ion channels - a class of (membrane) proteins of great physiological importance and potential as drug targets- the lack of adequate assay technologies is felt particularly strongly. The available, indirect, high throughput screening methodsfor ion channels clearly generate insufficient information. The best technology to study ion channel function and screen for compound interaction is the patch clamp technique, but patch clamping suffers from low throughput, which is not acceptable for drug screening. A first step towards a solution is presented here. The nano patch clamp technology, which is based on a planar, microstructured glass chip, enables automatic whole cell patch clamp measurements. The Port-a-Patch is an automated electrophysiology workstation, which uses planar patch clamp chips. This approach enables high quality and high content ion channel and compound evaluation on a one-cell-at-a-time basis. The presented automation of the patch process and its scalability to an array format are the prerequisites for any higher throughput electrophysiology instruments.

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2005 – Cytotoxicity of colloidal CdSe and CdSe/ZnS nanoparticles
Port-a-Patch Publication in Nano Letters (2005) Authors: Kirchner C., Liedl T., Kudera S., Pellegrino T., Munoz J. A., Gaub H.E., Stoelzle S., Fertig N., Parak W.J.

Cytotoxicity of CdSe and CdSe/ZnS nanoparticles has been investigated for different surface modifications such as coating with mercaptopropionic acid, silanization, and polymer coating. For all cases, quantitative values for the onset of cytotoxic effects in serum-free culture media are given. These values are correlated with  microscope images in which the uptake of the particles by the cells has been investigated. Our data suggest that in addition to the release of toxic Cd2+ ions from the particles also their surface chemistry, in particular their stability toward aggregation, plays an important role for cytotoxic effects. Additional patch clamp experiments investigate effects of the particles on currents through ion channels.

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2003 – Simultaneous optical and electrical recording of single gramicidin channels
Port-a-Patch Publication in Biophysical Journal (2003) Authors: Borisenko V., Lougheed L., Hesse J., Fuereder-Kitzmueller E., Fertig N., Behrends, J.C., Woolley G.A., Schuetz G.J.

We report here an approach for simultaneous fluorescence imaging and electrical recording of single ion channels in planar bilayer membranes. As a test case,  fluorescently labeled (Cy3 and Cy5) gramicidin derivatives were imaged at the single-molecule level using far-field illumination and cooled CCD camera detection.  Gramicidin monomers were observed to diffuse in the plane of the membrane with a diffusion coefficient of 3.3 x 10-8 cm2s-1. Simultaneous electrical recording detected gramicidin homodimer (Cy3/Cy3, Cy5/Cy5) and heterodimer (Cy3/Cy5) channels. Heterodimer formation was observed optically by the appearance of a fluorescence resonance energy transfer (FRET) signal (irradiation of Cy3, detection of Cy5). The number of FRET signals was significantly smaller than the number of Cy3 signals (Cy3 monomers plus Cy3homodimers) as expected. The number of FRET signals increased with increasing channel activity. In numerous cases the appearance of a FRET signal was observed to correlate with a channel opening event detected electrically. The heterodimers also diffused in the plane of the membrane with a diffusion coefficient of 3.0 x 10-8 cm2s-1. These experiments demonstrate the feasibility of simultaneous optical and electrical detection of structural changes in single ion channels as well as suggesting strategies for improving the reliability of such measurements.

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2012 – Natural and artificial ion channels for biosensing platforms
Port-a-Patch, Patchliner, SyncroPatch 96 ((a predecessor model of SyncroPatch 384PE) and Vesicle Prep Pro Publication in Analytical and Bioanalytical Chemistry (2012) Authors: Steller L., Kreir M., Salzer R.

The single-molecule selectivity and specificity of the binding process together with the expected intrinsic gain factor obtained when utilizing flow through a channel have attracted the attention of analytical chemists for two decades. Sensitive and selective ion channel biosensors for high-throughput screening are having an increasing impact on modern medical care, drug screening, environmental monitoring, food safety, and biowarefare control. Even virus antigens can be detected by ion channel biosensors. The study of ion channels and other transmembrane proteins is expected to lead to the development of new medications and therapies for a wide range of illnesses. From the first attempts to use membrane proteins as the receptive part of a sensor, ion channels have been engineered as chemical sensors. Several other types of peptidic or nonpeptidic channels have been investigated. Various gating mechanisms have been implemented in their pores. Three technical problems had to be solved to achieve practical biosensors based on ion channels: the fabrication of stable lipid bilayer membranes, the incorporation of a receptor into such a structure, and the marriage of the modified membrane to a transducer. The current status of these three areas of research, together with typical applications of ion-channel biosensors, are discussed in this review.

