Automated Patch Clamp Research Articles

Use of automated patch clamp in cardiac safety assessment: Past, present & future perspectives

There is no doubt that automated patch clamp (APC) technology has revolutionized research in biomedical science. High throughput ion channel screening is now an integral part of the development and safety profiling of the majority of new chemical entities currently developed to address unmet medical needs. The increased throughput it provides has significantly improved the ability to overcome the time-consuming, low throughput bottlenecks resulting from the more conventional manual patch clamp method, considered the ‘gold standard’, for studying ion channel function and pharmacology. While systems offering the luxury of automation have only been commercially available for two decades, the road leading to this new technology is long and rich in seminal, hands-on, studies dating back as far as the 18th century. So where does this technology currently stand, and what will it look like in the future? In the current article, we review the scientific history leading to the development of APC systems, examine key drivers in the rapid development of this technology (such as failed ion channel programmes and the issue of drug-induced hERG inhibition and QT interval prolongation), highlight key capabilities and finally provide some perspective on the current and future impact of the technology on cardiac safety assessment and biomedical science.

B-AB13-05 HIGH-THROUGHPUT PATCH CLAMP OF INDEL AND MISSENSE VARIANTS IN SCN5A

Variants in SCN5A are associated with congenital arrhythmias including Brugada Syndrome (BrS; decreased channel function) and type 3 Long QT Syndrome (LQT3; increased channel function). A majority of SCN5A variants are currently classified as variants of uncertain significance (VUS). Thus, clinicians are unable to make genotype-guided medical decisions for carriers of these variants. We aimed to clinically (re)classify and identify the mechanisms underlying SCN5A insertion/deletion (indel) and missense variants reported in cases of BrS and/or LQT3. Candidate BrS/LQT3-associated indel (n=23) and LQT3-associated missense (n=64) variants were selected based on prevalence in reported cases and their low frequency in the population database gnomAD. Suspected-benign variants (n=25) were selected which were present at least 15 times in gnomAD but absent from published cases. We created stable HEK293T lines expressing these variants, validated their SCN5A expression by flow cytometry, and studied them with automated patch clamping. For each variant, 6 parameters were measured, including peak and late current, inactivation time, and recovery from inactivation, in at least 20 cells. 10/23 in-frame indel variants had partial or complete loss of function (LoF, <50% peak current), 6 of which have previously been found in at least 1 case of BrS. 4/23 indel variants had at least one gain of function (GoF) feature, including elevated peak or late current, slower inactivation time and faster recovery from inactivation; all 4 have been found in at least 1 case of LQT3. 22/64 suspected LQT3-associated missense variants had at least one GoF feature. In contrast, only 2/25 suspected benign variants had GoF features. This result indicates that GoF features are readily captured by automated patch-clamp and are specific for LQT3-associated variants. Using the missense and indel data, 33 variants were reclassified from VUS to likely pathogenic. High-throughput patch clamp can identify loss and gain of function variants in SCN5A. In-frame indels are a substantial contributor to SCN5A-mediated arrhythmia risk. Missense variants in SCN5A associated with LQT3 can display multiple electrophysiological defects.

The role of the SARS-CoV-2 envelope protein as a pH-dependent cation channel.

The role of the SARS-CoV-2 envelope protein as a pH-dependent cation channel.

High-Throughput Screening of Transient Receptor Potential Channel 1 Ligands in the Light of the Bioluminescence Resonance Energy Transfer Technique.

