Publications by authors named "Adam P Hill"

48 Publications

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

Wellcome Open Res 2020 2;5:152. Epub 2021 Nov 2.

Department of Medical Physiology, Division of Heart and Lungs, University Medical Centre Utrecht, Utrecht, 3584 CX, The Netherlands.

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 Ca containing external solution - so that a CaF 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 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.
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http://dx.doi.org/10.12688/wellcomeopenres.15968.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8591515.2PMC
November 2021

Pathophysiological metabolic changes associated with disease modify the proarrhythmic risk profile of drugs with potential to prolong repolarisation.

Br J Pharmacol 2021 Nov 26. Epub 2021 Nov 26.

Victor Chang Cardiac Research Institute, Sydney, Australia.

Background And Purpose: Hydroxychloroquine, chloroquine and azithromycin are three drugs that were proposed to treat COVID-19. While concern already existed around their proarrhythmic potential there is little data regarding how altered physiological states encountered in patients such as febrile state, electrolyte imbalances or acidosis might change their risk profiles.

Experimental Approach: Potency of hERG block was measured using high-throughput electrophysiology in the presence of variable environmental factors. These potencies informed simulations to predict population risk profiles. Effects on cardiac repolarisation were verified in human induced pluripotent stem cell-derived cardiomyocytes from multiple individuals.

Key Results: Chloroquine and hydroxychloroquine blocked hERG with IC of 1.47±0.07 μM and 3.78±0.17 μM respectively, indicating proarrhythmic risk at concentrations effective against SARS-CoV-2 in vitro. Hypokalaemia and hypermagnesemia increased potency of chloroquine and hydroxychloroquine, indicating increased proarrhythmic risk. Acidosis significantly reduced potency of all drugs, whereas increased temperature decreased potency of chloroquine and hydroxychloroquine against hERG but increased potency for azithromycin. In silico simulations demonstrated that proarrhythmic risk was increased by female sex, hypokalaemia and heart failure, and identified specific genetic backgrounds associated with emergence of arrhythmia.

Conclusion And Implications: Our study demonstrates how proarrhythmic risk can be exacerbated by metabolic changes and pre-existing disease. More broadly, the study acts as a blueprint for how high-throughput in vitro screening, combined with in silico simulations can help guide both preclinical screening and clinical management of patients in relation to drugs with potential to prolong repolarisation.
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http://dx.doi.org/10.1111/bph.15757DOI Listing
November 2021

Heterozygous variant phenotyping using Flp-In HEK293 and high-throughput automated patch clamp electrophysiology.

Biol Methods Protoc 2021 19;6(1):bpab003. Epub 2021 Mar 19.

Victor Chang Cardiac Research Institute, 405 Liverpool Street, Darlinghurst, New South Wales 2010, Australia.

is one of the 59 medically actionable genes recommended by the American College of Medical Genetics for reporting of incidental findings from clinical genomic sequencing. However, half of the reported variants in the ClinVar database are classified as variants of uncertain significance. In the absence of strong clinical phenotypes, there is a need for functional phenotyping to help decipher the significance of variants identified incidentally. Here, we report detailed methods for assessing the molecular phenotype of any missense variant. The key components of the assay include quick and cost-effective generation of a bi-cistronic vector to co-express Wild-type (WT) and any variant allele, generation of stable Flp-In HEK293 cell lines and high-throughput automated patch clamp electrophysiology analysis of channel function. Stable cell lines take 3-4 weeks to produce and can be generated in bulk, which will then allow up to 30 variants to be phenotyped per week after 48 h of channel expression. This high-throughput functional genomics assay will enable a much more rapid assessment of the extent of loss of function of any variant.
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http://dx.doi.org/10.1093/biomethods/bpab003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8046900PMC
March 2021

Conserved Role of the Large Conductance Calcium-Activated Potassium Channel, K1.1, in Sinus Node Function and Arrhythmia Risk.

Circ Genom Precis Med 2021 04 25;14(2):e003144. Epub 2021 Feb 25.

Victor Chang Cardiac Research Institute, Darlinghurst (V.N.-K., C.L., A.-K.A., C.D.C., A.J., I.G.H., M.S., M.O., G.T., R.J., R.P.H., A.P.H., D.F.).

Background: encodes the α-subunit of the large-conductance Ca-activated K channel, K1.1, and lies within a linkage interval for atrial fibrillation (AF). Insights into the cardiac functions of K1.1 are limited, and has not been investigated as an AF candidate gene.

