The occurrence of life-threatening cardiac arrhythmia induced by certain drugs is frequently preceded by an extended duration of cardiac action potentials (AP), which are often associated with minor proarrhythmic fluctuations in the membrane potential. The configuration and temporal progression of the repolarizing segment of the AP plays a crucial role in determining whether arrhythmia will occur or not. Microelectrode arrays (MEA), a well-established tool in research and cardiac safety pharmacology, provide a straightforward method for observing the effects of cardiotoxic compounds through extracellular field potentials (FP). However, despite the significant utility of MEA, the waveform of the FP does not provide a clear representation of the original AP shape. This limitation is due to the extracellular recording principle and the inherent alternating current (AC) filtering that results from it. To overcome this challenge, the IntraCell device (Foresee Biosystems) has been evaluated. This innovative device can repeatedly breach the membrane of cardiomyocytes that are cultivated on top of the MEA electrodes at various cultivation time points. This is achieved through the use of a highly focused nanosecond laser beam. The process of laser poration transforms the electrophysiological signal from FP to intracellular-like APs, referred here as laser-induced APs (liAP), and facilitates the recording of transcellular voltage deflections. Amplitudes increased by 4.1 ± 0.41 (n = 20, range 1.34–8.83) times, analyzed from a randomly picked subset of recordings, resulting in amplitudes between 7 and 22 mV. Although the overall pharmacological sensitivity appeared unaltered when challenged with commonly used tool compounds (e.g. Nifedipine IC50: FP 0.282 ± 0.05 μM, n = 3; LiAP 0.164 ± 0.04 μM n = 3; E4031 prolongation compared to control @ 0.01 μM FP 1.4 ± 0.29 times n = 3; LiAP 4.16 ± 0.55 times n = 3), this access to the intracellular environment allows for a more accurate depiction of the AP shape and a more sensitive and precise classification of proarrhythmic potentials compared to standard MEA recordings. This novel system represents a groundbreaking extension to the existing electrophysiological methods. It allows for a more accurate evaluation of cardiotoxic effects while retaining all the benefits of MEA-based recordings. These benefits include ease of use, the ability to conduct acute and chronic experiments, and the capacity for signal propagation analysis, among others. Therefore, this new device offers a significant advancement in the field of cardiac safety pharmacology and has the potential to greatly enhance our understanding of drug-induced cardiac arrhythmias.

Current treatments against organophosphate poisoning (OPP) do not directly address effects mediated by the overstimulation of nicotinic acetylcholine receptors (nAChR). Non-oxime bispyridinium compounds (BPC) promote acetylcholine esterase-independent recovery of organophosphate-induced paralysis. Here, we test the hypothesis that they act by positive modulatory action on nAChRs. Using two-electrode voltage clamp analysis in combination with mutagenesis and molecular docking analysis, the potency and molecular mode of action of a series of nine BPCs was investigated on human α7 and muscle-type nAChRs expressed in Xenopus laevis oocytes. The investigated BPCs inhibited α7 and/or muscle-type nAChRs with IC50 values in the high nanomolar to high micromolar range. Further analysis of the most potent analogues revealed a noncompetitive, voltage-dependent inhibition. Co-application with the α7-selective positive allosteric modulator PNU120596 and generation of α7/5HT3 receptor chimeras excluded direct interaction with the PNU120596 binding site and binding to the extracellular domain of the α7 nAChR, suggesting that they act as open channel blockers (OCBs). Molecular docking supported by mutagenesis localized the BPC binding area in the outer channel vestibule between the extracellular and transmembrane domains. Analysis of BPC action on other cation-selective channels suggests a rather nonspecific inhibition of pentameric cation channels. BPCs have been shown to ameliorate organophosphate-induced paralysis in vitro and in vivo. Our data support molecular action as OCBs at α7 and muscle-type nAChRs and suggest that their positive physiological effects are more complex than anticipated and require further investigation.

