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 has contributed to major advances in the characterization of ion channels. The recent development of high throughput patch-clamp provides a new momentum to the field. However, whole-cell voltage-clamp technique presents certain limits that need to be considered for robust data generation. One major caveat is that current amplitude profoundly impacts the precision of the analyzed characteristics of the ion current under study. For voltagegated channels, the higher the current amplitude is, the less precise the characteristics of voltagedependence are. Similarly, in ion channel pharmacology, the characteristics of dose-response curves are hindered by high current amplitudes. In addition, the recent development of high throughput patch-clamp technique is often associated with the generation of stable cell lines demonstrating high current amplitudes. It is therefore critical to set the limits for current amplitude recordings to avoid inaccuracy in the characterization of channel properties or drug actions, such limits being different from one channel to another. In the present study, we use kinetic models of a voltage-gated sodium channel and a voltage-gated potassium channel to edict simple guidelines for good practice of whole-cell voltage-clamp recordings. words

A need for exhaustive and standardized characterization of ion channels activity. The case of KV11.1

“…is responsible for a major variability in the observed biophysical parameters (Lei et al, 2020). We also mathematically modeled the variability of the biophysical parameters of a voltage-dependent potassium channel, studied in conventional and high-throughput patch-clamp channels (Montnach et al, 2021). From the results we obtained, we proposed to reduce such variability, by limiting maximal current amplitude and series resistance.…”

“…Our model also pinpoints that a phenomenon observed in several K v channels and named "delayed repolarization" is in fact an artifactual property due to insufficient voltage-clamp. This strongly suggests that, in the case of transient transfection, the protocol has to be finely tuned to prevent currents larger than 10 nA (Montnach et al, 2021).…”

FHF2 phosphorylation and regulation of native myocardial NaV1.5 channels

“…Leak currents were always < 300 pA at HP (-120 mV), and were corrected offline. Cells exhibiting peak current amplitudes < 500 or > 5000 pA were excluded from analyses of biophysical properties because of errors associated with leak or voltage-clamp (Montnach et al, 2021), respectively, but were conserved in analyses of peak current density to avoid bias in evaluation of current densities.…”