The funnel-web spider venom derived single knot peptide Hc3a modulates acid-sensing ion channel 1a desensitisation.

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

Using automated patch clamp electrophysiology platforms in ion channel drug discovery: an industry perspective.

INTRODUCTION: Automated patch clamp (APC) is now well established as a mature technology for ion channel drug discovery in academia, biotech and pharma companies, and in contract research organizations (CRO), for a variety of applications including channelopathy research, compound screening, target validation and cardiac safety testing. AREAS COVERED: Ion channels are an important class of drugged and approved drug targets. The authors present a review of the current state of ion channel drug discovery along with new and exciting developments in ion channel research involving APC. This includes topics such as native and iPSC-derived cells in ion channel drug discovery, channelopathy research, organellar and biologics in ion channel drug discovery. EXPERT OPINION: It is our belief that APC will continue to play a critical role in ion channel drug discovery, not only in 'classical' hit screening, target validation and cardiac safety testing, but extending these applications to include high throughput organellar recordings and optogenetics. In this way, with advancements in APC capabilities and applications, together with high resolution cryo-EM structures, ion channel drug discovery will be re-invigorated, leading to a growing list of ion channel ligands in clinical development.

Development of ASIC1a ligand-gated ion channel drug screening assays across multiple automated patch clamp platforms.

Human acid-sensing ion channels (ASIC) are ligand-gated ionotropic receptors expressed widely in peripheral tissues as well as sensory and central neurons and implicated in detection of inflammation, tissue injury, and hypoxia-induced acidosis. This makes ASIC channels promising targets for drug discovery in oncology, pain and ischemia, and several modulators have progressed into clinical trials. We describe the use of hASIC1a as a case study for the development and validation of low, medium and high throughput automated patch clamp (APC) assays suitable for the screening and mechanistic profiling of new ligands for this important class of ligand-gated ion channel. Initial efforts to expand on previous manual patch work describing an endogenous hASIC1a response in HEK cells were thwarted by low current expression and unusual pharmacology, so subsequent work utilized stable hASIC1a CHO cell lines. Ligand-gated application protocols and screening assays on the Patchliner, QPatch 48, and SyncroPatch 384 were optimized and validated based on pH activation and nM-µM potency of reference antagonists (e.g., Amiloride, Benzamil, Memantine, Mambalgin-3, A-317567, PcTx1). By optimizing single and stacked pipette tip applications available on each APC platform, stable pH-evoked currents during multiple ligand applications enabled cumulative EC50 and IC50 determinations with minimized receptor desensitization. Finally, we successfully demonstrated for the first time on an APC platform the ability to use current clamp to implement the historical technique of input resistance tracking to measure ligand-gated changes in membrane conductance on the Patchliner platform.

There is no F in APC: Using physiological fluoride-free solutions for high throughput automated patch clamp experiments.

Fluoride has been used in the internal recording solution for manual and automated patch clamp experiments for decades because it helps to improve the seal resistance and promotes longer lasting recordings. In manual patch clamp, fluoride has been used to record voltage-gated Na (NaV) channels where seal resistance and access resistance are critical for good voltage control. In automated patch clamp, suction is applied from underneath the patch clamp chip to attract a cell to the hole and obtain a good seal. Since the patch clamp aperture cannot be moved to improve the seal like the patch clamp pipette in manual patch clamp, automated patch clamp manufacturers use internal fluoride to improve the success rate for obtaining GΩ seals. However, internal fluoride can affect voltage-dependence of activation and inactivation, as well as affecting internal second messenger systems and therefore, it is desirable to have the option to perform experiments using physiological, fluoride-free internal solution. We have developed an approach for high throughput fluoride-free recordings on a 384-well based automated patch clamp system with success rates >40% for GΩ seals. We demonstrate this method using hERG expressed in HEK cells, as well as NaV1.5, NaV1.7, and KCa3.1 expressed in CHO cells. We describe the advantages and disadvantages of using fluoride and provide examples of where fluoride can be used, where caution should be exerted and where fluoride-free solutions provide an advantage over fluoride-containing solutions.

