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

Mathematical models of ion channels, which constitute indispensable components of action potential models, are commonly constructed by fitting to whole-cell patch-clamp data. In a previous study, we fitted cell-specific models to hERG1a (Kv11.1) recordings simultaneously measured using an automated high-throughput system, and studied cell-cell variability by inspecting the resulting model parameters. However, the origin of the observed variability was not identified. Here, we study the source of variability by constructing a model that describes not just ion current dynamics, but the entire voltage-clamp experiment. The experimental artefact components of the model include: series resistance, membrane and pipette capacitance, voltage offsets, imperfect compensations made by the amplifier for these phenomena, and leak current. In this model, variability in the observations can be explained by either cell properties, measurement artefacts, or both. Remarkably, by assuming that variability arises exclusively from measurement artefacts, it is possible to explain a larger amount of the observed variability than when assuming cell-specific ion current kinetics. This assumption also leads to a smaller number of model parameters. This result suggests that most of the observed variability in patch-clamp data measured under the same conditions is caused by experimental artefacts, and hence can be compensated for in post-processing by using our model for the patch-clamp experiment. This study has implications for the question of the extent to which cell-cell variability in ion channel kinetics exists, and opens up routes for better correction of artefacts in patch-clamp data. This article is part of the theme issue 'Uncertainty quantification in cardiac and cardiovascular modelling and simulation'

A nonlinear and time-dependent leak current in the presence of calcium fluoride patch-clamp seal enhancer [version 1; peer review: 2 approved with reservations]

Automated patch-clamp platforms are widely used and vital tools in both academia and industry to enable high-throughput studies such as drug screening. A leak current to ground occurs whenever the seal between a pipette and cell (or internal solution and cell in high-throughput machines) is not perfectly insulated from the bath (extracellular) solution. Over 1 GΩ seal resistance between pipette and bath solutions is commonly used as a quality standard for manual patch work. With automated platforms it can be difficult to obtain such a high seal resistance between the intra- and extra-cellular solutions. One suggested method to alleviate this problem is using an F− containing internal solution together with a Ca2+ containing external solution — so that a CaF2 crystal forms when the two solutions meet which ‘plugs the holes’ to enhance the seal resistance. However, we observed an unexpected nonlinear-in-voltage and time-dependent current using these solutions on an automated patch-clamp platform. We performed manual patch-clamp experiments with the automated patch-clamp solutions, but no biological cell, and observed the same nonlinear time-dependent leak current. The current could be completely removed by washing out F− ions to leave a conventional leak current that was linear and not time-dependent. We therefore conclude fluoride ions interacting with the CaF2 crystal are the origin of the nonlinear time-dependent leak current. The consequences of such a nonlinear and time-dependent leak current polluting measurements should be considered carefully if it cannot be isolated and subtracted

Experimental Mapping of the Canine KCNJ2 and KCNJ12 Gene Structures and Functional Analysis of the Canine KIR2.2 ion Channel

For many model organisms traditionally in use for cardiac electrophysiological studies, characterization of ion channel genes is lacking. We focused here on two genes encoding the inward rectifier current, KCNJ2 and KCNJ12, in the dog heart. A combination of RT-PCR, 5′-RACE, and 3′-RACE demonstrated the status of KCNJ2 as a two exon gene. The complete open reading frame (ORF) was located on the second exon. One transcription initiation site was mapped. Four differential transcription termination sites were found downstream of two consensus polyadenylation signals. The canine KCNJ12 gene was found to consist of three exons, with its ORF located on the third exon. One transcription initiation and one termination site were found. No alternative splicing was observed in right ventricle or brain cortex. The gene structure of canine KCNJ2 and KCNJ12 was conserved amongst other vertebrates, while current GenBank gene annotation was determined as incomplete. In silico translation of KCN12 revealed a non-conserved glycine rich stretch located near the carboxy-terminus of the KIR2.2 protein. However, no differences were observed when comparing dog with human KIR2.2 protein upon ectopic expression in COS-7 or HEK293 cells with respect to subcellular localization or electrophysiological properties

Dog KCNJ2 and KCNJ12 genes

For many model organisms traditionally in use for cardiac electrophysiological studies, characterization of ion channel genes is lacking. We focused here on two genes encoding the inward rectifier current, KCNJ2 and KCNJ12, in the dog heart. A combination of RT-PCR, 5′-RACE, and 3′-RACE demonstrated the status of KCNJ2 as a two exon gene. The complete open reading frame (ORF) was located on the second exon. One transcription initiation site was mapped. Four differential transcription termination sites were found downstream of two consensus polyadenylation signals. The canine KCNJ12 gene was found to consist of three exons, with its ORF located on the third exon. One transcription initiation and one termination site were found. No alternative splicing was observed in right ventricle or brain cortex. The gene structure of canine KCNJ2 and KCNJ12 was conserved amongst other vertebrates, while current GenBank gene annotation was determined as incomplete. In silico translation of KCN12 revealed a non-conserved glycine rich stretch located near the carboxy-terminus of the KIR2.2 protein. However, no differences were observed when comparing dog with human KIR2.2 protein upon ectopic expression in COS-7 or HEK293 cells with respect to subcellular localization or electrophysiological properties

Leak current, even with gigaohm seals, can cause misinterpretation of stem cell-derived cardiomyocyte action potential recordings