Ion channels are attractive drug targets for many therapeutic applications. However, high-throughput screening (HTS) of drug candidates is difficult and remains very expensive. We thus assessed the suitability of the bioluminescence resonance energy transfer (BRET) technique as a new HTS method for ion-channel studies by taking advantage of our recently characterized intra- and intermolecular BRET probes targeting the transient receptor potential vanilloid type 1 (TRPV1) ion channel. These BRET probes monitor conformational changes during TRPV1 gating and subsequent coupling with calmodulin, two molecular events that are intractable using reference techniques such as automated calcium assay (ACA) and automated patch-clamp (APC). We screened the small-sized Prestwick chemical library, encompassing 1200 compounds with high structural diversity, using either intra- and intermolecular BRET probes or ACA. Secondary screening of the detected hits was done using APC. Multiparametric analysis of our results shed light on the capability of calmodulin inhibitors included in the Prestwick library to inhibit TRPV1 activation by capsaicin. BRET was the lead technique for this identification process. Finally, we present data exemplifying the use of intramolecular BRET probes to study other transient receptor potential (TRP) channels and non-TRPs ion channels. Knowing the ease of use of BRET biosensors and the low cost of the BRET technique, these assays may advantageously be included for extending ion-channel drug screening. SIGNIFICANCE STATEMENT: This study screened a chemical library against TRPV1 ion channel using bioluminescence resonance energy transfer (BRET) molecular probes and compared the results with the ones obtained using reference techniques such as automated calcium assay and automated patch-clamp. Multiparametric analysis of our results shed light on the capability of calmodulin antagonists to inhibit chemical activation of TRPV1 and indicates that BRET probes may advantageously be included in ion channel drug screening campaigns.

Use of automated patch clamp in cardiac safety assessment: past, present and future perspectives

There is no doubt that automated patch clamp (APC) technology has revolutionized research in biomedical science. High throughput ion channel screening is now an integral part of the development and safety profiling of the majority of new chemical entities currently developed to address unmet medical needs. The increased throughput it provides has significantly improved the ability to overcome the time-consuming, low throughput bottlenecks resulting from the more conventional manual patch clamp method, considered the ‘gold standard’, for studying ion channel function and pharmacology. While systems offering the luxury of automation have only been commercially available for two decades, the road leading to this new technology is long and rich in seminal, hands-on, studies dating back as far as the 18th century. So where does this technology currently stand, and what will it look like in the future? In the current article, we review the scientific history leading to the development of APC systems, examine key drivers in the rapid development of this technology (such as failed ion channel programmes and the issue of drug-induced hERG inhibition and QT interval prolongation), highlight key capabilities and finally provide some perspective on the current and future impact of the technology on cardiac safety assessment and biomedical science.

Transfection methods for high-throughput cellular assays of voltage-gated calcium and sodium channels involved in pain.

Chemical transfection is broadly used to transiently transfect mammalian cells, although often associated with cellular stress and membrane instability, which imposes challenges for most cellular assays, including high-throughput (HT) assays. In the current study, we compared the effectiveness of calcium phosphate, FuGENE and Lipofectamine 3000 to transiently express two key voltage-gated ion channels critical in pain pathways, CaV2.2 and NaV1.7. The expression and function of these channels were validated using two HT platforms, the Fluorescence Imaging Plate Reader FLIPRTetra and the automated patch clamp QPatch 16X. We found that all transfection methods tested demonstrated similar effectiveness when applied to FLIPRTetra assays. Lipofectamine 3000-mediated transfection produced the largest peak currents for automated patch clamp QPatch assays. However, the FuGENE-mediated transfection was the most effective for QPatch assays as indicated by the superior number of cells displaying GΩ seal formation in whole-cell patch clamp configuration, medium to large peak currents, and higher rates of accomplished assays for both CaV2.2 and NaV1.7 channels. Our findings can facilitate the development of HT automated patch clamp assays for the discovery and characterization of novel analgesics and modulators of pain pathways, as well as assisting studies examining the pharmacology of mutated channels.