The suitability of high throughput automated patch clamp for physiological applications.

Although automated patch clamp (APC) devices have been around for many years and have become an integral part of many aspects of drug discovery, high throughput instruments with gigaohm seal data quality are relatively new. Experiments where a large number of compounds are screened against ion channels are ideally suited to high throughput APC, particularly when the amount of compound available is low. Here we evaluate different APC approaches using a variety of ion channels and screening settings. We have performed a screen of 1920 compounds on GluN1/GluN2A NMDA receptors for negative allosteric modulation using both the SyncroPatch 384 and FLIPR. Additionally, we tested the effect of 36 arthropod venoms on NaV 1.9 using a single 384-well plate on the SyncroPatch 384. As an example for mutant screening, a range of acid-sensing ion channel variants were tested and the success rate increased through fluorescence-activated cell sorting (FACS) prior to APC experiments. Gigaohm seal data quality makes the 384-format accessible to recording of primary and stem cell-derived cells on the SyncroPatch 384. We show recordings in voltage and current clamp modes of stem cell-derived cardiomyocytes. In addition, the option of intracellular solution exchange enabled investigations into the effects of intracellular Ca2+ and cAMP on TRPC5 and HCN2 currents, respectively. Together, these data highlight the broad applicability and versatility of APC platforms and also outlines some limitations of the approach. KEY POINTS: High throughput automated patch clamp (APC) can be used for a variety of applications involving ion channels. Lower false positive rates were achieved using automated patch clamp versus a fluorometric imaging plate reader (FLIPR) in a high throughput compound screen against NMDA receptors.  Genetic variants and mutations can be screened on a single 384-well plate to reduce variability of experimental parameters. Intracellular solution can be perfused to investigate effects of ions and second messenger systems without the need for excised patches. Primary cells and stem cell-derived cells can be used on high throughput APC with reasonable success rates for cell capture, voltage clamp measurements and action potential recordings in current clamp mode.

Reliable identification of cardiac conduction abnormalities in drug discovery using automated patch clamp II: Best practices for Nav1.5 peak current in a high throughput screening environment.

INTRODUCTION: For reliable identification of cardiac safety risk, compounds should be screened for activity on cardiac ion channels in addition to hERG, including NaV1.5 and CaV1.2. We identified different parameters that might affect IC50s of compounds on NaV1.5 peak and late currents recorded using automated patch clamp (APC) and suggest outlines for best practices. METHODS: APC instruments SyncroPatch 384 and Patchliner were used to record NaV1.5 peak and late current. Up to 24 CiPA compounds were used to investigate effects of voltage protocol, holding potential (-80 mV or - 95 mV) and temperature (23 ± 1 °C or 36 ± 1 °C) on IC50 values on hNaV1.5 overexpressed in HEK or CHO cells either as frozen cells or running cultures. RESULTS: The IC50s of 18 compounds on the NaV1.5 peak current recorded on the SyncroPatch 384 using the CiPA step-ramp protocol correlated well with the literature. The use of frozen or cultured cells did not affect IC50s but voltage protocol and holding potential did cause differences in IC50 values. Temperature can affect Vhalf of inactivation and also compound potency. A compound incubation time of 5-6 min was sufficient for most compounds, however slow acting compounds such as terfenadine required longer to reach maximum effect. DISCUSSION: We conclude that holding potential, voltage protocol and temperature can affect IC50 values and recommend the use of the CiPA step-ramp protocol at physiological temperature to record NaV1.5 peak and late currents for cardiac safety. Further recommendations include: a minimum compound incubation time of 5 min, a replicate number of 4 and the use of positive and negative controls for reliable IC50s.

Electrophysiology on Channel-Forming Proteins in Artificial Lipid Bilayers: Next-Generation Instrumentation for Multiple Recordings in Parallel.