OBJECTIVE:Gain of function variants in the sodium-activated potassium channel KCNT1 have been associated with pediatric epilepsy disorders. Here, we systematically examine a spectrum of KCNT1 variants and establish their impact on channel function in multiple cellular systems.METHODS:KCNT1 variants identified from published reports and genetic screening of pediatric epilepsy patients were expressed in Xenopus oocytes and HEK cell lines. Variant impact on current magnitude, current-voltage relationships, and sodium ion modulation were examined.RESULTS:We determined basic properties of KCNT1 in Xenopus oocyte and HEK systems, including the role of extra- and intracellular sodium in regulating KCNT1 activity. The most common six KCNT1 variants demonstrated strong gain of function (GOF) effects on one or more channel properties. Analysis of 36 total variants identified phenotypic heterogeneity but a strong tendency for pathogenic variants to exert GOF effects on channel properties. By controlling intracellular sodium, we demonstrate that multiple pathogenic KCNT1 variants modulate channel voltage dependence by altering the sensitivity to sodium ions.SIGNIFICANCE:This study represents the largest systematic functional examination of KCNT1 variants to date. We both confirm previously reported GOF channel phenotypes and expand the number of variants with in vitro GOF effects. Our data provide further evidence that novel KCNT1 variants identified in epilepsy patients lead to disease through generalizable GOF mechanisms including increases in current magnitude and/or current-voltage relationships.

Life-threatening drug-induced cardiac arrhythmia is often preceded by prolonged cardiac action potentials (AP), commonly accompanied by small proarrhythmic membrane potential fluctuations. The shape and time course of the repolarizing fraction of the AP can be pivotal for the presence or absence of arrhythmia. Microelectrode arrays (MEA) allow easy access to cardiotoxic compound effects via extracellular field potentials (FP). Although a powerful and well-established tool in research and cardiac safety pharmacology, the FP waveform does not allow to infer the original AP shape due to the extracellular recording principle and the resulting intrinsic alternating current (AC) filtering. A novel device, described here, can repetitively open the membrane of cardiomyocytes cultivated on top of the MEA electrodes at multiple cultivation time points, using a highly focused nanosecond laser beam. The laser poration results in transforming the electrophysiological signal from FP to intracellular-like APs (laser-induced AP, liAP) and enables the recording of transcellular voltage deflections. This intracellular access allows a better description of the AP shape and a better and more sensitive classification of proarrhythmic potentials than regular MEA recordings. This system is a revolutionary extension to the existing electrophysiological methods, permitting accurate evaluation of cardiotoxic effect with all advantages of MEA-based recordings (easy, acute, and chronic experiments, signal propagation analysis, etc.).

Aspidasept (Pep19-2.5) and its derivative Pep19-4LF ("Aspidasept II") are anti-infective and anti-inflammatory synthetic polypeptides currently in development for application against a variety of moderate to severe bacterial infections that could lead to systemic inflammation, as in the case of severe sepsis and septic shock, as well as application to non-systemic diseases in the case of skin and soft tissue infections (SSTI). In the present study, Aspidasept and Aspidasept II and their part structures were analysed with respect to their toxic behavior in different established models against a variety of relevant cells, and in electrophysiological experiments targeting the hERG channel according to ICH S7B. Furthermore, the effects in mouse models of neurobiological behavior and the local lymph node according to OECD test guideline 429 were investigated, as well as a rat model of repeated dose toxicology according to ICH M3. The data provide conclusive information about potential toxic effects, thus specifying a therapeutic window for the application of the peptides. Therefore, these data allow us to define Aspidasept concentrations for their use in clinical studies as parenteral application.