Unattended Drug Discovery Screening Workflow in a 384-Well Automated Patch Clamp System

Automated patch clamp (APC) electrophysiology is an established and accepted technique in most safety testing laboratories. Recently published cross-site studies [1] highlight the need for standardized protocols for reliable results, for example, for Nav1.5 recordings. A standardization of protocols should be backed up by a robust screening assay before a screening workflow is implemented in a lab. Challenging state-of-the-art APC instrumentation with full-day unattended runs and probing them for reliable results was the goal of this study. We used a previously validated assay methodology [2] and challenged it with significant HTS deliverables (success rate, number of data points, Z′). Experimental runs were executed and 4-point dose-response curves of 7 reference compounds, amongst them bepridil and verapamil, were recorded on Nav1.5 in CHO cells. Positive and negative controls were also performed to determine false positive and negative rates and Z’ values were calculated to determine reliability of the assay. Rundown of 0.3% DMSO as vehicle control solution was found to be 5.5 ± 0.3% (n = 364 wells) and success rates for completed experiments were > 93% for every plate. In a full working day 224 IC50s derived from 112 full concentration response curves (4 point) with n = 6 replicates were generated. Z’ values of 0.91 ± 0.004 for a 1st peak Nav amplitude and 0.89 ± 0.006 for the 2nd peak identifying state-dependent inhibition prove low variability of the data. Our statistical measures of assay performance over a typical full working day in a HT ion channel screen showed that the 384-well APC system used in our study (SyncroPatch 384i) delivers reproducible and reliable results for the human Nav1.5 channel. [1] Kramer et al. Sci Rep 10 (2020). https://doi.org/10.1038/s41598-020-62344-w [2] Brinkwirth et al., JPTM (2020), https://doi.org/10.1016/j.vascn.2020.106884

Activation and Inhibition of Assay-Ready TRPA1 and TRPV Cells: An Automated Patch Clamp Study

Transient Receptor Potential (TRP) channels are widely distributed throughout the mammalian central and peripheral nervous systems. They can be directly activated by ligands, heat or cold and mechano-stimulation, and are important targets in drug discovery for the treatment of pain, respiratory diseases, cancer, immune disorders and others. Here, we studied the responses of TRPA1, TRPV1, TRPV3 and TRPV4 assay-ready and cultured cells activated using a variety of stimuli on automated patch clamp (APC) systems.

Validation of an ASIC1a Ligand-Gated Assay on an Automated Patch Clamp Platform and its Use for Novel Ligand Screening

There is growing interest in automated patch clamp assays for ligand-gated targets, many of which are expressed throughout the nervous system and underlie complex neurological diseases. The Acid-Sensing Ion Channel (ASIC) family comprises combinations of ASIC1-4 proteins that form cation-selective channels in the central and peripheral nervous system. ASIC1a proteins are involved in synaptic plasticity, and implicated in stroke, ischemia and pain. Small molecules and toxins modulate ASIC1a channels, and fragments can bind to ASIC3 channels (Wolkenberg et al., 2010), so we screened a diverse low molecular weight library for novel ASIC1a modulators. All data was generated on the gigaseal QPatch48 system (Sophion) using coated metal pipetting tips and a CHO cell line stably expressing human ASIC1a. Currents were elicited from a holding potential of −60 mV by rapid application of saline or compound-containing solutions of varied pH. EC50 of pH activation and IC50 of test compound inhibition were determined by cumulative applications to the same cell. A proprietary low MW library was tested at 200 μM (N=2) and active hits were confirmed by cumulative IC50 testing (N=3). We validated the pharmacology of the hASIC1a cell line using toxins (e.g. IC50 of 120 nM for Mambalgin-3), small molecules (Benzamil IC50 of 4.0 μM), and clinical development compounds, which paved the way to screen a proprietary library of low molecular weight compounds in an effort to find chemical building blocks for the design of novel and efficacious ASIC1a modulators. A hit rate of 3.0% was achieved which included several novel scaffolds with low μM potency, which can be used in biophysical binding assays and structure-based drug design to develop ligand-efficient modulators of the human ASIC1a channel.

Heterozygous KCNH2 variant phenotyping using Flp-In HEK293 and high-throughput automated patch clamp electrophysiology.