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2019 – Role of High‐Throughput Electrophysiology in Drug Discovery
SyncroPatch 384PE (a predecessor model of the SyncroPatch 384i), Patchliner and Port-a-Patch review article in Current Protocols in Pharmacology (2019) Authors: Liu C., Li T., Chen J.

Due to their important physiological functions, ion channels are key therapeutic targets for a variety of disorders. However, electrophysiological assessment of ion channel activity is technically challenging and has been a bottleneck in the discovery of drugs that modulate channel function. To address this issue, automated patch clamp platforms have been developed with improved throughput and broader applications. An overview of the current status of high‐throughput electrophysiology and its applications in drug discovery is provided.

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2020 – Automated Patch Clamp in Drug Discovery: major breakthroughs and innovation in the last decade
SyncroPatch 384PE (a predecessor model of the SyncroPatch 384i instrument), Patchliner and Port-a-Patch Publication in Expert Opinion on Drug Discovery (2020) Authors: Obergrussberger A., Friis S., Brüggemann A., Fertig N.

Patch-clamp electrophysiology remains an important technique in studying ion channels; indeed, it is still considered the gold standard since it was first described by Neher and Sakmann in the 1970s [1]. Ion channels are integral membrane proteins which allow ion current flow across the cell membrane. They are involved in almost all physiological processes, and their malfunction underlies many disease states, making them important pharmacological targets. Conventional patch clamp is a very information-rich technique, but it requires skilled personnel to perform experiments, and typically, only one experiment can be performed at a time. In the late 1990s and early 2000s, the field of ion-channel research was revolutionized by the development of the automated patch-clamp (APC) technique. The most successful approach involved replacing the patch-clamp pipette with a planar substrate (for review, see [2]), making the experiments easier to perform and offering the option for recording multiple cells in parallel. In the last two decades, much has changed in the field of ion-channel drug discovery and APC, with increased throughput and enhanced simplicity. We summarize the main changes in the last decade and attempt to look into the future of what’s to come.

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2011 – State-of-the-art automated patch clamp devices: heat activation, action potentials, and high throughput in ion channel screening
Port-a-Patch, Patchliner and SyncroPatch 96 (a predecessor model of SyncroPatch 384PE) Publication in Frontiers in Pharmacology (2011) Authors: Stoelzle S., Obergrussberger A., Brüggemann A., Haarmann C., George M., Kettenhofen R., Fertig N.

Ion channels are essential in a wide range of cellular functions and their malfunction underlies many disease states making them important targets in drug discovery. The availability of standardized cell lines expressing ion channels of interest lead to the development of diverse automated patch clamp (APC) systems with high-throughput capabilities. These systems are now available for drug screening, but there are limitations in the application range. However, further development of existing devices and introduction of new systems widen the range of possible experiments and increase throughput. The addition of well controlled and fast solution exchange, temperature control and the availability of the current clamp mode are required to analyze standard cell lines and excitable cells such as stem cell-derived cardiomyocytes in a more physiologically relevant environment. Here we describe two systems with different areas of applications that meet the needs of drug discovery researchers and basic researchers alike. The here utilized medium throughput APC device is a planar patch clamp system capable of recording up to eight cells simultaneously. Features such as temperature control and recordings in the current clamp mode are described here. Standard cell lines and excitable cells such as stem cell-derived cardiomyocytes have been used in the voltage clamp and current clamp modes with the view to finding new drug candidates and safety testing methods in a more physiologically relevant environment. The high-throughput system used here is a planar patch clamp screening platform capable of recording from 96 cells in parallel and offers a throughput of 5000 data points per day. Full dose response curves can be acquired from individual cells reducing the cost per data point. The data provided reveals the suitability and relevance of both APC platforms for drug discovery, ion channel research, and safety testing.

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2012 – HTS techniques for patch clamp-based ion channel screening – economy and advances
Port-a-Patch, Patchliner and SyncroPatch 96 (a predecessor model of SyncroPatch 384PE) Publication in Expert Opinion on Drug Discovery (2012) Authors: Farre C. and Fertig N.

