scholarly journals Rapid characterisation of hERG channel kinetics I: using an automated high-throughput system

2019 ◽  
Author(s):  
Chon Lok Lei ◽  
Michael Clerx ◽  
David J. Gavaghan ◽  
Liudmila Polonchuk ◽  
Gary R. Mirams ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Single Cell ◽  
Voltage Clamp ◽  
High Throughput ◽  
Rapid Development ◽  
Herg Channel ◽  
Model Parameters ◽  
Channel Kinetics ◽  
Automated Patch Clamp ◽  
High Information

ABSTRACTPredicting how pharmaceuticals may affect heart rhythm is a crucial step in drug-development, and requires a deep understanding of a compound’s action on ion channels.In vitrohERG-channel current recordings are an important step in evaluating the pro-arrhythmic potential of small molecules, and are now routinely performed using automated high-throughput patch clamp platforms. These machines can execute traditional voltage clamp protocols aimed at specific gating processes, but the array of protocols needed to fully characterise a current is typically too long to be applied in a single cell. Shorter high-information protocols have recently been introduced which have this capability, but they are not typically compatible with high-throughput platforms. We present a new high-information 15 s protocol to characterise hERG (Kv11.1) kinetics, suitable for both manual and high-throughput systems. We demonstrate its use on the Nanion SyncroPatch 384PE, a 384 well automated patch clamp platform, by applying it to CHO cells stably expressing hERG1a. From these recordings we construct 124 cell-specific variants/parameterisations of a hERG model at 25 °C. A further 8 independent protocols are run in each cell, and are used to validate the model predictions. We then combine the experimental recordings using a hierarchical Bayesian model, which we use to quantify the uncertainty in the model parameters, and their variability from cell to cell, which we use to suggest reasons for the variability. This study demonstrates a robust method to measure and quantify uncertainty, and shows that it is possible and practical to use high-throughput systems to capture full hERG channel kinetics quantitatively and rapidly.Statement of SignificanceWe present a method for high-throughput characterisation of hERG potassium channel kinetics, via fitting a mathematical model to results of over one hundred single cell patch clamp measurements collected simultaneously on an automated voltage clamp platform. The automated patch clamp data are used to parameterise a mathematical ion channel model fully, opening a new era of automated and rapid development of mathematical models from quick and cheap experiments. The method also allows ample data for independent validation of the models and enables us to study experimental variability and propose its origins. In future the method can be applied to characterise changes to hERG currents in different conditions, for instance at different temperatures (see Part II of the study) or under mutations or the action of pharmaceuticals; and should be easily adapted to study many other currents.

scholarly journals High-throughput Evaluation of Epilepsy-associated KCNQ2 Variants Reveals Functional and Pharmacological Heterogeneity

2021 ◽  
Author(s):  
Carlos G Vanoye ◽  
Reshma R Desai ◽  
Zhigang Ji ◽  
Sneha Adusumilli ◽  
Nirvani Jairam ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Cell Line ◽  
Voltage Clamp ◽  
Functional Properties ◽  
High Throughput ◽  
Dominant Negative ◽  
Loss Of Function ◽  
Patch Clamp Recording ◽  
Pathogenic Variants ◽  
Automated Patch Clamp