Methods: The gene was sequenced in 118 patients with familial AF. The role of K1.1 in normal cardiac structure and function was evaluated in humans, mice, zebrafish, and fly. A novel variant was functionally characterized.

Results: A complex variant was identified in 1 kindred with AF. To evaluate potential disease mechanisms, we first evaluated the distribution of K1.1 in normal hearts using immunostaining and immunogold electron microscopy. K1.1 was seen throughout the atria and ventricles in humans and mice, with strong expression in the sinus node. In an ex vivo murine sinoatrial node preparation, addition of the K1.1 antagonist, paxilline, blunted the increase in beating rate induced by adrenergic receptor stimulation. Knockdown of the K1.1 ortholog, , in zebrafish embryos resulted in sinus bradycardia with dilatation and reduced contraction of the atrium and ventricle. Genetic inactivation of the K1.1 ortholog, , systemically or in adult stages, also slowed the heartbeat and produced fibrillatory cardiac contractions. Electrophysiological characterization of -deficient flies revealed bursts of action potentials, reflecting increased events of fibrillatory arrhythmias. Flies with cardiac-specific overexpression of the human mutant also showed increased heart period and bursts of action potentials, similar to the K1.1 loss-of-function models.

Conclusions: Our data point to a highly conserved role of K1.1 in sinus node function in humans, mice, zebrafish, and fly and suggest that K1.1 loss of function may predispose to AF.
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http://dx.doi.org/10.1161/CIRCGEN.120.003144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8058291PMC
April 2021

Arrhythmogenic effects of ultra-long and bistable cardiac action potentials.

PLoS Comput Biol 2021 02 16;17(2):e1008683. Epub 2021 Feb 16.

Victor Chang Cardiac Research Institute, Darlinghurst, NSW, Australia.

Contemporary accounts of the initiation of cardiac arrhythmias typically rely on after-depolarizations as the trigger for reentrant activity. The after-depolarizations are usually triggered by calcium entry or spontaneous release within the cells of the myocardium or the conduction system. Here we propose an alternative mechanism whereby arrhythmias are triggered autonomously by cardiac cells that fail to repolarize after a normal heartbeat. We investigated the proposal by representing the heart as an excitable medium of FitzHugh-Nagumo cells where a proportion of cells were capable of remaining depolarized indefinitely. As such, those cells exhibit bistable membrane dynamics. We found that heterogeneous media can tolerate a surprisingly large number of bistable cells and still support normal rhythmic activity. Yet there is a critical limit beyond which the medium is persistently arrhythmogenic. Numerical analysis revealed that the critical threshold for arrhythmogenesis depends on both the strength of the coupling between cells and the extent to which the abnormal cells resist repolarization. Moreover, arrhythmogenesis was found to emerge preferentially at tissue boundaries where cells naturally have fewer neighbors to influence their behavior. These findings may explain why atrial fibrillation typically originates from tissue boundaries such as the cuff of the pulmonary vein.
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http://dx.doi.org/10.1371/journal.pcbi.1008683DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7909657PMC
February 2021

Co-expression of calcium and hERG potassium channels reduces the incidence of proarrhythmic events.

Cardiovasc Res 2021 08;117(10):2216-2227

University of New South Wales, Sydney, Kensington, NSW 2052, Australia.

Aims: Cardiac electrical activity is extraordinarily robust. However, when it goes wrong it can have fatal consequences. Electrical activity in the heart is controlled by the carefully orchestrated activity of more than a dozen different ion conductances. While there is considerable variability in cardiac ion channel expression levels between individuals, studies in rodents have indicated that there are modules of ion channels whose expression co-vary. The aim of this study was to investigate whether meta-analytic co-expression analysis of large-scale gene expression datasets could identify modules of co-expressed cardiac ion channel genes in human hearts that are of functional importance.

Methods And Results: Meta-analysis of 3653 public human RNA-seq datasets identified a strong correlation between expression of CACNA1C (L-type calcium current, ICaL) and KCNH2 (rapid delayed rectifier K+ current, IKr), which was also observed in human adult heart tissue samples. In silico modelling suggested that co-expression of CACNA1C and KCNH2 would limit the variability in action potential duration seen with variations in expression of ion channel genes and reduce susceptibility to early afterdepolarizations, a surrogate marker for proarrhythmia. We also found that levels of KCNH2 and CACNA1C expression are correlated in human-induced pluripotent stem cell-derived cardiac myocytes and the levels of CACNA1C and KCNH2 expression were inversely correlated with the magnitude of changes in repolarization duration following inhibition of IKr.

