TMEM175 is an AKT-activated lysosomal potassium- and proton-permeable channel that functions to dissipate voltage and pH gradients generated by the V-type H+-ATPase. Loss-of-function (LOF) variants in TMEM175 have been identified as genetic risk factors for Parkinson's disease (PD), highlighting the potential of small-molecule activators as a novel therapeutic strategy for this disease. We developed a high-throughput screening assay using HEK-293 cells stably overexpressing TMEM175 at the cell surface and screened 960 FDA-approved drugs for TMEM175 potentiators. The screen identified 71 activators, including the cysteinyl leukotriene 1 receptor (CysLT1R) antagonists, pranlukast and montelukast. Because HEK-293 cells lack CysLT1R expression, we suspected these drugs may be direct channel activators. Fluorescence and automated patch clamp assays were used to evaluate the dose-dependency of pranlukast, montelukast, zafirlukast, and the known TMEM175 activator, DCPIB. These experiments revealed rank-order potencies and efficacies of DCPIB ~ zafirlukast > montelukast >> pranlukast. DCPIB, zafirlukast, and pranlukast activated TMEM175 independently of AKT activation, whereas the AKT inhibitor MK2206 partially inhibited montelukast-dependent TMEM175 activation. Computer modeling revealed a conformation-dependent solvent-accessible cavity near T119 and H449 that could participate in drug-induced activation, prompting us to examine these sites with mutagenesis. Not only did T119A and H449A mutations decrease apparent potencies of DCPIB, zafirlukast, and montelukast, but the T119A mutation produced a constitutively open channel phenotype. This study adds zafirlukast to the short list of moderately potent TMEM175 activators and identifies a region of the channel that contributes to activation gating.

In vitro functional modeling of genetic variants has revolutionized our understanding of which variants can cause cardiac disorders, providing insights into their molecular underpinnings. This review provides an overview of high-throughput methods used for the functional assessment of variants implicated in inherited cardiac diseases. Advances in gene-editing technology now enable the efficient generation of cells expressing individual genetic variants or libraries of variants for robust functional studies. We discuss innovative assays that can evaluate dozens or hundreds of variants sequentially. For example, the electrophysiological properties of numerous cardiac ion channel variants in genes linked to inherited arrhythmias can be characterized using automated patch clamping. The mechanical properties of cardiomyocytes expressing candidate cardiomyopathy variants can be assessed using techniques such as atomic force microscopy, traction force microscopy, and impedance-based methods. Multiplexed assays of variant effect are an emerging family of techniques that use gene-specific or general assays, combined with next-generation sequencing, to characterize hundreds or thousands of pooled genetic variants. We examine the key advantages and limitations of each method and outline future goals for the field. Innovative in vitro studies of cardiac genetic variants will enhance our understanding of variant-disease relationships and improve diagnosis, screening, and treatment options for these disorders.

Tetrodotoxins (TTXs) pose significant food safety risks due to their potent neurotoxicity. Growing concerns about the impact of these toxins on public health have driven the development of new detection methods, with immunoassays showing strong potential. However, limited knowledge of the cross-reactivity of anti-TTX antibodies with analogues may compromise the reliability of these assays in food safety applications. To address this, cross-reactivity factors (CRFs) for five TTX analogues (i.e., 11-norTTX-6(S)-ol, 11-deoxyTTX, 6,11-dideoxyTTX, 5,11-dideoxyTTX, and 5,6,11-trideoxyTTX) were assessed using a magnetic bead-based immunoassay. In parallel, the antibody's ability to neutralise the toxicity of TTX analogues was evaluated in Neuro-2a cells using automated patch clamp, a single-cell biosensing platform specifically designed for in vitro toxicity assessment and characterisation. Antibody cross-reactivity towards the tested analogues correlated with their relative toxicity, enabling a selective detection of the most hazardous compounds. These findings highlight the dual role of molecular structure in dictating both toxicological potency and immunological recognition, and support the use of immunoassays as effective tools for TTX monitoring in food safety applications.