Introduction: Ten years ago, the first publication appeared showing patch clamp recordings performed on a planar glass chip instead of using a conventional patch clamp pipette. “Going planar” proved to revolutionize ion channel drug screening as we know it, by allowing high quality measurements of ion channels and their effectors at a higher throughput and at the same time de-skilling the highly laborious technique. Over the years, platforms evolved in response to user requirements regarding experimental features, data handling plus storage, and suitable target diversity. Areas covered: This article gives a snapshot image of patch clamp-based ion channel screening with focus on platforms developed to meet requirements of high-throughput screening environments. The commercially available platforms are described, along with their benefits and drawbacks in ion channel drug screening. Expert opinion: Automated patch clamp (APC) platforms allow faster investigation of a larger number of ion channel active compounds or cell clones than previously possible. Since patch clamp is the only method allowing direct, real-time measurements of ion channel activity, APC holds the promise of picking up high quality leads, where they otherwise would have been overseen using indirect methods. In addition, drug candidate safety profiling can be performed earlier in the drug discovery process, avoiding late-phase compound withdrawal due to safety liability issues, which is highly costly and inefficient.

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2010 – Renaissance of ion channel research and drug discovery by patch clamp automation
Port-a-Patch, Patchliner and SyncroPatch 96 (a predecessor model of SyncroPatch 384PE)  Publication in Future Medical Chemistry (2010) Authors: Farre C. and Fertig N.

Ion channels are highly intriguing biophysical entities that play an incredibly subtle role in the concerted actions in which they are involved, and that also have a crucial impact on inter- and intra-cellular communication. They respond to numerous kinds of stimuli and play a decisive role in the vitality of all living organisms. Ion channels are involved in the function of the cardiovascular and nervous systems and their malfunction underlies numerous diseases and indications. For exactly these reasons, ion channels have for decades been, and are still, the subject of in-depth research into a very broad range of important therapeutic areas. As membrane-bound proteins they are highly ‘druggable’ targets, being readily accessible to small molecules that are capable of fine tuning ion channel function by pharmacological modulation. Approximately 15% of the most successful drugs target ion channels, although ion channels have traditionally been difficult to screen due to a lack of adequate assays. Many of the marketed ion channel drugs were actually not discovered in rational drug-discovery programs, but rather empirically and by serendipity since the available ion channel-screening techniques typically confer a tradeoff between high content and high throughput.

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2016 – Coupling Data Mining and Laboratory Experiments to Discover Drug Interactions Causing QT Prolongation
Patchliner Publication in Journal of the American College of Cardiology (2016) Authors: Lorberbaum T., Sampson K.J., Chang J.B., Iyer V., Woosley R.L., Kass R.S., Tatonetti N.P.

Background:QT interval-prolonging drug-drug interactions (QT-DDIs) may increase the risk of life-threatening arrhythmia. Despite guidelines for testing from regulatory agencies, these interactions are usually discovered after drugs are marketed and may go undiscovered for years.Objectives:Using a combination of adverse event reports, electronic health records (EHR), and laboratory experiments, the goal of this study was to develop a data-driven pipeline for discovering QT-DDIs.Methods:1.8 million adverse event reports were mined for signals indicating a QT-DDI. Using 1.6 million electrocardiogram results from 380,000 patients in our institutional EHR, these putative interactions were either refuted or corroborated. In the laboratory, we used patch-clamp electrophysiology to measure the human ether-à-go-go-related gene (hERG) channel block (the primary mechanism by which drugs prolong the QT interval) to evaluate our top candidate.Results:Both direct and indirect signals in the adverse event reports provided evidence that the combination of ceftriaxone (a cephalosporin antibiotic) and lansoprazole (a proton-pump inhibitor) will prolong the QT interval. In the EHR, we found that patients taking both ceftriaxone and lansoprazole had significantly longer QTc intervals (up to 12 ms in white men) and were 1.4 times more likely to have a QTc interval above 500 ms. In the laboratory, we found that, in combination and at clinically relevant concentrations, these drugs blocked the hERG channel. As a negative control, we evaluated the combination of lansoprazole and cefuroxime (another cephalosporin), which lacked evidence of an interaction in the adverse event reports. We found no significant effect of this pair in either the EHR or in the electrophysiology experiments. Class effect analyses suggested this interaction was specific to lansoprazole combined with ceftriaxone but not with other cephalosporins.Conclusions:Coupling data mining and laboratory experiments is an efficient method for identifying QT-DDIs. Combination therapy of ceftriaxone and lansoprazole is associated with increased risk of acquired long QT syndrome.