Conclusion: Meta-analytic approaches of multiple independent human gene expression datasets can be used to identify gene modules that are important for regulating heart function. Specifically, we have verified that there is co-expression of CACNA1C and KCNH2 ion channel genes in human heart tissue, and in silico analyses suggest that CACNA1C-KCNH2 co-expression increases the robustness of cardiac electrical activity.
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http://dx.doi.org/10.1093/cvr/cvaa280DOI Listing
August 2021

A constricted opening in Kir channels does not impede potassium conduction.

Nat Commun 2020 06 15;11(1):3024. Epub 2020 Jun 15.

Structural Biology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, 3052, Australia.

The canonical mechanistic model explaining potassium channel gating is of a conformational change that alternately dilates and constricts a collar-like intracellular entrance to the pore. It is based on the premise that K ions maintain a complete hydration shell while passing between the transmembrane cavity and cytosol, which must be accommodated. To put the canonical model to the test, we locked the conformation of a Kir K channel to prevent widening of the narrow collar. Unexpectedly, conduction was unimpaired in the locked channels. In parallel, we employed all-atom molecular dynamics to simulate K ions moving along the conduction pathway between the lower cavity and cytosol. During simulations, the constriction did not significantly widen. Instead, transient loss of some water molecules facilitated K permeation through the collar. The low free energy barrier to partial dehydration in the absence of conformational change indicates Kir channels are not gated by the canonical mechanism.
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http://dx.doi.org/10.1038/s41467-020-16842-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7295778PMC
June 2020

Pharmacological activation of IKr in models of long QT Type 2 risks overcorrection of repolarization.

Cardiovasc Res 2020 07;116(8):1434-1445

Victor Chang Cardiac Research Institute, 405 Liverpool Street, Darlinghurst, New South Wales 2010, Australia.

Aims: Current treatment for congenital long QT syndrome Type 2 (cLQTS2), an electrical disorder that increases the risk of life-threatening cardiac arrhythmias, is aimed at reducing the incidence of arrhythmia triggers (beta-blockers) or terminating the arrhythmia after onset (implantable cardioverter-defibrillator). An alternative strategy is to target the underlying disease mechanism, which is reduced rapid delayed rectifier current (IKr) passed by Kv11.1 channels. Small molecule activators of Kv11.1 have been identified but the extent to which these can restore normal cardiac signalling in cLQTS2 backgrounds remains unclear. Here, we examined the ability of ICA-105574, an activator of Kv11.1 that impairs transition to the inactivated state, to restore function to heterozygous Kv11.1 channels containing either inactivation enhanced (T618S, N633S) or expression deficient (A422T) mutations.

Methods And Results: ICA-105574 effectively restored Kv11.1 current from heterozygous inactivation enhanced or expression defective mutant channels in heterologous expression systems. In a human-induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model of cLQTS2 containing the expression defective Kv11.1 mutant A422T, cardiac repolarization, estimated from the duration of calcium transients in isolated cells and the rate corrected field potential duration (FPDc) in culture monolayers of cells, was significantly prolonged. The Kv11.1 activator ICA-105574 was able to reverse the prolonged repolarization in a concentration-dependent manner. However, at higher doses, ICA-105574 produced a shortening of the FPDc compared to controls. In vitro and in silico analysis suggests that this overcorrection occurs as a result of a temporal redistribution of the peak IKr to much earlier in the plateau phase of the action potential, which results in early repolarization.

Conclusion: Kv11.1 activators, which target the primary disease mechanism, provide a possible treatment option for cLQTS2, with the caveat that there may be a risk of overcorrection that could itself be pro-arrhythmic.
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http://dx.doi.org/10.1093/cvr/cvz247DOI Listing
July 2020

High-throughput phenotyping of heteromeric human ether-à-go-go-related gene potassium channel variants can discriminate pathogenic from rare benign variants.

Heart Rhythm 2020 03 23;17(3):492-500. Epub 2019 Sep 23.

Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales, Australia; St Vincent's Clinical School, UNSW Sydney, Darlinghurst, New South Wales, Australia. Electronic address:

Background: KCNH2 encodes the human ether-à-go-go-related gene potassium channel, which passes the rapid delayed rectifier potassium current. Loss-of-function variants in KCNH2 cause long QT syndrome type 2, which is associated with a markedly increased risk of cardiac arrhythmias. The majority of rare KCNH2 variants, however, are likely to be benign.