Brugada Syndrome (BrS) is an inherited arrhythmia disorder that causes an elevated risk of sudden cardiac death. Approximately 20% of patients with BrS have rare variants in SCN5A, which encodes the cardiac sodium channel NaV1.5. Genetic workup of BrS is often complicated by SCN5A variants of uncertain significance (VUS) and/or incomplete penetrance. This study deployed an SCN5A-BrS functional assay at cohort scale to facilitate the implementation of genetic and precision medicine. All 252 missense and in-frame insertion/deletion SCN5A variants from a previously published large cohort of BrS cases (n = 3335 patients) were analysed using a calibrated high-throughput automated patch-clamp (APC) assay. Variant functional Z-scores were assigned evidence levels ranging from BS3_moderate (normal function) to PS3_strong (loss-of-function), as defined by American College of Medical Genetics and Genomics criteria. Functional evidence was combined with population frequency, hotspot, case counts, protein-length changes, and in silico predictions. Odds ratios of BrS case-control enrichment and penetrance for BrS were calculated from variant frequencies in the BrS cohort and in gnomAD. Most variants (146/252) were functionally abnormal (Z ≤ -2), with 100 having severe loss-of-function (Z ≤ -4). Functional evidence enabled the reclassification of 110 of 225 VUS; 104 to likely pathogenic and 6 to likely benign. SCN5A variants with loss-of-function were mainly localized to the transmembrane domains, especially the regions comprising the central pore. SCN5A variant penetrance was proportional to the severity of loss-of-function; variants with Z ≤ -6 had penetrance of 24.5% (15.9%-37.7% CI) and an odds ratio of 501 for BrS. This cohort-scale APC dataset stratifies SCN5A variants found in BrS patients into normal function 'bystander' variants that have a low risk of BrS and loss-of-function variants that have a high risk for BrS. Functional data can be integrated with other criteria to reclassify a substantial fraction of VUS. The dataset helps clarify the SCN5A-BrS relationship and will improve the diagnosis and clinical management of BrS probands and their families.

Introduction: Genetic data are transforming preventative cardiology by identifying individuals at risk of disease before sudden manifestations. Brugada Syndrome (BrS) is an inherited arrhythmia disorder that causes an elevated risk of sudden cardiac death. Approximately 20% of patients with BrS have rare variants in SCN5A , which encodes the cardiac sodium channel Na V 1.5. Genetic workup of BrS, and analysis of secondary findings, is often complicated by SCN5A variants of uncertain significance (VUS) and/or incomplete penetrance. Research Question: What is the additive value of variant functional testing when applied across a cohort of patients undergoing evaluation of potential BrS? Methods: We comprehensively studied 252 missense and in-frame insertion/deletion SCN5A variants from a previously published large cohort of BrS cases (n=3,335 patients) using a calibrated high-throughput automated patch clamp (APC) assay. Variant functional Z -scores were assigned evidence levels ranging from BS3_moderate (normal function) to PS3_strong (loss-of-function), as defined by American College of Medical Genetics and Genomics criteria. Functional evidence was combined with population frequency, hot-spot, case counts, protein length changes, and in silico predictions. Odds ratios of BrS case-control enrichment and penetrance for BrS were calculated from variant frequencies in the BrS cohort and in gnomAD. Results: Most variants (146/252) were functionally abnormal ( Z ≤ -2), with 100 having severe loss-of-function ( Z ≤ -4). Functional evidence enabled the reclassification of 110 of 225 VUS; 104 to likely pathogenic and 6 to likely benign. SCN5A variants with loss-of-function were mainly localized to the transmembrane domains, especially the regions comprising the central pore. SCN5A variant penetrance was proportional to the severity of loss-of-function; variants with Z ≤ -6 had penetrance of 24.5% (15.9 – 37.7% CI) and an odds ratio of 501 for BrS. Conclusions: This cohort-scale APC dataset stratifies SCN5A variants found in BrS patients into normal function “bystander” variants that have a low risk for BrS and loss-of-function variants that have a high risk for BrS. Functional data can be integrated with other criteria to reclassify a substantial fraction of VUS and identifies variants with higher penetrance among a secondary findings population. We anticipate this dataset will improve the diagnosis and clinical management of BrS probands and their families.