Artificial lipid bilayers have been used for several decades to study channel-forming pores and ion channels in membranes. Until recently, the classical two-chamber setups have been primarily used for studying the biophysical properties of pore forming proteins. Within the last 10 years, instruments for automated lipid bilayer measurements have been developed and are now commercially available. This chapter focuses on protein purification and reconstitution of channel-forming proteins into lipid bilayers using a classic setup and on the commercially available systems, the Orbit mini and Orbit 16.

Reliable identification of cardiac liability in drug discovery using automated patch clamp: Benchmarking best practices and calibration standards for improved proarrhythmic assessment.

INTRODUCTION: Screening compounds for activity on the hERG channel using patch clamp is a crucial part of safety testing. Automated patch clamp (APC) is becoming widely accepted as an alternative to manual patch clamp in order to increase throughput whilst maintaining data quality. In order to standardize APC experiments, we have investigated the effects on IC50 values under different conditions using several devices across multiple sites. METHODS: APC instruments SyncroPatch 384i, SyncroPatch 384PE and Patchliner, were used to record hERG expressed in HEK or CHO cells. Up to 27 CiPA compounds were used to investigate effects of voltage protocol, incubation time, labware and time between compound preparation and experiment on IC50 values. RESULTS: All IC50 values of 21 compounds recorded on the SyncroPatch 384PE correlated well with IC50 values from the literature (Kramer et al., 2013) regardless of voltage protocol or labware, when compounds were used immediately after preparation, but potency of astemizole decreased if prepared in Teflon or polypropylene (PP) compound plates 2-3 h prior to experiments. Slow acting compounds such as dofetilide, astemizole, and terfenadine required extended incubation times of at least 6 min to reach steady state and therefore, stable IC50 values. DISCUSSION: Assessing the influence of different experimental conditions on hERG assay reliability, we conclude that either the step-ramp protocol recommended by CiPA or a standard 2-s step-pulse protocol can be used to record hERG; a minimum incubation time of 5 min should be used and although glass, Teflon, PP or polystyrene (PS) compound plates can be used for experiments, caution should be taken if using Teflon, PS or PP vessels as some adsorption can occur if experiments are not performed immediately after preparation. Our recommendations are not limited to the APC devices described in this report, but could also be extended to other APC devices.

Automated Dynamic Clamp for Simulation of IK1 in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes in Real Time Using Patchliner Dynamite8.

Current in vitro assays typically poorly predict cardiac liability as they focus on single ion channels overexpressed in cell lines. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), on the other hand, provide a unique opportunity for drug testing on human cardiomyocytes using high-throughput systems. However, these cells can differ from adult cardiomyocytes in their ion channel expression and, therefore, electrophysiologic properties. One of the main challenges of hiPSC-CMs is the physiologic expression of ion channels such as the inward rectifiers (e.g., Kir2.1-2.3), which conduct the cardiac inward rectifier potassium current (IK1 ). IK1 is one of the primary contributors in maintaining a stable resting membrane potential in cardiac cells, which is essential for excitability. This is only expressed in low levels, or sometimes not at all, in hiPSC-CMs as shown by patch clamp studies. Dynamic clamp is a method of electronically introducing ion currents (e.g., IK1 ) into cells to compensate for the lack of endogenous expression, thus offering the potential to record more stable action potentials in hiPSC-CMs. In this article, we describe the method of using hiPSC-CMs on an automated patch clamp device (Patchliner) coupled with the automated dynamic clamp add-on (Dynamite8 ). We describe protocols for optimized cell handling and harvesting for use on the Patchliner and the steps required for automated execution of experiments and data analysis in dynamic clamp mode. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Recording action potential pharmacology from human induced pluripotent stem cell-derived cardiomyocytes in automated patch clamp combined with dynamic clamp to introduce simulated IK1 and compensate seal resistance Support Protocol 1: Cardiomyocyte plating and culture Support Protocol 2: Cell harvesting and dissociation Alternate Protocol: Recording action potential pharmacology at physiologic temperatures.