Objective: The purpose of this study was to develop a high-throughput assay for discriminating pathogenic from benign KCNH2 variants.

Methods: Nonsynonymous homozygous KCNH2 variants stably expressed in Flp-In human embryonic kidney 293 cell lines were phenotyped using an automated patch-clamp platform and a cell surface enzyme-linked immunosorbent assay. Functional phenotyping of heterozygous KCNH2 variants stably expressed in Flp-In human embryonic kidney 293 cell lines using a bicistronic vector was performed using an automated patch-clamp platform.

Results: In homozygous KCNH2 variant cell lines, discrepancies between current density and cell surface expression levels measured using an enzyme-linked immunosorbent assay can be explained by changes in gating properties of the variant channels. For the 30 heterozygous KCNH2 variant cell lines studied, the assay correctly predicted the ClinVar ascribed classification for 17/17 pathogenic/likely pathogenic/benign variants. Of the 13 pore-domain variants studied, 11 had a dominant-negative expression defect while the remaining 2 had enhanced inactivation gating, resulting in a dominant-negative phenotype.

Conclusion: High-throughput electrophysiological phenotyping of heterozygous KCNH2 variants can accurately distinguish between dominant-negative, haploinsufficient loss-of-function, and benign variants. This assay will help with future classification of KCNH2 variants.
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http://dx.doi.org/10.1016/j.hrthm.2019.09.020DOI Listing
March 2020

Introduction to the Australian Society for Biophysics.

Authors:
Adam P Hill

Biophys Rev 2019 Jun 8;11(3):253-254. Epub 2019 May 8.

Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales, Australia.

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http://dx.doi.org/10.1007/s12551-019-00519-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6557942PMC
June 2019

Protocol-Dependent Differences in IC Values Measured in Human Ether-Á-Go-Go-Related Gene Assays Occur in a Predictable Way and Can Be Used to Quantify State Preference of Drug Binding.

Mol Pharmacol 2019 05 15;95(5):537-550. Epub 2019 Feb 15.

Victor Chang Cardiac Research Institute (W.L., M.J.W., M.D.P., J.I.V., A.P.H.) and St Vincent's Clinical School (W.L., M.J.W., M.D.P., J.I.V., A.P.H.), University of New South Wales, Darlinghurst, New South Wales, Australia

Current guidelines around preclinical screening for drug-induced arrhythmias require the measurement of the potency of block of voltage-gated potassium channel subtype 11.1 (K11.1) as a surrogate for risk. A shortcoming of this approach is that the measured IC of K11.1 block varies widely depending on the voltage protocol used in electrophysiological assays. In this study, we aimed to investigate the factors that contribute to these differences and to identify whether it is possible to make predictions about protocol-dependent block that might facilitate the comparison of potencies measured using different assays. Our data demonstrate that state preferential binding, together with drug-binding kinetics and trapping, is an important determinant of the protocol dependence of K11.1 block. We show for the first time that differences in IC measured between protocols occurs in a predictable way, such that machine-learning algorithms trained using a selection of simple voltage protocols can indeed predict protocol-dependent potency. Furthermore, we also show that the preference of a drug for binding to the open versus the inactivated state of K11.1 can also be inferred from differences in IC values measured between protocols. Our work therefore identifies how state preferential drug binding is a major determinant of the protocol dependence of IC values measured in preclinical K11.1 assays. It also provides a novel method for quantifying the state dependence of K11.1 drug binding that will facilitate the development of more complete models of drug binding to K11.1 and improve our understanding of proarrhythmic risk associated with compounds that block K11.1.
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http://dx.doi.org/10.1124/mol.118.115220DOI Listing
May 2019

Experimentally Validated Pharmacoinformatics Approach to Predict hERG Inhibition Potential of New Chemical Entities.

Front Pharmacol 2018 19;9:1035. Epub 2018 Sep 19.

Research Center for Modeling and Simulation, National University of Science and Technology, Islamabad, Pakistan.