Hundreds of KCNQ2 variants have been identified by genetic testing of children with early onset epilepsy and/or developmental disability. Voltage-clamp recording from heterologous cells has proved useful for establishing deleterious functional effects of KCNQ2 variants, but procedures adapting these assays for standardized, higher throughput data collection and reporting are lacking. In this study, we employed automated patch clamp recording to assess in parallel the functional and pharmacological properties of 79 missense and 2 in-frame deletion variants of KCNQ2. Among the variants we studied were a training set of 18 pathogenic variants previously studied by voltage-clamp recording, 24 mostly rare population variants, and 39 disease-associated variants with unclear functional effects. Variant KCNQ2 subunits were transiently expressed in a cell line stably expressing KCNQ3 to reconstitute the physiologically relevant channel complex. Variants with severe loss-of-function were also co-expressed 1:1 with WT KCNQ2 in the KCNQ3 cell line to mimic the heterozygous genotype and assess dominant-negative behavior. In total, we analyzed electrophysiological data recorded from 9,480 cells. The functional properties of WT KCNQ2/KCNQ3 channels and pharmacological responses to known blockers and activators determined by automated patch clamp recording were highly concordant with previous findings. Similarly, functional properties of 18 known pathogenic variants largely matched previously published results and the validated automated patch clamp assay. Many of the 39 previously unstudied disease-associated KCNQ2 variants exhibited prominent loss-of-function and dominant-negative effects, providing strong evidence in support of pathogenicity. All variants, exhibit response to retigabine (10 μM), although there were differences in maximal responses. Variants within the ion selectivity filter exhibited the weakest responses whereas retigabine had the strongest effect on gain-of-function variants in the voltage-sensor domain. Our study established a high throughput method to detect deleterious functional consequences of KCNQ2 variants. We demonstrated that dominant-negative loss-of-function is a common mechanism associated with missense KCNQ2 variants but this does not occur with rare population variation in this gene. Importantly, we observed genotype-dependent differences in the response of KCNQ2 variants to retigabine.

scholarly journals Accounting for variability in ion current recordings using a mathematical model of artefacts in voltage-clamp experiments

2020 ◽  
Vol 378 (2173) ◽  
pp. 20190348 ◽  
Author(s):  
Chon Lok Lei ◽  
Michael Clerx ◽  
Dominic G. Whittaker ◽  
David J. Gavaghan ◽  
Teun P. de Boer ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Voltage Clamp ◽  
Ion Current ◽  
Model Parameters ◽  
Whole Cell Patch Clamp ◽  
Patch Clamp Experiment ◽  
Ion Channel Kinetics ◽  
Voltage Clamp Experiment ◽  
Model Variability ◽  
Cell Cell

Mathematical models of ion channels, which constitute indispensable components of action potential models, are commonly constructed by fitting to whole-cell patch-clamp data. In a previous study, we fitted cell-specific models to hERG1a (Kv11.1) recordings simultaneously measured using an automated high-throughput system, and studied cell-cell variability by inspecting the resulting model parameters. However, the origin of the observed variability was not identified. Here, we study the source of variability by constructing a model that describes not just ion current dynamics, but the entire voltage-clamp experiment. The experimental artefact components of the model include: series resistance, membrane and pipette capacitance, voltage offsets, imperfect compensations made by the amplifier for these phenomena, and leak current. In this model, variability in the observations can be explained by either cell properties, measurement artefacts, or both. Remarkably, by assuming that variability arises exclusively from measurement artefacts, it is possible to explain a larger amount of the observed variability than when assuming cell-specific ion current kinetics. This assumption also leads to a smaller number of model parameters. This result suggests that most of the observed variability in patch-clamp data measured under the same conditions is caused by experimental artefacts, and hence can be compensated for in post-processing by using our model for the patch-clamp experiment. This study has implications for the question of the extent to which cell-cell variability in ion channel kinetics exists, and opens up routes for better correction of artefacts in patch-clamp data. This article is part of the theme issue ‘Uncertainty quantification in cardiac and cardiovascular modelling and simulation’.

High-Throughput Electrophysiology Screen Revealed Cardiotoxicity of Strychnine by Selectively Targeting hERG Channel

2018 ◽  
Vol 46 (08) ◽  
pp. 1825-1840 ◽  
Author(s):  
Taiyi Wang ◽  
Xiaonan Chen ◽  
Jiahui Yu ◽  
Qunqun Du ◽  
Jie Zhu ◽  
...  
Keyword(s):  
Herbal Medicine ◽  
Patch Clamp ◽  
High Throughput ◽  
Chinese Herbal Medicine ◽  
Large Scale ◽  
Cardiac Toxicity ◽  
Herg Channel ◽  
Chinese Herbal ◽  
Cell Vitality