An update on the advancing high-throughput screening techniques for patch clamp-based ion channel screens: implications for drug discovery.

INTRODUCTION: Automated patch clamp (APC) devices have become commonplace in many industrial and academic labs. Their ease-of-use and flexibility have ensured that users can perform routine screening experiments and complex kinetic experiments on the same device without the need for months of training and experience. APC devices are being developed to increase throughput and flexibility. Areas covered: Experimental options such as temperature control, internal solution exchange and current clamp have been available on some APC devices for some time, and are being introduced on other devices. A comprehensive review of the literature pertaining to these features for the Patchliner, QPatch and Qube and data for these features for the SyncroPatch 384/768PE, is given. In addition, novel features such as dynamic clamp on the Patchliner and light stimulation of action potentials using channelrhodosin-2 is discussed. Expert opinion: APC devices will continue to play an important role in drug discovery. The instruments will be continually developed to meet the needs of HTS laboratories and for basic research. The use of stem cells and recordings in current clamp mode will increase, as will the development of complex add-ons such as dynamic clamp and optical stimulation on high throughput devices.

A Hybrid Model for Safety Pharmacology on an Automated Patch Clamp Platform: Using Dynamic Clamp to Join iPSC-Derived Cardiomyocytes and Simulations of Ik1 Ion Channels in Real-Time.

An important aspect of the Comprehensive In Vitro Proarrhythmia Assay (CiPA) proposal is the use of human stem cell-derived cardiomyocytes and the confirmation of their predictive power in drug safety assays. The benefits of this cell source are clear; drugs can be tested in vitro on human cardiomyocytes, with patient-specific genotypes if needed, and differentiation efficiencies are generally excellent, resulting in a virtually limitless supply of cardiomyocytes. There are, however, several challenges that will have to be surmounted before successful establishment of hSC-CMs as an all-round predictive model for drug safety assays. An important factor is the relative electrophysiological immaturity of hSC-CMs, which limits arrhythmic responses to unsafe drugs that are pro-arrhythmic in humans. Potentially, immaturity may be improved functionally by creation of hybrid models, in which the dynamic clamp technique joins simulations of lacking cardiac ion channels (e.g., IK1) with hSC-CMs in real-time during patch clamp experiments. This approach has been used successfully in manual patch clamp experiments, but throughput is low. In this study, we combined dynamic clamp with automated patch clamp of iPSC-CMs in current clamp mode, and demonstrate that IK1 conductance can be added to iPSC-CMs on an automated patch clamp platform, resulting in an improved electrophysiological maturity.

Safety pharmacology studies using EFP and impedance.

INTRODUCTION: While extracellular field potential (EFP) recordings using multi-electrode arrays (MEAs) are a well-established technique for monitoring changes in cardiac and neuronal function, impedance is a relatively unexploited technology. The combination of EFP, impedance and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has important implications for safety pharmacology as functional information about contraction and field potentials can be gleaned from human cardiomyocytes in a beating monolayer. The main objectives of this study were to demonstrate, using a range of different compounds, that drug effects on contraction and electrophysiology can be detected using a beating monolayer of hiPSC-CMs on the CardioExcyte 96. METHODS: hiPSC-CMs were grown as a monolayer on NSP-96 plates for the CardioExcyte 96 (Nanion Technologies) and recordings were made in combined EFP and impedance mode at physiological temperature. The effect of the hERG blockers, E4031 and dofetilide, hERG trafficking inhibitor, pentamidine, ß-adrenergic receptor agonist, isoproterenol, and calcium channel blocker, nifedipine, was tested on the EFP and impedance signals. RESULTS: Combined impedance and EFP measurements were made from hiPSC-CMs using the CardioExcyte 96 (Nanion Technologies). E4031 and dofetilide, known to cause arrhythmia and Torsades de Pointes (TdP) in humans, decreased beat rate in impedance and EFP modes. Early afterdepolarization (EAD)-like events, an in vitro marker of TdP, could also be detected using this system. Isoproterenol and nifedipine caused an increase in beat rate. A long-term study (over 30h) of pentamidine, a hERG trafficking inhibitor, showed a concentration and time-dependent effect of pentamidine. DISCUSSION: In the light of the new Comprehensive in Vitro Proarrhythmia Assay (CiPA) initiative to improve guidelines and standardize assays and protocols, the use of EFP and impedance measurements from hiPSCs may become critical in determining the proarrhythmic risk of potential drug candidates. The combination of EFP offering information about cardiac electrophysiology, and impedance, providing information about contractility from the same area of a synchronously beating monolayer of human cardiomyocytes in a 96-well plate format has important implications for future cardiac safety testing.

