Dataset Information

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

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.

SUBMITTER: Montnach J

PROVIDER: S-EPMC7870888 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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

Montnach Jérôme J Lorenzini Maxime M Lesage Adrien A Simon Isabelle I Nicolas Sébastien S Moreau Eléonore E Marionneau Céline C Baró Isabelle I De Waard Michel M Loussouarn Gildas G

Scientific reports 20210208 1

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-g ...[more]

PMID: 33558601

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Project description:Experimental records of single molecules or ion channels from fluorescence microscopy and patch-clamp electrophysiology often include high-frequency noise and baseline fluctuations that are not generated by the system under investigation and have to be removed. Moreover, multiple channels or conductance levels can be present at a time in the data that need to be quantified to accurately understand the behavior of the system. Manual procedures for removing these fluctuations and extracting conducting states or multiple channels are laborious, prone to subjective bias, and likely to hinder the processing of often very large data sets. We introduce a maximal likelihood formalism for separating signal from a noisy and drifting background such as fluorescence traces from imaging of elementary Ca2+ release events called puffs arising from clusters of channels, and patch-clamp recordings of ion channels. Parameters such as the number of open channels or conducting states, noise level, and background signal can all be optimized using the expectation-maximization algorithm. We implement our algorithm following the Baum-Welch approach to expectation-maximization in the portable Java language with a user-friendly graphical interface and test the algorithm on both synthetic and experimental data from the patch-clamp electrophysiology of Ca2+ channels and fluorescence microscopy of a cluster of Ca2+ channels and Ca2+ channels with multiple conductance levels. The resulting software is accurate, fast, and provides detailed information usually not available through manual analysis. Options for visual inspection of the raw and processed data with key parameters are provided, in addition to a range of statistics such as the mean open probabilities, mean open times, mean close times, dwell-time distributions for different number of channels open or conductance levels, amplitude distribution of all opening events, and number of transitions between different number of open channels or conducting levels in asci format with a single click.

Dataset Information

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

Publications

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

Similar Datasets

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