Several neonicotinoids have recently been shown to activate the nicotinic acetylcholine receptor (nAChR) on human neurons. Moreover, imidacloprid (IMI) and other members of this pesticide family form a set of diverse metabolites within crops. Among these, desnitro-imidacloprid (DN-IMI) is of special toxicological interest, as there is evidence (i) for human dietary exposure to this metabolite, (ii) and that DN-IMI is a strong trigger of mammalian nicotinic responses. We set out here to quantify responses of human nAChRs to DN-IMI and an alternative metabolite, IMI-olefin. To evaluate toxicological hazards, these data were then compared to those of IMI and nicotine. Ca2+-imaging experiments on human neurons showed that DN-IMI exhibits an agonistic effect on nAChRs at sub-micromolar concentrations (equipotent with nicotine) while IMI-olefin activated the receptors less potently (in a similar range as IMI). Direct experimental data on the interaction with defined receptor subtypes were obtained by heterologous expression of various human nAChR subtypes in Xenopus laevis oocytes and measurement of the transmembrane currents evoked by exposure to putative ligands. DN-IMI acted on the physiologically important human nAChR subtypes α7, α3β4, and α4β2 (high-sensitivity variant) with similar potency as nicotine. IMI and IMI-olefin were confirmed as nAChR agonists, although with 2–3 orders of magnitude lower potency. Molecular docking studies, using receptor models for the α7 and α4β2 nAChR subtypes supported an activity of DN-IMI similar to that of nicotine. In summary, these data suggest that DN-IMI functionally affects human neurons similar to the well-established neurotoxicant nicotine by triggering α7 and several non-α7 nAChRs.

© 2020 The Author(s) The authors regret that one affiliation address is mistaken in the published paper. Matthew Bridgland-Taylor's affiliation was incorrectly listed as Clinical Pharmacology & Safety Sciences, R&D, AstraZeneca, Cambridge, United Kingdom. The correct affiliation is Clinical Pharmacology & Safety Sciences, BioPharmaceuticals R&D, AstraZeneca, Cambridge, United Kingdom. The authors would like to apologise for any inconvenience caused.

Prediction of drug toxicity on the human nervous system still relies mainly on animal experiments. Here, we developed an alternative system allowing assessment of complex signaling in both individual human neurons and on the network level. The LUHMES cultures used for our approach can be cultured in 384-well plates with high reproducibility. We established here high-throughput quantification of free intracellular Ca2+ concentrations [Ca2+]i as broadly applicable surrogate of neuronal activity and verified the main processes by patch clamp recordings. Initially, we characterized the expression pattern of many neuronal signaling components and selected the purinergic receptors to demonstrate the applicability of the [Ca2+]i signals for quantitative characterization of agonist and antagonist responses on classical ionotropic neurotransmitter receptors. This included receptor sub-typing and the characterization of the anti-parasitic drug suramin as modulator of the cellular response to ATP. To exemplify potential studies on ion channels, we characterized voltage-gated sodium channels and their inhibition by tetrodotoxin, saxitoxin and lidocaine, as well as their opening by the plant alkaloid veratridine and the food-relevant marine biotoxin ciguatoxin. Even broader applicability of [Ca2+]i quantification as an end point was demonstrated by measurements of dopamine transporter activity based on the membrane potential-changing activity of this neurotransmitter carrier. The substrates dopamine or amphetamine triggered [Ca2+]i oscillations that were synchronized over the entire culture dish. We identified compounds that modified these oscillations by interfering with various ion channels. Thus, this new test system allows multiple types of neuronal signaling, within and between cells, to be assessed, quantified and characterized for their potential disturbance.

Introduction: hERG block potency is widely used to calculate a drug's safety margin against its torsadogenic potential. Previous studies are confounded by use of different patch clamp electrophysiology protocols and a lack of statistical quantification of experimental variability. Since the new cardiac safety paradigm being discussed by the International Council for Harmonisation promotes a tighter integration of nonclinical and clinical data for torsadogenic risk assessment, a more systematic approach to estimate the hERG block potency and safety margin is needed. Methods: A cross-industry study was performed to collect hERG data on 28 drugs with known torsadogenic risk using a standardized experimental protocol. A Bayesian hierarchical modeling (BHM) approach was used to assess the hERG block potency of these drugs by quantifying both the inter-site and infra-site variability. A modeling and simulation study was also done to evaluate protocol-dependent changes in hERG potency estimates. Results: A systematic approach to estimate hERG block potency is established. The impact of choosing a safety margin threshold on torsadogenic risk evaluation is explored based on the posterior distributions of hERG potency estimated by this method. The modeling and simulation results suggest any potency estimate is specific to the protocol used. Discussion: This methodology can estimate hERG block potency specific to a given voltage protocol. The relationship between safety margin thresholds and torsadogenic risk predictivity suggests the threshold should be tailored to each specific context of use, and safety margin evaluation may need to be integrated with other information to form a more comprehensive risk assessment.