Automated Patch Clamp Meets High-Throughput Screening: 384 Cells Recorded in Parallel on a Planar Patch Clamp Module.

We have developed an automated patch clamp module for high-throughput ion channel screening, recording from 384 cells simultaneously. The module is incorporated into a laboratory pipetting robot and uses a 384-channel pipettor head for application of cells and compounds. The module contains 384 amplifier channels for fully parallel recordings using a digital amplifier. Success rates for completed experiments (1- to 4-point concentration-response curves for cells satisfying defined quality control parameters) of greater than 85% have been routinely achieved with, for example, HEK, CHO, and RBL cell lines expressing hNaV1.7, hERG, Kir2.1, GABA, or glutamate receptors. Pharmacology experiments are recorded and analyzed using specialized software, and the pharmacology of hNaV1.7 and hERG is described. Fast external solution exchange rates of <50 ms are demonstrated using Kir2.1. Short exposure times are achieved by stacking the external solutions inside the pipette of the robot to minimize exposure of the ligand on the receptor. This ensures that ligand-gated ion channels, for example, GABA and glutamate described in this report, can be reproducibly recorded. Stem cell-derived cardiomyocytes have also been used with success rates of 52% for cells that have a seal resistance of >200 MΩ, and recordings of voltage-gated Na+ and Ca2+ are shown.

Novel screening techniques for ion channel targeting drugs.

Ion channels are integral membrane proteins that regulate the flux of ions across the cell membrane. They are involved in nearly all physiological processes, and malfunction of ion channels has been linked to many diseases. Until recently, high-throughput screening of ion channels was limited to indirect, e.g. fluorescence-based, readout technologies. In the past years, direct label-free biophysical readout technologies by means of electrophysiology have been developed. Planar patch-clamp electrophysiology provides a direct functional label-free readout of ion channel function in medium to high throughput. Further electrophysiology features, including temperature control and higher-throughput instruments, are continually being developed. Electrophysiological screening in a 384-well format has recently become possible. Advances in chip and microfluidic design, as well as in cell preparation and handling, have allowed challenging cell types to be studied by automated patch clamp. Assays measuring action potentials in stem cell-derived cardiomyocytes, relevant for cardiac safety screening, and neuronal cells, as well as a large number of different ion channels, including fast ligand-gated ion channels, have successfully been established by automated patch clamp. Impedance and multi-electrode array measurements are particularly suitable for studying cardiomyocytes and neuronal cells within their physiological network, and to address more complex physiological questions. This article discusses recent advances in electrophysiological technologies available for screening ion channel function and regulation.

Automated Patch Clamp Analysis of nAChα7 and Na(V)1.7 Channels.

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 (Na(V)1.7) and nicotinic acetylcholine α7 receptors (nAChα7R). Provided herein are protocols for recording activation and inactivation kinetics of Na(V)1.7, and activation and allosteric modulation of nAChα7R.

State-of-the-Art Automated Patch Clamp Devices: Heat Activation, Action Potentials, and High Throughput in Ion Channel Screening.

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.