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2011 – Cardiac Toxicity
Patchliner and Port-a-Patch book chapter in ADMET for Medicinal Chemists: A Practical Guide (2011) Authors: Kettenhofen R., Schwengberg S.
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2014 – Automated Patch Clamp Analysis of nAChα7 and NaV1.7 Channels
Port-a-Patch and Patchliner Publication in Current Protocols in Pharmacology (2014) Authors: Obergrussberger A., Haarmann C., Rinke I., Becker N., Guinot D., Brueggemann A., Stoelzle-Feix S., George M., Fertig N.

Automated patch clamp devices are now commonly used for studying ion channels. A useful modification of this approach is the replacement of the glass pipet with a thin planar glass layer with a small hole in the middle. Planar patch clamp devices, such as the three described in this unit, are overtaking glass pipets in popularity because they increase throughput, are easier to use, provide for the acquisition of high-quality and information-rich data, and allow for rapid perfusion and temperature control. Covered in this unit are two challenging targets in drug discovery: voltage-gated sodium subtype 1.7 (NaV1.7) and nicotinic acetylcholine α7 receptors (nAChα7R). Provided herein are protocols for recording activation and inactivation kinetics of NaV1.7, and activation and allosteric modulation of nAChα7R.

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2010 – Cor.At Cardiomyocytes: Primary-like Cardiomyocytes for Manual and Automated Electrophysiological Screening
Port-a-Patch and Patchliner Publication in Lonza Resource Notes (2010) Authors: Kettenhofen R., Stölzle S.

Cor.At® Cardiomyocytes are derived from mouse embryonic stem cells (mESC). During differentiation of the mESC, about 5% of all cells develop into cardiomyocytes. Using transgenic mESC with the puromycin resistance cassette under the control of the cardiac α-myosin heavy chain-(MHC) promoter, 99.9% pure Cor.At® Cardiomyocytes can be selected from the large amount of noncardiac myocyte cell population by the application of puromycin. For long-term storage, Cor.At® Cells are deep frozen as single cell suspensions in liquid nitrogen or -150°C deep freezers. Quality control strategies are implemented to guarantee lot-to-lot reproducibility and uniformity of functional properties of Cor.At® Cardiomyocytes for a storage period of at least 12 months. Thawed Cor.At®  Cardiomyocytes readily form spontaneously and synchronously contracting monolayers overnight. Seeded in low density on cover slips, Cor.At® Cardiomyocytes can be applied to manual patch clamp for the recording of action potentials as well as all three typical cardiac ion currents INa, ICa,L and IK (data not shown). Additionally, single cell suspensions of pre-cultured  Cor.At® Cardiomyocytes can be readily analyzed with very high success rates in automated patch clamp systems like the Port-a-Patch® and Patchliner® from Nanion Technologies GmbH, Munich, Germany, as well as in other automated patch clamp systems (data not shown). The uniqueness of both Nanion systems is their capability to record action potentials in the current clamp mode and the possibility to perform the recordings at physiological temperature in addition to the standard measurements of ion currents in the voltage clamp mode.

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2011 – Automated Patch Clamp on mESC-Derived Cardiomyocytes for Cardiotoxicity Prediction
Patchliner and Port-a-Patch Publication in Journal of Biological Chemistry (2011) Authors: Stoelzle S., Haythornthwaite A., Kettenhofen R., Kolossov E., Bohlen H., George M., Brüggemann A., Fertig N.

Cardiovascular side effects are critical in drug development and have frequently led to late-stage project terminations or even drug withdrawal from the market. Physiologically relevant and predictive assays for cardiotoxicity are hence strongly demanded by the pharmaceutical industry. To identify a potential impact of test compounds on ventricular repolarization, typically a variety of ion channels in diverse heterologously expressing cells have to be investigated. Similar to primary cells, in vitro–generated stem cell–derived cardiomyocytes simultaneously express cardiac ion channels. Thus, they more accurately represent the native situation compared with cell lines overexpressing only a single type of ion channel. The aim of this study was to determine if stem cell–derived cardiomyocytes are suited for use in an automated patch clamp system. The authors show recordings of cardiac ion currents as well as action potential recordings in readily available stem cell–derived cardiomyocytes. Besides monitoring inhibitory effects of reference compounds on typical cardiac ion currents, the authors revealed for the first time drug-induced modulation of cardiac action potentials in an automated patch clamp system. The combination of an in vitro cardiac cell model with higher throughput patch clamp screening technology allows for a cost-effective cardiotoxicity prediction in a physiologically relevant cell system.