KCNH2 is one of the 59 medically actionable genes recommended by the American College of Medical Genetics for reporting of incidental findings from clinical genomic sequencing. However, half of the reported KCNH2 variants in the ClinVar database are classified as variants of uncertain significance. In the absence of strong clinical phenotypes, there is a need for functional phenotyping to help decipher the significance of variants identified incidentally. Here, we report detailed methods for assessing the molecular phenotype of any KCNH2 missense variant. The key components of the assay include quick and cost-effective generation of a bi-cistronic vector to co-express Wild-type (WT) and any KCNH2 variant allele, generation of stable Flp-In HEK293 cell lines and high-throughput automated patch clamp electrophysiology analysis of channel function. Stable cell lines take 3–4 weeks to produce and can be generated in bulk, which will then allow up to 30 variants to be phenotyped per week after 48 h of channel expression. This high-throughput functional genomics assay will enable a much more rapid assessment of the extent of loss of function of any KCNH2 variant.

Advancing Ion Channel Research with Automated Patch Clamp (APC) Electrophysiology Platforms.

Since its development on the cusp of the new millennium, automated patch clamp (APC) technology has matured over the last two decades. The increased throughput it afforded promised a new paradigm in ion channel recordings: It offered the potential to overcome the time-consuming, low-throughput bottleneck arising from manual patch clamp (MPC) investigations. This chapter highlights the advances in technology, showing how APC platforms have 'democratised' ion channel recordings, lowering the technical bar whilst substantially raising throughput. It will describe the background of the seminal first-generation and updates on advances in second-generation platforms. Furthermore, the chapter summarises the advances APC has made in ion channel studies, including finding new tool compounds and medicines. New functionality and applications on APC platforms give ion channel researchers flexible tools to study ion channels with high quality and high throughput.

Abstract 15438: Comprehensive Structural, Molecular, and Electrophysiological Maturation of Human Induced Pluripotent Stem Cell Derived Atrial Cardiomyocytes to Serve as a Platform to Model Atrial Fibrillation

Introduction: Increasingly, human induced pluripotent stem cells (hiPSC) faithfully recapitulate human models of arrhythmias. However, enhancing hiPSC-derived atrial cardiomyocyte (aCM) maturity is vital as modeling mature CMs will provide insights into cellular mechanisms of atrial fibrillation (AF) and signaling pathways critical to atrial development Hypothesis: Combinatorial conditioning of hiPSC-aCMs with biochemical cues (T3, IGF-1, dexamethasone; TID), fatty acids (FA; oleic/palmitic acid), and acute electrical stimulation (ES) at increasing intensity over 45 days comprehensively enhances structural, molecular, and electrophysiological (EP) maturity of hiPSC-aCMs Methods: HiPSCs generated from patient specific peripheral blood mononuclear cells were differentiated into aCMs using retinoic acid and glucose starvation. Maturity of atrial iPSC-CMs was enhanced using TID, FA, and acute ES for the final 4 weeks of culture. Structural (immunofluorescence; transmission EM), molecular (qPCR; RNAseq), and EP (patch clamping; multielectrode array; high throughput automated patch clamping) maturity is assessed and compared to untreated hiPSC-aCMs and adult human aCMs harvested from the same patient (optimal maturity) Results: We showed improved hiPSC-aCM structural maturity with TID, FA, and ES ( Fig. 1A ). EP maturity also displayed more hyperpolarized resting membrane potential (RMP; Fig. 1B ), and improved upstroke velocity, action potential duration (APD), and amplitude (not shown). Expression of ion channels, and calcium handling and structural proteins is significantly improved ( Fig. 1C ) Conclusions: Combinatorial conditioning with TID, FA, and ES markedly improved structural, molecular, and EP maturity of hiPSC-aCMs. Our findings will serve as a platform to model AF, elucidate underlying cellular mechanisms, and identify novel therapeutic targets for a personalized, mechanism based approach to treat this common condition

Optogenetics and Optical Tools in Automated Patch Clamping.