Background and AimsHuman induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have become an essential tool to study arrhythmia mechanisms. Much of the foundational work on these cells, and the computational models built from the resultant data, has overlooked the contribution of seal-leak current on the immature and heterogeneous phenotype that has come to define these cells. The aim of this study is to understand the effect of seal-leak current on recordings of action potential (AP) morphology.MethodsAPs were recorded in human iPSC-CMs using patch clamp and simulated using previously published mathematical models.ResultsOur in silico and in vitro studies demonstrate how seal-leak current depolarises APs, substantially affecting their morphology, even with seal resistances (Rseal) above 1 GΩ. We show that compensation of this leak current is difficult due to challenges with obtaining accurate measures of Rseal during an experiment. Using simulation, we show that Rseal measures: 1) change during an experiment, invalidating the use of pre-rupture values, and 2) are polluted by the presence of transmembrane currents at every voltage. Finally, we posit that the background sodium current in baseline iPSC-CM models imitates the effects of seal-leak current and is increased to a level that masks the effects of seal-leak current on iPSC-CMs.ConclusionBased on these findings, we make recommendations to improve iPSC-CM AP data acquisition, interpretation, and model-building. Taking these recommendations into account will improve our understanding of iPSC-CM physiology and the descriptive ability of models built from such data

A nonlinear and time-dependent leak current in the presence of calcium fluoride patch-clamp seal enhancer

Automated patch-clamp platforms are widely used and vital tools in both academia and industry to enable high-throughput studies such as drug screening. A leak current to ground occurs whenever the seal between a pipette and cell (or internal solution and cell in high-throughput machines) is not perfectly insulated from the bath (extracellular) solution. Over 1 G seal resistance between pipette and bath solutions is commonly used as a quality standard for manual patch work. With automated platforms it can be difficult to obtain such a high seal resistance between the intra- and extra-cellular solutions. One suggested method to alleviate this problem is using an F containing internal solution together with a Ca 2+ containing external solution so that a CaF 2 crystal forms when the two solutions meet which plugs the holes' to enhance the seal resistance. However, we observed an unexpected nonlinear-in-voltage and time-dependent current using these solutions on an automated patch-clamp platform. We performed manual patch-clamp experiments with the automated patch-clamp solutions, but no biological cell, and observed the same nonlinear time-dependent leak current. The current could be completely removed by washing out F ions to leave a conventional leak current that was linear and not time-dependent. We therefore conclude fluoride ions interacting with the CaF 2 crystal are the origin of the nonlinear time-dependent leak current. The consequences of such a nonlinear and time-dependent leak current polluting measurements should be considered carefully if it cannot be isolated and subtracted

Recording ten-fold larger IKr conductances with automated patch clamping using equimolar Cs+ solutions

Background: The rapid delayed rectifier potassium current (IKr) is important for cardiac repolarization and is most often involved in drug-induced arrhythmias. However, accurately measuring this current can be challenging in human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes because of its small current density. Interestingly, the ion channel conducting IKr, hERG channel, is not only permeable to K+ ions but also to Cs+ ions when present in equimolar concentrations inside and outside of the cell.Methods: In this study, IhERG was measured from Chinese hamster ovary (CHO)-hERG cells and hiPSC-CM using either Cs+ or K+ as the charge carrier. Equimolar Cs+ has been used in the literature in manual patch-clamp experiments, and here, we apply this approach using automated patch-clamp systems. Four different (pre)clinical drugs were tested to compare their effects on Cs+- and K+-based currents.Results: Using equimolar Cs+ solutions gave rise to approximately ten-fold larger hERG conductances. Comparison of Cs+- and K+-mediated currents upon application of dofetilide, desipramine, moxifloxacin, or LUF7244 revealed many similarities in inhibition or activation properties of the drugs studied. Using equimolar Cs+ solutions gave rise to approximately ten-fold larger hERG conductances. In hiPSC-CM, the Cs+-based conductance is larger compared to the known K+-based conductance, and the Cs+ hERG conductance can be inhibited similarly to the K+-based conductance.Conclusion: Using equimolar Cs+ instead of K+ for IhERG measurements in an automated patch-clamp system gives rise to a new method by which, for example, quick scans can be performed on effects of drugs on hERG currents. This application is specifically relevant when such experiments are performed using cells which express small IKr current densities in combination with small membrane capacitances

Uncertainty and variability in models of the cardiac action potential: Can we build trustworthy models?

Cardiac electrophysiology models have been developed for over 50 years, and now include detailed descriptions of individual ion currents and sub-cellular calcium handling. It is commonly accepted that there are many uncertainties in these systems, with quantities such as ion channel kinetics or expression levels being difficult to measure or variable between samples. Until recently, the original approach of describing model parameters using single values has been retained, and consequently the majority of mathematical models in use today provide point predictions, with no associated uncertainty. In recent years, statistical techniques have been developed and applied in many scientific areas to capture uncertainties in the quantities that determine model behaviour, and to provide a distribution of predictions which accounts for this uncertainty. In this paper we discuss this concept, which is termed uncertainty quantification, and consider how it might be applied to cardiac electrophysiology models. We present two case studies in which probability distributions, instead of individual numbers, are inferred from data to describe quantities such as maximal current densities. Then we show how these probabilistic representations of model parameters enable probabilities to be placed on predicted behaviours. We demonstrate how changes in these probability distributions across data sets offer insight into which currents cause beat-to-beat variability in canine APs. We conclude with a discussion of the challenges that this approach entails, and how it provides opportunities to improve our understanding of electrophysiology