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

Abstract

1 Affiliations removed for initial submission as per guidelines. 6 ABSTRACT Predicting how pharmaceuticals may affect heart rhythm is a crucial step in drug-development, and requires a 7 deep understanding of a compound's action on ion channels. In vitro hERG-channel current recordings are an important step in 8 evaluating the pro-arrhythmic potential of small molecules, and are now routinely performed using automated high-throughput 9 patch clamp platforms. These machines can execute traditional voltage clamp protocols aimed at specific gating processes, 10 but the array of protocols needed to fully characterise a current is typically too long to be applied in a single cell. Shorter 11 high-information protocols have recently been introduced which have this capability, but they are not typically compatible with 12 high-throughput platforms. We present a new high-information 15 s protocol to characterise hERG (Kv11.1) kinetics, suitable for 13 both manual and high-throughput systems. We demonstrate its use on the Nanion SyncroPatch 384PE, a 384 well automated 14 patch clamp platform, by applying it to CHO cells stably expressing hERG1a. From these recordings we construct 124 cell-specific 15 variants/parameterisations of a hERG model at 25 • C. A further 8 independent protocols are run in each cell, and are used to 16 validate the model predictions. We then combine the experimental recordings using a hierarchical Bayesian model, which we 17 use to quantify the uncertainty in the model parameters, and their variability from cell to cell, which we use to suggest reasons 18 for the variability. This study demonstrates a robust method to measure and quantify uncertainty, and shows that it is possible 19 and practical to use high-throughput systems to capture full hERG channel kinetics quantitatively and rapidly. 20 21 We present a method for high-throughput characterisation of hERG potassium channel kinetics, via fitting a mathematical 22 model to results of over one hundred single cell patch clamp measurements collected simultaneously on an automated voltage 23 clamp platform. The automated patch clamp data are used to parameterise a mathematical ion channel model fully, opening a 24 new era of automated and rapid development of mathematical models from quick and cheap experiments. The method also 25 allows ample data for independent validation of the models and enables us to study experimental variability and propose its 26 origins. In future the method can be applied to characterise changes to hERG currents in different conditions, for instance 27 at different temperatures (see Part II of the study) or under mutations or the action of pharmaceuticals; and should be easily 28 adapted to study many other currents. Statement of Significance 30The human Ether-à-go-go-Related Gene (hERG) is of great interest in the field of cardiac safety pharmacology. hERG encodes 31 the pore-forming alpha subunit of the ion channel Kv11.1 which conducts the rapid delayed rectifier potassium current, I Kr 32(1)...

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

“…(11)), E-4031 subtracted, and passed a semi-automated quality control. The recordings, and parameter values derived from them, showed good agreement with earlier work using manual patch, for details on the methods please see Lei et al Lei et al (2019b).…”

“…To model the dynamics of the current of interest, I ion , the assumptions behind Eqs. (1)-(2) can be removed to instead model the entire voltage-clamp experiment and amplifier compensations with Lei et al (2019b); 100-400 pF has been measured for individual cardiomyocytes in various species Satoh et al (1996) (∼ 190 pF for human Sakakibara et al (1993)); and can be as high as 100 nF for large cells such as Xenopus oocytes Schmitt and Koepsell (2002 Table 1: Glossary of symbols and parameters. We also denote a (machine or post-processing) estimate of a parameter X as X * , and the error in the estimate of the same parameter as X † .…”

“…Note the excellent predictions for V m and its deviation from V cmd . The series of action potential-like waveforms were concatenated from those in a previous study Lei et al (2019b), they are composed of linear ramps and steps to permit use on high-throughput machines that cannot clamp to arbitrary waveforms. Magnifications of the protocols are shown later in Figure 7C-F; they include a delayed afterdepolarization (DAD)-like protocol, an early afterdepolarization (EAD)-like protocol, and action-potential-like protocols with different beating frequencies.…”

“…After experimentally validating the mathematical model of the full voltage-clamp experiment with two electrical model cells, it is now applied to experimental data from real cells. Here, a high-throughput dataset from a previous publication is used Lei et al (2019b). The dataset contains 124 voltage-clamp recordings of the potassium current that flows through hERG (Kv11.1) channels (I Kr ) measured with a staircase protocol and eight other independent protocols.…”

“…High levels of variability in kinetics have been observed in many studies Bekkers et al (1990); Finkel et al (2006); Feigenspan et al (2010); Golowasch (2014); Santillo et al (2014); Altomare et al (2015); Annecchino and Schultz (2018). A previous study Lei et al (2019b) analysed hERG current kinetics recorded simultaneously in 124 cells using an automated high-throughput patch-clamp machine. The experiments used Chinese hamster ovary (CHO) cells stably expressing hERG1a.…”