Although the efficacy and the health care advantages of Chinese herbal medicine (CHM) have become increasingly recognized worldwide, the potential side effects and toxicity still restrict its broader application. This study established and applied an integrated platform anchored on automatic patch clamp system to screen and evaluate a collection of CHM extracts, compositions and monomeric compounds for in vitro cardiac toxicity. Of 1036 CHM samples screened, 2.79% significantly inhibited hERG channel activity. Among them, Strychnine was identified for the first time as a potent hERG inhibitor with an IC[Formula: see text] of [Formula: see text]M in comparison to that of Dofetilide at [Formula: see text]M and Quinidine at [Formula: see text]M. Langendorff-perfusion experiments confirmed that strychnine increased QT interphase from [Formula: see text][Formula: see text]ms to [Formula: see text][Formula: see text]ms and decreased heart rates from [Formula: see text][Formula: see text]bmp to [Formula: see text][Formula: see text]bmp in isolated rat hearts. The cardiac toxicity effect of strychnine appears to be specific to hERG channel since an in vitro multiplex imaging analysis showed that it did not affect cellular phenotypes such as cell vitality, nucleus area, mitochondria mass and function, nor intracellular calcium in rat primary myocytes. This integrated high-throughput hERG patch clamp and high-content multi-parameter imaging cardiac toxicity screen approach should be useful for large-scale preclinical evaluation of complex Chinese herbal medicine.

scholarly journals Accounting for variability in ion current recordings using a mathematical model of artefacts in voltage-clamp experiments

2019 ◽  
Author(s):  
Chon Lok Lei ◽  
Michael Clerx ◽  
Dominic G. Whittaker ◽  
David J. Gavaghan ◽  
Teun P. de Boer ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Voltage Clamp ◽  
Ion Current ◽  
Model Parameters ◽  
Whole Cell Patch Clamp ◽  
Patch Clamp Experiment ◽  
Ion Channel Kinetics ◽  
Voltage Clamp Experiment ◽  
Model Variability ◽  
Cell Cell

AbstractMathematical models of ion channels, which constitute indispensable components of action potential models, are commonly constructed by fitting to whole-cell patch-clamp data. In a previous study we fitted cell-specific models to hERG1a (Kv11.1) recordings simultaneously measured using an automated high-throughput system, and studied cell-cell variability by inspecting the resulting model parameters. However, the origin of the observed variability was not identified. Here we study the source of variability by constructing a model that describes not just ion current dynamics, but the entire voltage-clamp experiment. The experimental artefact components of the model include: series resistance, membrane and pipette capacitance, voltage offsets, imperfect compensations made by the amplifier for these phenomena, and leak current. In this model, variability in the observations can be explained by either cell properties, measurement artefacts, or both. Remarkably, by assuming that variability arises exclusively from measurement artefacts, it is possible to explain a larger amount of the observed variability than when assuming cell-specific ion current kinetics. This assumption also leads to a smaller number of model parameters. This result suggests that most of the observed variability in patch-clamp data measured under the same conditions is caused by experimental artefacts, and hence can be compensated for in post-processing by using our model for the patch-clamp experiment. This study has implications for the question of the extent to which cell-cell variability in ion channel kinetics exists, and opens up routes for better correction of artefacts in patch-clamp data.

scholarly journals A Yoda1-Based Approach to Investigate Piezo1 Channels in Red Blood Cells Using Automated Patch Clamp Technology

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1031-1031
Author(s):  
Maria Giustina Rotordam ◽  
Elisa Fermo ◽  
Nadine Becker ◽  
Wilma Barcellini ◽  
Andrea Brüggemann ◽  
...  
Keyword(s):  
Shear Stress ◽  
Patch Clamp ◽  
Ion Channel ◽  
Red Blood Cells ◽  
High Throughput ◽  
Blood Cells ◽  
Screening Assay ◽  
Whole Cell ◽  
Automated Patch Clamp ◽  
Induced Currents