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2008 – Ion channel screening – automated patch clamp on the rise
Port-a-Patch and Patchliner Publication in Drug Discovery Today (2008)   Authors: Farre C., George M., Brüggemann A., Fertig N.

Ion channel proteins are of major importance for the human physiology and thus highly attractive molecular drug targets. Large-scale ion channel screening of wanted and unwanted drug effects is required, but has been limited by the lack of adequate screening technology, because available methods put a tradeoff between high-throughput and high-information content. The advent of automated patch clamp platforms has revolutionized ion channel screening, enabling investigations from a more functional perspective at a much higher throughput. The current status of automated patch clamp platforms, their strengths and drawbacks as well as future developments are reviewed.

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2009 – Port-a-Patch and Patchliner: High fidelity electrophysiology for secondary screening and safety pharmacology
Port-a-Patch and Patchliner Publication in Combinatorial Chemistry & High Throughput Screening (2009) Authors: Farre C., Haythornthwaite A., Haarmann C., Stoelzle S., Kreir M., George M., Brüggemann A., Fertig N.

Ion channel dysfunction is known to underlie several acute and chronic disorders and, therefore, ion channels have gained increased interest as drug targets. During the past decade, ion channel screening platforms have surfaced that enable high throughput drug screening from a more functional perspective. These two factors taken together have further inspired the development of more refined screening platforms, such as the automated patch clamp platforms described in this article. Approximately four years ago, Nanion introduced its entry level device for automated patch clamping - the Port-a-Patch. With this device, Nanion offers the world’s smallest patch-clamp workstation, whilst greatly simplifying the experimental procedures. This makes the patch clamp technique accessible to researchers and technicians regardless of previous experience in electrophysiology. The same flexibility and high data quality is achieved in a fully automated manner with the Patchliner, Nanion’s higher throughput patch clamp workstation. The system utilizes a robotic liquid handling environment for fully automated application of solutions, cells and compounds. The NPC-16 chips come in a sophisticated, yet simplistic, microfluidic cartridge, which allow for fast and precise perfusion. In this way, full concentration response curves are easily obtained. The Port-a-Patch and Patchliner workstations from Nanion are valuable tools for target validation, secondary screening and safety pharmacology (for example hERG and NaV1.5 safety screening). They are widely used in drug development efforts by biotechnological and pharmaceutical companies, as well as in basic and applied biophysical research within academia.

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2007 – Automated ion channel screening: patch clamping made easy
Port-a-Patch and Patchliner Publication in Expert Opinion Therapeutic Targets (2007) Authors: Farre C., Stoelzle S., Haarman C., George M., Brueggemann A., Fertig N.

Efficient high resolution techniques are required for screening efforts and research targeting ion channels. The conventional patch clamp technique, a high resolution but low efficiency technique, has been established for 25 years. Recent advances have opened up new possibilities for automated patch clamping. This new technology meets the need of drug developers for higher throughput and facilitates new experimental approaches in ion channel research. Specifically, Nanion’s electrophysiology workstations, the Port-a-Patch and the Patchliner, have been successfully introduced as high-quality automated patch clamp platforms for industry as well as academic users. Both platforms give high quality patch clamp recordings, capable of true giga-seals and stable recordings, accessible to the user without the need for years of practical training. They also offer sophisticated experimental possibilities, such as accurate and fast ligand application, temperature control and internal solution exchange. This article describes the chip-based patch clamp technology and its usefulness in ion channel drug screening and academic research.

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2007 – Planar Patch Clamping
Port-a-Patch and Patchliner book chapter in "Patch Clamp Analysis – Advanced Techniques", Series: Neuromethods (2007) Authors: Behrends, J.C., Fertig, N.

The technique of patch clamping can be seen in retrospect as a combination of two separate lines of development that both originated in the 1960s and 1970s. The classical biophysics of the nerve impulse had by then been established in the squid giant axon using a combination of (1) voltage clamping with axial wire electrodes and (2) internal perfusion or dialysis. This combination had given experimenters control of both the electrical and the chemical gradients governing membrane ion flux. The problem of the day was to extend this type of analysis to smaller, noncylindrical, cellular structures (such as neuronal somata) that would not allow insertion of metal wires, let alone tolerate any of the procedures used for internal perfusion or dialysis of squid axons. While intracellular glass microelectrodes afforded intracellular electrical access to most cellular somata, two independent electrodes for current passing and voltage recording, respectively, were initially necessary, until time-sharing systems made single-microelectrode voltage clamping possible. Even then, however, two severe problems remained: (1) spatially nonuniform voltage control (the so-called space-clamp problem), and (2) the lack of control over intracellular ionic composition.

