Computer modeling of whole-cell voltage-clamp analyses to delineate\xa0guidelines for good practice of manual and automated patch-clamp

Jérôme Montnach,Céline Marionneau,Isabelle Baró,Eléonore Moreau,Adrien Lesage,Sébastien Nicolas,Isabelle Simon,Maxime Lorenzini,Gildas Loussouarn,Michel De Waard

doi:10.1038/s41598-021-82077-8

Abstract

The patch-clamp technique and more recently the high throughput patch-clamp technique have contributed to major advances in the characterization of ion channels. However, the whole-cell voltage-clamp technique presents certain limits that need to be considered for robust data generation. One major caveat is that increasing current amplitude profoundly impacts the accuracy of the biophysical analyses of macroscopic ion currents under study. Using mathematical kinetic models of a cardiac voltage-gated sodium channel and a cardiac voltage-gated potassium channel, we demonstrated how large current amplitude and series resistance artefacts induce an undetected alteration in the actual membrane potential and affect the characterization of voltage-dependent activation and inactivation processes. We also computed how dose–response curves are hindered by high current amplitudes. This is of high interest since stable cell lines frequently demonstrating high current amplitudes are used for safety pharmacology using the high throughput patch-clamp technique. It is therefore critical to set experimental limits for current amplitude recordings to prevent inaccuracy in the characterization of channel properties or drug activity, such limits being different from one channel type to another. Based on the predictions generated by the kinetic models, we draw simple guidelines for good practice of whole-cell voltage-clamp recordings.

Highlights

The patch-clamp technique and more recently the high throughput patch-clamp technique have contributed to major advances in the characterization of ion channels
The patch-clamp technique has contributed to major advances in the characterization of ion channel biophysical properties and pharmacology, thanks to the versatility of the readouts: (i) unitary currents allowing the study of a single channel conductance, open probability and kinetics, and (ii) whole-cell currents allowing characterization of a population of channels, their pharmacology, the macroscopic properties of the gates, and the gating kinetics[1,2]
We developed a simple model, using published kinetic models of ion currents, to simulate and describe such a caveat

Summary

Introduction

The patch-clamp technique and more recently the high throughput patch-clamp technique have contributed to major advances in the characterization of ion channels. The actual voltage applied to the cell membrane ( Vm) is different than the voltage clamped by the amplifier and applied between the two electrodes (pipette and bath electrodes, V cmd) This leads for example to an erroneous characterization of a channel voltage-dependent activation process. This caveat was described early on when the patch-clamp technique was developed[3]. We extensively witness new publications that report ionic currents in the range of several nA, that undoubtedly have led to incorrect voltage clamp and erroneous conclusions On, this problem was partially solved by the development of amplifiers with the capacity to add a potential equivalent to the lost one (VS), a function which is called RS compensation[4]. Compensation rarely reaches 100% and some highthroughput systems have limited compensation abilities, to avoid over-compensation and consequent current oscillation that can lead to seal disruption

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