This study highlights the complementarity of automated patch-clamp (APC) and manual patch-clamp (MPC) approaches to describe the electrophysiological properties of eighteen Cav3.1 calcium channel variants associated with various neurological conditions. Current density was measured efficiently for all variants in APC experiments, with four variants (p.V184G, p.N1200S, p.S1263A and p.D2242N) showing elevated current densities, compared to wild-type Cav3.1 channel, while six variants (p.M197R, p.V392M, p.F956del, p.I962N, p.I1412T, and p.G1534D) displayed reduced current densities, and were therefore preferentially studied using MPC. The electrophysiological properties were well preserved in APC (e.g., inactivation and deactivation kinetics, steady-state properties), with only the APC-MPC correlation for activation kinetics being less robust. In addition, neuronal modeling, using a deep cerebellar neuron (DCN) environment, revealed that most of the variants localized to the intracellular gate (S5 and S6 segments) could increase DCN spike frequencies. This DCN firing was highly dependent on current density and further pointed to the gain-of-function (GOF) properties of p.A961T and p.M1531V, the two recurrent variants associated with Spinocerebellar Ataxia type-42 with Neurodevelopmental Deficit (SCA42ND). Action-potential (AP) clamp experiments performed using cerebellar and thalamic neuron activities further established the GOF properties of p.A961T and p.M1531V variants. Overall, this study demonstrates that APC is well-suited for high-throughput analysis of Cav3.1 channel variants, and that MPC complements APC for characterizing low-expression variants. Furthermore, in silico modeling and AP clamp experiments reveal that the gain- or loss-of-function properties of the variants are determined by how the Cav3.1 channel decodes the electrophysiological context of a neuron.

The Nav1.7 voltage-gated sodium channel recently emerged as a candidate target for developing analgesic drugs due to its contribution to nociception and association with different pain disorders. Despite extensive research, no selective modulator of Nav1.7 has been put into clinic yet. Meanwhile, some non-selective sodium channel blockers, such as lidocaine and mexiletine (Mex), have been used for treating peripheral neuropathy and Nav1.7-related pain syndromes. The development of selective molecules targeting Nav1.7 channels requires robust preclinical tests integrating efficient electrophysiological screening with proper cell models. Here, we used the automated patch clamp platform, Patchliner (Nanion Technologies), and two different cell lines, the human rhabdomyosarcoma TE671 cells endogenously expressing Nav1.7 and HEK293 cells stably expressing heterologous human Nav1.7, to compare the biophysical properties and state-dependent effect of Mex on Nav1.7 channels. Our results show that in voltage-dependent conditions, Mex similarly blocked sodium currents in both cell lines (IC50: 226 ± 16 and 227 ± 14 μM, at -140 mV; 15 ± 1 and 12 ± 1 μM, at -70 mV, for TE671 and HEK293 cells). Likewise, Mex exerted a comparable tonic (0.3 Hz) and use-dependent (20 Hz) block in TE671 and in HEK293 cells (IC50: 96 ± 4 vs 114 ± 9 μM; IC50: 22 ± 2 and 18 ± 2 μM, at 0.3 Hz and 20 Hz). Moreover, Mex negatively shifted the voltage-dependence of inactivation of Nav1.7 channels in a dose-dependent manner in both cell lines. In conclusion, we confirmed the state-dependency of Mex block on Nav1.7 using automated patch clamp and validated two cell lines expressing Nav1.7 as suitable models for drug screening. This approach can help developing Nav1.7 modulators for the treatment of pain disorders.

The real-time, cost-effective detection of marine toxins like tetrodotoxin (TTX) remains a significant challenge for the scientific community. Traditional methods, including cell-based assays (CBAs), high-performance liquid chromatography (HPLC), and automated patch clamp (APC), are time-consuming, requiring expensive lab-based equipment and highly trained personnel. Enzyme-linked immunosorbent assays (ELISAs), lateral flow assays (LFAs), and immunosensors may not be suitable for toxin analogues. Thus, a simplified approach has been developed in this study, which involves the electrophysiological and electrochemical interrogation of N2a cells grown on ITO-coated glass electrodes by measuring extracellular field potentials (EFP) in conjunction with whole-cell patch clamp recordings and electrochemical impedance spectroscopy (EIS) measurements both before and after incubation with TTX. The ITO substrate proved biocompatible and non-toxic for N2a cells. TTX exposure caused 102% inhibition in EFP values at 300 nM, confirmed by sodium current inhibition of 93% at 300 nM and 22% at 1 nM in patch clamp studies (IC50 = 6.7 nM). EIS measurements indicated concentration-dependent impedance changes in the range of 6–300 nM. This research aims to provide a proof-of-concept for integration of electrophysiological and electrochemical approaches to simplify toxin detection systems.