The hERG (human ether-a-go-go-related gene) encoded potassium ion (K) channel plays a major role in cardiac repolarization. Drug-induced blockade of hERG has been a major cause of potentially lethal ventricular tachycardia termed Torsades de Pointes (TdPs). Therefore, we presented a pharmacoinformatics strategy using combined ligand and structure based models for the prediction of hERG inhibition potential (IC) of new chemical entities (NCEs) during early stages of drug design and development. Integrated GRid-INdependent Descriptor (GRIND) models, and lipophilic efficiency (LipE), ligand efficiency (LE) guided template selection for the structure based pharmacophore models have been used for virtual screening and subsequent hERG activity (pIC) prediction of identified hits. Finally selected two hits were experimentally evaluated for hERG inhibition potential (pIC) using whole cell patch clamp assay. Overall, our results demonstrate a difference of less than ±1.6 log unit between experimentally determined and predicted hERG inhibition potential (IC) of the selected hits. This revealed predictive ability and robustness of our models and could help in correctly rank the potency order (lower μM to higher nM range) against hERG.
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http://dx.doi.org/10.3389/fphar.2018.01035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6176658PMC
September 2018

Endocardial TRPC-6 Channels Act as Atrial Mechanosensors and Load-Dependent Modulators of Endocardial/Myocardial Cross-Talk.

JACC Basic Transl Sci 2017 Oct 30;2(5):575-590. Epub 2017 Oct 30.

Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales, Australia.

Mechanoelectrical feedback may increase arrhythmia susceptibility, but the molecular mechanisms are incompletely understood. This study showed that mechanical stretch altered the localization, protein levels, and function of the cation-selective transient receptor potential channel (TRPC)-6 in atrial endocardial cells in humans, pigs, and mice. In endocardial/myocardial cross-talk studies, addition of media from porcine atrial endocardium (AE) cells altered the calcium (Ca) transient characteristics of human-induced pluripotent stem cell-derived cardiomyocytes. These changes did not occur with media from stretched AE cells. Our data suggested that endocardial TRPC-6-dependent paracrine signaling may modulate myocardial Ca homeostasis under basal conditions and protect against stretch-induced atrial arrhythmias.
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http://dx.doi.org/10.1016/j.jacbts.2017.05.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6058914PMC
October 2017

The Temperature Dependence of Kinetics Associated with Drug Block of hERG Channels Is Compound-Specific and an Important Factor for Proarrhythmic Risk Prediction.

Mol Pharmacol 2018 07 4;94(1):760-769. Epub 2018 May 4.

Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales, Australia and St Vincent's Clinical School, University of NSW, Darlinghurst, New South Wales, Australia

Current mandated preclinical tests for drug-induced proarrhythmia are very sensitive, but not sufficiently specific. This has led to concern that there is a high attrition rate of potentially safe drugs that could have been beneficial to patients. The comprehensive in vitro proarrhythmia initiative has proposed new metrics based around in silico risk predictions, which are informed, among other things, by measures of human ether-à-go-go-related gene channel (hERG) block kinetics. However, high-throughput patch-clamp systems set to collect these data largely operate at ambient temperature, whereas the simulations for risk prediction are carried out at physiologic temperature. The aims of this study were to: 1) determine to what degree kinetics of drug block of hERG are temperature-dependent, 2) assess the impact of any temperature dependence of drug binding kinetics on repolarization in silico, and 3) identify whether a common set of Q scalars can be used to extrapolate kinetic data gathered at ambient to physiologic temperatures for use in in silico proarrhythmic risk prediction. We show that, for a range of drugs, kinetics of block are temperature-dependent and, furthermore, that the degree of temperature dependence is different for each drug. As a result, no common set of Q scalars could describe the observed range of temperature dependencies. These results suggest that if accurate physiologic temperature models of the kinetics of drug binding are important for in silico risk prediction, the in vitro data should be acquired at physiologic temperature.
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http://dx.doi.org/10.1124/mol.117.111534DOI Listing
July 2018

Sinusoidal voltage protocols for rapid characterisation of ion channel kinetics.

J Physiol 2018 05 17;596(10):1813-1828. Epub 2018 Apr 17.

Centre for Mathematical Medicine & Biology, School of Mathematical Sciences, University of Nottingham, Nottingham, NG7 2RD, UK.

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.

Abstract: Understanding 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.
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http://dx.doi.org/10.1113/JP275733DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5978315PMC
May 2018

Structural Dynamics of the MscL C-terminal Domain.

Sci Rep 2017 12 8;7(1):17229. Epub 2017 Dec 8.