Automated patch clamp (APC) instruments enable efficient evaluation of electrophysiologic effects of drugs on human cardiac currents in heterologous expression systems. Differences in experimental protocols, instruments, and dissimilar site procedures affect the variability of IC50 values characterizing drug block potency. This impacts the utility of APC platforms for assessing a drug's cardiac safety margin. We determined variability of APC data from multiple sites that measured blocking potency of 12 blinded drugs (with different levels of proarrhythmic risk) against four human cardiac currents (hERG [IKr], hCav1.2 [L-Type ICa], peak hNav1.5, [Peak INa], late hNav1.5 [Late INa]) with recommended protocols (to minimize variance) using five APC platforms across 17 sites. IC50 variability (25/75 percentiles) differed for drugs and currents (e.g., 10.4-fold for dofetilide block of hERG current and 4-fold for mexiletine block of hNav1.5 current). Within-platform variance predominated for 4 of 12 hERG blocking drugs and 4 of 6 hNav1.5 blocking drugs. hERG and hNav1.5 block. Bland-Altman plots depicted varying agreement across APC platforms. A follow-up survey suggested multiple sources of experimental variability that could be further minimized by stricter adherence to standard protocols. Adoption of best practices would ensure less variable APC datasets and improved safety margins and proarrhythmic risk assessments.

Event Abstract Back to Event Development of a microelectrode array (MEA) based neurotoxicity assay for detecting the seizurogenic activity of novel drug candidates Dominik Loser1, 2, 3*, Timm Danker2, Clemens Möller1, Anita Niedworok3 and Udo Kraushaar3 1 Hochschule Albstadt-Sigmaringen, Life Sciences Faculty, Germany 2 NMI Technologie Transfer GmbH, Germany 3 Natural and Medical Sciences Institute, Germany During the early phase of drug development, the identification of possible side effects of novel drug candidates on the neuronal activity is very important. Until now most of the neurotoxicity assays are based on in vivo or in vitro animal models, which are problematic in terms of ethical issues and predictivity for humans. Over the last few years the continuous improvement of human iPSC derived neurons has increased their importance in this field, since they offer a great opportunity for the investigation of compound effects directly on a complex in vitro system of human origin. By combining these cells with the microelectrode array (MEA) technique we can investigate the functional neurotoxicity of novel drug candidates on a neuronal network and thereby detect their seizurogenic activity. In order to achieve this goal, we examined the effects of different coating and plating conditions on the development of the electrical activity of the human iPSC derived neurons (GlutaNeurons, Cellular Dynamics International CDI, US) that were cultured on 24-well glass Multiwell-MEAs (Multi Channel Systems MCS, GER). We tested three different coatings: 0.1% PEI, 0.07% PEI, which was diluted in a ready-to-use borate buffer, and the 0.07% PEI coating in combination with a pre-dotting with Laminin (10 µg/ml). The cells were dotted on the electrode fields of the MEAs in medium with a Laminin concentration of 10 µg/ml. The results showed no major differences between the tested coatings. Therefore, and because the handling of the 0.07% PEI coating is easier compared to the other tested coatings, we continued the study with the 0.07% PEI coating and explored the effects of different plating conditions. We examined the influences of a lower and a higher Laminin concentration in the dotting medium (10 µg/ml and 100 µg/ml) and in the medium (1 µg/ml and 33.3 µg/ml) that was used to fill-up the wells after the cells attached to the bottom of the wells. The cells that were plated in the higher Laminin concentration showed an earlier outgrowth of neurites as well as an earlier increase of the electrical activity and the occurrence of synchronous bursting compared to the cells plated in the lower Laminin concentration. The results suggest that there is a difference in the development of the cells in the tested plating conditions. The higher Laminin concentration led to an earlier outgrowth of neurites which could have favored an earlier formation of a neuronal network and thereby an earlier occurrence of synchronous burst activity. These findings have to be further investigated to establish a reliable neurotoxicity assay for detecting the seizurogenic activity of novel drug candidates in the future. Acknowledgements The authors would like to thank Dr. Sabine Lange (CDI) for providing the cells and useful information as well as Multi Channel Systems for providing the Glass Multiwell MEAs. This work was partially funded by the Baden-Württemberg Ministry of Science, Research and Art. Keywords: Assay development, human iPSC derived neurons, microelectrode array, seizurogenic compounds, Neurotoxicity Conference: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays, Reutlingen, Germany, 4 Jul - 6 Jul, 2018. Presentation Type: Oral Presentation Topic: Assay development Citation: Loser D, Danker T, Möller C, Niedworok A and Kraushaar U (2019). Development of a microelectrode array (MEA) based neurotoxicity assay for detecting the seizurogenic activity of novel drug candidates. Conference Abstract: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays. doi: 10.3389/conf.fncel.2018.38.00016 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2018; Published Online: 17 Jan 2019. * Correspondence: Mr. Dominik Loser, Hochschule Albstadt-Sigmaringen, Life Sciences Faculty, Albstadt, Germany, dominik.loser@nmi.de Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Dominik Loser Timm Danker Clemens Möller Anita Niedworok Udo Kraushaar Google Dominik Loser Timm Danker Clemens Möller Anita Niedworok Udo Kraushaar Google Scholar Dominik Loser Timm Danker Clemens Möller Anita Niedworok Udo Kraushaar PubMed Dominik Loser Timm Danker Clemens Möller Anita Niedworok Udo Kraushaar Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Manual patch clamp, the gold standard of electrophysiology, represents a powerful and versatile toolbox to stimulate, modulate, and record ion channel activity from membrane fragments and whole cells. The electrophysiological readout can be combined with fluorescent or optogenetic methods and allows for ultrafast solution exchanges using specialized microfluidic tools. A hallmark of manual patch clamp is the intentional selection of individual cells for recording, often an essential prerequisite to generate meaningful data. So far, available automation solutions rely on random cell usage in the closed environment of a chip and thus sacrifice much of this versatility by design. To parallelize and automate the traditional patch clamp technique while perpetuating the full versatility of the method, we developed an approach to automation, which is based on active cell handling and targeted electrode placement rather than on random processes. This is achieved through an automated pipette positioning system, which guides the tips of recording pipettes with micrometer precision to a microfluidic cell handling device. Using a patch pipette array mounted on a conventional micromanipulator, our automated patch clamp process mimics the original manual patch clamp as closely as possible, yet achieving a configuration where recordings are obtained from many patch electrodes in parallel. In addition, our implementation is extensible by design to allow the easy integration of specialized equipment such as ultrafast compound application tools. The resulting system offers fully automated patch clamp on purposely selected cells and combines high-quality gigaseal recordings with solution switching in the millisecond timescale.