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2006 – Microchip technology for automated and parallel patch clamp recording
Port-a-Patch and Patchliner Publication in Small Journal (2006) Authors: Brüggemann A., Stoelzle S., George M., Behrends J.C., Fertig N.

The patch-clamp technique is the state-of-the-art technology for the study of a large class of membrane proteins called ion channels. Ion channels mediate electrical current flow, have crucial roles in cellular physiology, and are important drug targets. However, patch clamping is a laborious process requiring a skilled experimenter and is, therefore, not compatible with the high throughput needed in drug development. The solution for automated and parallel patch-clamp measurements that is provided by microchip technology is presented here.

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2015 – Antibiotic translocation through porins studied in planar lipid bilayers using parallel platforms
Orbit 16, Port-a-Patch and Vesicle Prep Pro Publication in Analyst (2015) Authors: Weichbrodt C., Bajaj H., Baaken G., Wang J., Guinot S., Kreir M, Behrends J.C., Winterhalter M., Fertig N.

In general, the method of choice to characterize the conductance properties of channel-forming bacterial porins is electrophysiology. Here, the classical method is to reconstitute single porins into planar lipid bilayers to derive functional information from the observed channel conductance. In addition to an estimated pore size, ion selectivity or transport properties in general are of importance. For the latter, measuring the ion current fluctuation can provide some information about the mode of transport of charged molecules penetrating the proteins. For instance, increasing the external voltage modifies the residence time in the channel: charged molecules with the ability to permeate through channels will travel faster whereas non-permeating molecules get pushed to the constriction zone with enhanced residence time. Here, we are interested in the ability of antibiotics to permeate channels and compare different techniques to reveal fast events.

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2023 – A bioengineered probiotic for the oral delivery of a peptide Kv1.3 channel blocker to treat rheumatoid arthritis
Port-a-Patch Publication in PNAS (2023) Authors: Wanga Y., Zhu D., Ortiz-Velez L.C., Perry J.L., Pennington M.W., Hyser J.M., Britton R.A., Beeton C.
Significance
New therapeutics that combine efficacy with limited side effects and can be delivered noninvasively are needed to adequately treat patients with rheumatoid arthritis (RA) and other autoimmune diseases. Kv1.3 channel-expressing CCR7 effector memory T (TEM) lymphocytes are significant players in the pathogenesis of multiple autoimmune diseases, and blocking Kv1.3 reduces disease severity in rat models of RA and patients with plaque psoriasis. However, peptide therapeutics require repeated injections, reducing patient compliance. We used a bioengineered Lactobacillus reuteri as an oral delivery method of a Kv1.3 blocker for immunomodulation in rat models of atopic dermatitis and RA. This study demonstrates a novel approach for the noninvasive delivery of peptide-based therapeutics for the oral treatment of chronic inflammatory diseases.
Abstract
Engineered microbes for the delivery of biologics are a promising avenue for the treatment of various conditions such as chronic inflammatory disorders and metabolic disease. In this study, we developed a genetically engineered probiotic delivery system that delivers a peptide to the intestinal tract with high efficacy. We constructed an inducible system in the probiotic Lactobacillus reuteri to secrete the Kv1.3 potassium blocker ShK-235 (LrS235). We show that LrS235 culture supernatants block Kv1.3 currents and preferentially inhibit human T effector memory (TEM) lymphocyte proliferation in vitro. A single oral gavage of healthy rats with LrS235 resulted in sufficient functional ShK-235 in the circulation to reduce inflammation in a delayed-type hypersensitivity model of atopic dermatitis mediated by TEM cells. Furthermore, the daily oral gavage of LrS235 dramatically reduced clinical signs of disease and joint inflammation in rats with a model of rheumatoid arthritis without eliciting immunogenicity against ShK-235. This work demonstrates the efficacy of using the probiotic L. reuteri as a novel oral delivery platform for the peptide ShK-235 and provides an efficacious strategy to deliver other biologics with great translational potential.

How can we help you?

Contact our specialist Dr. Patrick Mumm (Product Manager of the Port-a-Patch family). Patrick is delighted to help you:

patrick.mumm@nanion.de
or call: +49 89 2190 95-099
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