Abstract Piezo1 is a mechanosensitive ion channel supposed to regulate the volume and maintain the structural integrity in Red Blood Cells (RBCs), as gain-of-function mutations in this channel are associated to the RBC disease Hereditary Xerocytosis (Zarychanski et al. Blood 2012; Bae et al. Proceedings of the National Academy of Sciences 2013). Piezo1 is activated by several mechanical forces, including stretching, poking and shear stress and allows Ca2+ and other cations to enter the cell generating an electrical response. In 2015, it has been discovered that Piezo1 is sensitive to a small molecule, Yoda1 (Syeda et al. Elife 2015), which keeps the channel open and affects its inactivation kinetics. This finding has created new possibilities to elucidate Piezo1 gating mechanism and explore its functional significance in physiological and pathophysiological conditions. Here, we present a patient with a novel PIEZO1 mutation (R2110W) and a patch clamp-based high-throughput screening assay for Piezo1 activity. We established a protocol to detect functional Piezo1 mutations upon chemical stimulation by Yoda1, yet were not able to stimulate the channel via mechanical force, i.e. pressure steps and shear-stress. The assay was first developed on Neuro2A (N2A), a neuroblastoma cell-line endogenously expressing Piezo1 channels (kindly provided by Max-Delbrück Center, Berlin), due to larger abundance of Piezo1 channels in these cells. Initial experiments were performed on the Patchliner (Nanion Technologies GmbH, Munich), a medium-throughput automated patch clamp system able to record up to 8 cells at a time. Currents were elicited using a voltage ramp ranging from -100 to +80 mV for 300 ms, the holding potential was set to -60 mV. A significantly increased whole-cell current was observed upon 10 µM Yoda1 application in half of the recorded cells and the resulting Yoda1-induced currents were inhibited by 30 µM gadolinium chloride, a non-specific blocker of stretch-activated channels. The assay was then implemented on the SyncroPatch 384PE (Nanion Technologies GmbH, Munich), capable of recording up to 384 cells in parallel under identical experimental conditions, thus allowing for reliable statistical analysis. Yoda1 responding cells were selected based on strict quality control (QC) criteria, i.e. the seal resistance stability over time. In one example NPC-384 chip 140 out of 384 N2A cells (37%) passed the QC criteria and 85 cells (60% of the valid cells) were considered as Yoda1 responders. Finally, we investigated Piezo1 electrophysiological properties in healthy and patient RBCs carrying the novel PIEZO1 R2110W mutation. Similar to N2A cells, RBCs currents were analyzed and divided into Yoda1 responders and non-responders according to our QC criteria. The increase in whole-cell currents induced by Yoda1 application was significantly higher in patient compared to control RBCs, which was also reflected by a higher number of Yoda1 responders compared to control. Residue R2110W is structurally located in a gating sensitive area of the channel protein suggesting a gain-of-function. This would be in line with previously described mutations in PIEZO1 (Albuisson et al. Nature Communications 2013) and the mild form of anaemia observed in the patient. Furthermore, we excluded any involvement of Gardos channels in the Yoda1-induced currents by comparing measurements in the presence and absence of the specific Gardos channel inhibitor TRAM-34. Altogether, our work demonstrates that high-throughput patch clamping can provide a robust assay to study functional Piezo1 impairments in primary RBCs without expressing the mutated channel protein in a heterologous expression system. Our approach may be used to detect other channelopathies not only in RBCs and may serve as routine screening assay for diseases related to ion channel dysfunctions in general, complementary to gene sequencing. Disclosures No relevant conflicts of interest to declare.

scholarly journals Rapid Characterization of hERG Channel Kinetics I: Using an Automated High-Throughput System

2019 ◽  
Vol 117 (12) ◽  
pp. 2438-2454 ◽  
Author(s):  
Chon Lok Lei ◽  
Michael Clerx ◽  
David J. Gavaghan ◽  
Liudmila Polonchuk ◽  
Gary R. Mirams ◽  
...  
Keyword(s):  
High Throughput ◽  
Herg Channel ◽  
Channel Kinetics ◽  
Rapid Characterization
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