Objective Mutations in KCNQ2 and KCNQ3 , encoding the voltage‐gated potassium channels K V 7.2 and K V 7.3, are known to cause benign familial neonatal seizures mainly by haploinsufficiency. Here, we set out to determine the disease mechanism of 7 de novo missense KCNQ2 mutations that were recently described in patients with a severe epileptic encephalopathy including pharmacoresistant seizures and pronounced intellectual disability. Methods Mutations were inserted into the KCNQ2 cDNA. Potassium currents were recorded using 2‐microelectrode voltage clamping, and surface expression was analyzed by a biotinylation assay in cRNA‐injected Xenopus laevis oocytes. Results We observed a clear loss of function for all mutations. Strikingly, 5 of 7 mutations exhibited a drastic dominant‐negative effect on wild‐type K V 7.2 or K V 7.3 subunits, either by globally reducing current amplitudes (3 pore mutations) or by a depolarizing shift of the activation curve (2 voltage sensor mutations) decreasing potassium currents at the subthreshold level at which these channels are known to critically influence neuronal firing. One mutation significantly reduced surface expression. Application of retigabine, a recently marketed K V 7 channel opener, partially reversed these effects for the majority of analyzed mutations. Interpretation The development of severe epilepsy and cognitive decline in children carrying 5 of the 7 studied KCNQ2 mutations can be related to a dominant‐negative reduction of the resulting potassium current at subthreshold membrane potentials. Other factors such as genetic modifiers have to be postulated for the remaining 2 mutations. Retigabine or similar drugs may be used as a personalized therapy for this severe disease. Ann Neurol 2014;75:382–394