Assessing variability of hERG data generated using a mock action potential waveform and automated patch clamp platforms – A HESI-coordinated, multi-laboratory comparison of 28 drugs across 3 platforms

Temperature effect on hERG channel pharmacology measured using the qube automated patch clamp system

Piezo1 is a mechanosensitive non-selective cation channel. Genetic alterations of the channel result in a hematologic phenotype named Hereditary Xerocytosis. With Yoda1 and, more recently, Yoda2, compounds to increase the activity of Piezo1 have become available. However, their concrete effect depends on the nano environment of the channel and hence on the cell type. Here we compare the potency of Yoda1 and Yoda2 in red blood cells (RBCs). We investigate the effect of the compounds on direct channel activity using automated patch clamp, as well as the secondary effects of channel activation on signalling molecules and cellular response. In terms of signalling, we investigate the temporal response of the second messenger Ca2+, and in terms of cellular response, the activity of the Gárdos channel. The opening of the Gárdos channel leads to a hyperpolarisation of the RBCs, which is measured by the Macey–Bennekou–Egée (MBE) method. Although the interpretation of the data is not straightforward, we discuss the results in a physiological context and provide recommendations for the use of Yoda1 and Yoda2 to investigate RBCs.

ATP-sensitive potassium (KATP) channels are therapeutic targets for numerous metabolic, cardiovascular, and neurological disorders. Drug development for KATP channels requires electrophysiology assays for detailed compound characterization. Parallel automated patch clamp (APC) techniques offer considerable advantages over low-throughput manual patch clamp electrophysiology. Here, we characterized the functional properties and pharmacological sensitivity of heterologously expressed Kir6.2/SUR1 and Kir6.1/SUR2B using a SyncroPatch 384PE APC instrument. Ruptured-membrane and perforated-patch whole cell recordings in potassium fluoride and fluoride-free assay buffers and electrophysiology chips were evaluated for both subtypes. Effects of internal ATP and ADP, and magnesium (Mg2+) addition were also assessed. Kir6.2/SUR1 currents were constitutively active in all potassium fluoride-based recordings, insensitive to activation by the SUR1 agonist, VU0071063, and variably inhibited by glibenclamide. Success rates, current rundown, and glibenclamide sensitivity were associated with internal buffer composition. Recordings in fluoride-free buffers revealed a minor population of constitutively active Kir6.2/SUR1 currents and a larger population of currents exhibiting low basal activity and activation by VU0071063. Success rate and stability were associated with internal buffer composition. Kir6.1/SUR2B currents, which were most readily assayed in ruptured-membrane and potassium fluoride-based conditions, were stable, activatable with pinacidil, and inhibited by glibenclamide. Our study sheds new light on the behavior of Kir6.2/SUR1 and Kir6.1/SUR2B currents under available APC conditions and represents an important step toward developing truly high-throughput APC techniques for KATP.NEW & NOTEWORTHY Highly parallel automated patch clamp (APC) methods have revolutionized the way electrophysiology is performed in the pharmaceutical and biotechnology industries and increasingly in academic laboratories. Here, we characterized the functional and pharmacological properties of heterologously expressed Kir6.2/SUR1 and Kir6.1/SUR2B using a SyncroPatch 384PE APC instrument. The results of our studies highlight heretofore unappreciated effects of fluoride-base internal solutions on Kir6.2/SUR1 and provide foundational support for developing truly high-throughput electrophysiology methods for both drug targets.

The advent of human induced pluripotent stem cells (hiPSCs) and their differentiation into neurons and brain organoids has revolutionized our ability to model brain disorders in a human context. However, current technologies to assay the electrophysiological properties of human neurons in these models remain limited by throughput, as single-cell manual patch clamp is laborious and resource intensive. Here, we provide methods to perform high-throughput automated patch-clamp (APC) on hiPSC-derived neurons. We describe how to dissociate and perform voltage-clamp recordings on human neurons from three well-established protocols - 2D directed differentiation of cortical neurons, NGN2-induced neurons, and 3D cortical organoids - using the Nanion Syncropatch 384, a commercially available high-throughput APC system. Using this approach, we investigated the biophysical properties of voltage-gated sodium channels (VGSCs) and provide direct comparisons between manual and APC recordings across all three hiPSC-derived model systems. We demonstrate the capability of this automated system for pharmacological analysis of native human VGSC isoforms, which will enable compound screening approaches. Lastly, we provide methods to sort specific cellular populations within these hiPSC models using fluorescence-activated cell sorting (FACS) followed by APC. These methods and results provide a transformative and novel high-throughput technique for quantifying passive and active membrane properties in cell-type specific and/or genetically modified hiPSC-derived neurons.