Automated patch clamping (APC) has been used for almost two decades to increase the throughput of electrophysiological measurements, especially in preclinical safety screening of drug compounds. Typically, cells are suctioned onto holes in planar surfaces and a stronger subsequent suction allows access to a whole cell configuration for electrical measurement of ion channel activity. The development of optogenetic tools over a wide range of wavelengths (UV to IR) provides powerful tools for improving spatiotemporal control of in vivo and in vitro experiments and is emerging as a powerful means of investigating cell networks (neuronal), single cell transduction, and subcellular pathways.Combining APC and optogenetic tools paves the way for improved investigation and control of cell kinetics and provides the opportunity for collecting robust data for new and exciting applications and therapeutic areas. Here, we present an APC optogenetics capability on the Qube Opto 384 system including experiments on light activated ion channels and photoactivated ligands.

Perforated Whole-Cell Recordings in Automated Patch Clamp Electrophysiology.

The automated patch clamp (APC) technology is used for increasing the data throughput of electrophysiological measurements, especially in safety pharmacology and drug discovery. Typically, electrical access to the cells are obtained using standard whole-cell formation by rupturing the membrane, thereby causing a rapid washout of cytosolic components. In contrast the perforated whole-cell configuration provides electrical access to the cell interior while limiting intracellular wash-out. This method allows for recordings of ion channels that are gated by intracellular modulators (e.g., ATP, cyclic nucleotides, or Ca2+), prevents channel current "run down," and maintains a physiological membrane potential for action potential recordings. Here we present some practical approaches to the use of perforated patch clamp for APC recordings. Our findings from these high-throughput, data-rich measurements (e.g., defining optimized concentrations and practical recommendations for four different perforating agents) can be more broadly applied to perforated patch clamp experiments in general (automated and manual), improving success rates, experimental conditions, and applications.

Functional Characterization of KCNH2 genetic variants, encoding hERG potassium channel, as a clinically-relevant information for type 2 LQTS syndrome

hERG potassium channels are essential for normal cardiac electrical activity. Mutations in the hERG gene (KCNH2) cause long QT syndrome, a disorder that predisposes individuals to life-threatening arrhythmias. More than 1000 mutations in hERG sequence are described in databases and most of them remains to be characterized. The objective of the work is to determine the consequences of the variants identified in LQT2 patients on the expression and function of the hERG channel. An optimized sequence hERG fused to pHluorin tag was synthesized. By amplifying mutated overlapping fragments, and using the Gibson assembly strategy, we constructed hERG-pHluorin plasmids with missense variations. CHO cells were transfected by electroporation with the Maxcyte system or Fugene method, and studied after 40 hours. Confocal images and flow cytometry analysis were used to evaluate the percentage of transfected cells and to quantify the membrane channel expression, through pH sensitive pHluorin tag. Conventional or automated patch clamp were used to evaluate the hERG activity. In the present work, we constructed hERG plasmids carrying 48 variations identified in patients in France. Higher efficiency of transfection was observed with electroporation as compared to the Fugene method. The activity of hERG-pHluorin was validated in manual patch-clamp, the pHluorin tag does not interfere with channel activity. Characterization of several mutations in conventional patch clamp showed difference in current amplitude. Automated patch-clamp data was compared to manual patch-clamp, as an initial validation for high throughput screening of all constructed mutations. High-throughput characterization of KCNH2 genetic variants is relevant to discriminate mutants that affect hERG channel activity from variants with undetectable effects. This information will be indicated in a patient database, as a basis of personalized medicine.

Reliable identification of hERG liability in drug discovery by automated patch clamp

Reliable identification of hERG liability in drug discovery by automated patch clamp

Functional assessment of hNav1.x ion channels using state-dependent protocols on the QPatch HT automated patch clamp system

Functional assessment of hNav1.x ion channels using state-dependent protocols on the QPatch HT automated patch clamp system

Comparison of 5-HT3 binding and functional activity using the IonFlux™ HT automated patch clamp platform

Comparison of 5-HT3 binding and functional activity using the IonFlux™ HT automated patch clamp platform

Automated patch clamp system introducing simulated Ik1 into human iPSC-cardiomycoytes using dynamic clamp

Automated patch clamp system introducing simulated Ik1 into human iPSC-cardiomycoytes using dynamic clamp

Predicting proarrhythmic risk exclusively using automated patch clamp data

Predicting proarrhythmic risk exclusively using automated patch clamp data