Sinusoidal voltage protocols for rapid characterisation of ion channel kinetics

Abstract

Key points Ion current kinetics are commonly represented by current–voltage relationships, time constant–voltage relationships and subsequently mathematical models fitted to these. These experiments take substantial time, which means they are rarely performed in the same cell.Rather than traditional square‐wave voltage clamps, we fitted a model to the current evoked by a novel sum‐of‐sinusoids voltage clamp that was only 8 s long.Short protocols that can be performed multiple times within a single cell will offer many new opportunities to measure how ion current kinetics are affected by changing conditions.The new model predicts the current under traditional square‐wave protocols well, with better predictions of underlying currents than literature models. The current under a novel physiologically relevant series of action potential clamps is predicted extremely well.The short sinusoidal protocols allow a model to be fully fitted to individual cells, allowing us to examine cell–cell variability in current kinetics for the first time. AbstractUnderstanding the roles of ion currents is crucial to predict the action of pharmaceuticals and mutations in different scenarios, and thereby to guide clinical interventions in the heart, brain and other electrophysiological systems. Our ability to predict how ion currents contribute to cellular electrophysiology is in turn critically dependent on our characterisation of ion channel kinetics – the voltage‐dependent rates of transition between open, closed and inactivated channel states. We present a new method for rapidly exploring and characterising ion channel kinetics, applying it to the hERG potassium channel as an example, with the aim of generating a quantitatively predictive representation of the ion current. We fitted a mathematical model to currents evoked by a novel 8 second sinusoidal voltage clamp in CHO cells overexpressing hERG1a. The model was then used to predict over 5 minutes of recordings in the same cell in response to further protocols: a series of traditional square step voltage clamps, and also a novel voltage clamp comprising a collection of physiologically relevant action potentials. We demonstrate that we can make predictive cell‐specific models that outperform the use of averaged data from a number of different cells, and thereby examine which changes in gating are responsible for cell–cell variability in current kinetics. Our technique allows rapid collection of consistent and high quality data, from single cells, and produces more predictive mathematical ion channel models than traditional approaches.

Cross-site and cross-platform variability of automated patch clamp assessments of drug effects on human cardiac currents in recombinant cells

“…The implementation of the Milnes protocol might be best suited for manual patch techniques later in the drug discovery process conducted for risk assessments requiring more extensive characterization on a few candidate compounds (or for regulatory submissions). Beattie et al 20 and Lei et al 21,22 recently published voltage protocols more amenable to APC approaches to characterizing the kinetics of hERG block that might prove as suitable alternatives to the Milnes voltage clamp protocol.…”

“…Differences in the ratio of overexpressed current of interest (e.g., hERG) vs. endogenous background currents may lead to underestimates of blocking potency if background currents are not considered. For example, some authors prefer CHO cells over HEK cells as a hERG expression system based on generally lower background endogenous currents 20 . These differences may be corrected based on background currents measured at the end of each experiment in the presence of complete hERG block (e.g., with a maximal blocking concentration of a specific hERG blocking agent such as E-4031).…”

“…19 Novoheart, Vancouver, BC, Canada. 20 Health and Environmental Sciences Institute, Washington, DC, USA. 21 AbbVie, Chicago, IL, USA.…”

Sensitivity and Uncertainty Analysis of Two Human Atrial Cardiac Cell Models Using Gaussian Process Emulators

“…The rationale for this approach was an assumption that kinetic parameters are determined by biophysics, and so less prone to variation than the expression of ion channels, pumps, and exchangers. However, a detailed sensitivity analysis of I Kr dynamics in the Courtemanche model showed that these kinetic parameters influence APD (Chang et al, 2017), and other studies have highlighted difficulties in calibrating ion channel dynamics using traditional approaches as well as showing that different formulations can have an important effect on the magnitude and time course of an ion channel current (Beattie et al, 2018). In the present study our focus was on the action potential rather than Ca 2+ handling, and a detailed sensitivity analysis of the mechanisms of Ca 2+ storage, release, and uptake in each model would be a valuable extension to the work presented here.…”

Sensitivity and uncertainty analysis of two human atrial cardiac cell models using Gaussian process emulators

“…The rationale for this approach was an 501 assumption that kinetic parameters are determined by biophysics, and so less prone to 502 variation than the expression of ion channels, pumps, and exchangers. However, a 503 detailed sensitivity analysis of I Kr dynamics in the Courtemanche model showed that 504 these kinetic parameters influence APD [44], and other studies have highlighted 505 difficulties in calibrating ion channel dynamics using traditional approaches as well as 506 showing that different formulations can have an important effect on the magnitude and 507 time course of an ion channel current [45]. In the present study our focus was on the 508 action potential rather than Ca 2+ handling, and a detailed sensitivity analysis of the 509 mechanisms of Ca 2+ storage, release, and uptake in each model would be a valuable 510 extension to the work presented here.…”