The epithelial sodium channel (ENaC) is crucial for sodium absorption in several epithelial tissues including lung and kidney. Its involvement in various renal and pulmonary disorders makes ENaC a potential drug target. High-throughput screening using the automated patch-clamp (APC) technique appears to be a promising approach to discover novel ENaC modulators with (patho-)physiological and therapeutic implications. The aim of this methodological study was to establish APC measurements of ENaC-mediated currents. First, we confirmed functional expression of ENaC in a HEK293 cell line stably transfected with human αβγ-ENaC using conventional manual whole-cell patch-clamp recordings. For APC measurements, a standard enzymatic cell-detachment procedure was used to prepare single cell suspensions. This resulted in a high success rate of APC recordings with amiloride inhibitable ENaC currents. Using a γ-inhibitory peptide and the small molecule ENaC activator S3969, we demonstrate that APC recordings could reveal inhibitory as well as stimulatory effects on ENaC. Interestingly, the enzymatic cell-detachment protocol resulted in partial proteolytic ENaC activation. The portion of proteolytically activated channels could be reduced by prolonged incubation of suspended cells in cell culture medium. This recovery protocol enhanced the relative stimulatory effect of chymotrypsin, a prototypical serine protease known to cause proteolytic ENaC activation. Thus, this protocol may be particularly useful for identifying novel ENaC activators mimicking proteolytic channel activation. In conclusion, we have established a high-throughput screening method for the identification of novel ENaC activators and inhibitors using APC.

Using automated patch clamp for high throughput pharmacological characterization of cardiac action potentials

Protocol optimization to improve performance of an automated patch clamp system on assessing hERG activity for challenging compounds

Application of human induced pluripotent stem cell-derived nociceptors on an automated patch clamp system for mechanism of action-based high throughput de-risking of neuronal toxicity

Automated patch clamp for assessing Nav1.4 vs Nav1.5 effects of mexiletine: Implications for anti-myotonic action and safety pharmacology

Development and validation of a qube automated patch clamp hERG assay at physiological temperature

Objectiveγ‐Aminobutyric acid type A (GABAA) receptor positive allosteric modulators (PAMs) that lack α‐subunit selectivity, including benzodiazepines such as diazepam, exhibit antiseizure actions in animal models and in humans. ENX‐101 is a deuterated analog of the ⍺2,3,5‐selective GABAA receptor PAM L‐838,417. The purpose of this study was to characterize the α‐subunit selectivity of ENX‐101 and evaluate its antiseizure potential in preclinical seizure and epilepsy models.MethodsENX‐101 potentiation of GABA chloride current responses in cells expressing recombinant GABAA receptors were evaluated using an automated patch clamp assay. Antiseizure effects of ENX‐101 were examined in the mouse 6 Hz test at 32 and 44 mA, amygdala kindled rats, and Genetic Absence Epilepsy Rat from Strasbourg (GAERS).ResultsENX‐101 displayed partial PAM activity with respect to diazepam at GABAA receptors containing α2, α3, or α5 subunits but did not enhance GABA responses of GABAA receptors containing α1 subunits. ENX‐101 (30, 100, and 300 mg/kg, i.p.) and diazepam protected most animals in the 6 Hz model at 32 mA but was less effective at 44 mA. In amygdala kindled rats, ENX‐101 (1–100 mg/kg, p.o.) reduced behavioral seizure severity and afterdischarge duration in a dose‐dependent manner. ENX‐101 (0.075–100 mg/kg, p.o.) caused dose‐dependent, persistent (>130 min) inhibition of spontaneous spike‐and‐wave discharges (SWDs) in GAERS, whereas diazepam transiently inhibited discharges. ENX‐101 did not cause motor impairment, as measured by performance in the rotarod assay.SignificanceENX‐101 is an α2,α3,α5‐selective GABAA receptor PAM that has high potency and partial efficacy. The drug is highly effective in rodent seizure and epilepsy models. ENX‐101 is most potent in the GAERS model of absence epilepsy, and active in the 6 Hz model and amygdala kindled rats. These results demonstrate that a partial, subtype‐selective GABAA receptor PAM has activity in translationally validated preclinical epilepsy screening models. Clinical evaluation of ENX‐101 as a treatment for focal and generalized epilepsies is warranted.