Blockade of the cardiac ion channel coded by human ether-à-gogo-related gene (hERG) can lead to cardiac arrhythmia, which has become a major concern in drug discovery and development. Automated electrophysiological patch clamp allows assessment of hERG channel effects early in drug development to aid medicinal chemistry programs and has become routine in pharmaceutical companies. However, a number of potential sources of errors in setting up hERG channel assays by automated patch clamp can lead to misinterpretation of data or false effects being reported. This article describes protocols for automated electrophysiology screening of compound effects on the hERG channel current. Protocol details and the translation of criteria known from manual patch clamp experiments to automated patch clamp experiments to achieve good quality data are emphasized. Typical pitfalls and artifacts that may lead to misinterpretation of data are discussed. While this article focuses on hERG channel recordings using the QPatch (Sophion A/S, Copenhagen, Denmark) technology, many of the assay and protocol details given in this article can be transferred for setting up different ion channel assays by automated patch clamp and are similar on other planar patch clamp platforms.

Ligand gated ion channels like nACh Receptors show fast inactivation kinetics in the ms range. Therefore, state of the art investigation of receptor kinetics and drug action requires the combination of patch clamp recording with advanced ultra-fast compound application systems like the piezo-driven "I-Tube". So far, automated patch clamp systems have not been able to offer this combination. Here, we demonstrate automated ,,Θ-Tube" recordings on nACh receptors using the Patchserver, a new device based on automated pipette micro positioning, gigaseal and whole cell formation. Unlike concurrent approaches utilizing "planar" chip designs, the method retains important features of the classical patch clamp methodology, including the possibility to optically select individual cells to be patched. This also enables the use of transiently transfected cells qualified by fluorescent markers. The system offered compound applications to the cell membrane with millisecond exchange- and exposure times, which is crucial to investigate the kinetics of fast inactivating ligand gated ion channels. Thus, the new method offers a cost efficient approach to significantly enhanced throughput and data quality for research on fast inactivating ligand gated ion channels.

α-Conotoxins are subtype-selective nicotinic acetylcholine receptor (nAChR) antagonists. Although potent α3β2 nAChR-selective α-conotoxins have been identified, currently characterized α-conotoxins show no or only weak affinity for α4β2 nAChRs, which are, besides α7 receptors, the most abundant nAChRs in the mammalian brain. To identify the determinants responsible for this difference, we substituted selected amino acid residues in the ligand-binding domain of the α4 subunit by the corresponding residues in the α3 subunit. Two-electrode voltage clamp analysis of these mutants revealed increased affinity of α-conotoxins MII, TxIA, and [A10L]TxIA at the α4(R185I)β2 receptor. Conversely, α-conotoxin potency was reduced at the reverse α3(I186R)β2 mutant. Replacement of α4Arg185 by alanine, glutamate, and lysine demonstrated that a positive charge in this position prevents α-conotoxin binding. Combination of the R185I mutation with a P195Q mutation outside the binding site but in loop C completely transferred high α-conotoxin potency to the α4β2 receptor. Molecular dynamics simulations of homology models with docked α-conotoxin indicate that these residues control access to the α-conotoxin binding site.

We present a new approach of automating the "classical" patch clamp experiment based on cost efficient standard patch pipettes. Built on top of a standard patch clamp setup, the system enables recording of multiple cells in parallel. Pipette positioning, as well as seal and whole cell formation are automated. Unlike concurrent approaches utilizing "planar" chip designs, the method retains important features of the classical patch clamp methodology, including the possibility to optically select individual cells to be patched. This enables the use of transiently transfected cells qualified by fluorescent markers. The system also offers enhanced throughput for experiments which so far could only be addressed by tedious "manual" patch clamp. This includes piezo-driven, ultra-fast application of compounds to the cell membrane with millisecond exchange- and exposure times which is crucial to investigate the kinetics of fast inactivating ligand gated ion channels. Thus, the new method offers a cost efficient approach to significantly enhanced throughput in areas of neurobiological and neuropharmaceutical research which so far were not amenable to automation.