Department of Biochemistry and Molecular Biology, University of Chicago, 929 E 57th St, Chicago, Illinois, 60637, USA.

The large conductance mechanosensitive channel (MscL), acts as an osmoprotective emergency valve in bacteria by opening a large, water-filled pore in response to changes in membrane tension. In its closed configuration, the last 36 residues at the C-terminus form a bundle of five α-helices co-linear with the five-fold axis of symmetry. Here, we examined the structural dynamics of the C-terminus of EcMscL using site-directed spin labelling electron paramagnetic resonance (SDSL EPR) spectroscopy. These experiments were complemented with computational modelling including molecular dynamics (MD) simulations and finite element (FE) modelling. Our results show that under physiological conditions, the C-terminus is indeed an α-helical bundle, located near the five-fold symmetry axis of the molecule. Both experiments and computational modelling demonstrate that only the top part of the C-terminal domain (from the residue A110 to E118) dissociates during the channel gating, while the rest of the C-terminus stays assembled. This result is consistent with the view that the C-terminus functions as a molecular sieve and stabilizer of the oligomeric MscL structure as previously suggested.
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http://dx.doi.org/10.1038/s41598-017-17396-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5722894PMC
December 2017

Risk Assessment in Acquired Long QT Syndrome: The Devil Is in the Details.

Front Physiol 2017 16;8:934. Epub 2017 Nov 16.

Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Darlinghurst, NSW, Australia.

Acquired long QT syndrome, mostly as a result of drug block of the Kv11. 1 potassium channel in the heart, is characterized by delayed cardiac myocyte repolarization, prolongation of the T interval on the ECG, syncope and sudden cardiac death due to the polymorphic ventricular arrhythmia Torsade de Pointes (TdP). In recent years, efforts are underway through the Comprehensive proarrhythmic assay (CiPA) initiative, to develop better tests for this drug induced arrhythmia based in part on simulations of pharmacological disruption of repolarization. However, drug binding to Kv11.1 is more complex than a simple binary molecular reaction, meaning simple steady state measures of potency are poor surrogates for risk. As a result, there is a plethora of mechanistic detail describing the drug/Kv11.1 interaction-such as drug binding kinetics, state preference, temperature dependence and trapping-that needs to be considered when developing models for risk prediction. In addition to this, other factors, such as multichannel pharmacological profile and the nature of the ventricular cell models used in simulations also need to be considered in the search for the optimum approach. Here we consider how much of mechanistic detail needs to be included for models to accurately predict risk and further, how much of this detail can be retrieved from protocols that are practical to implement in high throughout screens as part of next generation of preclinical drug screening approaches?
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http://dx.doi.org/10.3389/fphys.2017.00934DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5696636PMC
November 2017

Recent advances in understanding and prevention of sudden cardiac death.

F1000Res 2017 31;6:1614. Epub 2017 Aug 31.

St Vincent's Clinical School, University of New South Wales, Darlinghurst, Australia.

There have been tremendous advances in the diagnosis and treatment of heart disease over the last 50 years. Nevertheless, it remains the number one cause of death. About half of heart-related deaths occur suddenly, and in about half of these cases the person was unaware that they had underlying heart disease. Genetic heart disease accounts for only approximately 2% of sudden cardiac deaths, but as it typically occurs in younger people it has been a particular focus of activity in our quest to not only understand the underlying mechanisms of cardiac arrhythmogenesis but also develop better strategies for earlier detection and prevention. In this brief review, we will highlight trends in the recent literature focused on sudden cardiac death in genetic heart diseases and how these studies are contributing to a broader understanding of sudden death in the community.
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http://dx.doi.org/10.12688/f1000research.11855.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5583740PMC
August 2017

Measuring kinetics and potency of hERG block for CiPA.

J Pharmacol Toxicol Methods 2017 Sep 10;87:99-107. Epub 2017 Feb 10.

Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, 405 Liverpool Street, Sydney, NSW 2010, Australia; St. Vincent's Clinical School, University of New South Wales, Sydney, NSW 2052, Australia. Electronic address:

Introduction: The Comprehensive in vitro Proarrhythmic Assay (CiPA) aims to update current cardiac safety testing to better evaluate arrhythmic risk. A central theme of CiPA is the use of in silico approaches to risk prediction incorporating models of drug binding to hERG. To parameterize these models, accurate in vitro measurement of potency and kinetics of block is required. The Ion Channel Working Group was tasked with: i) selecting a protocol that could measure kinetics of block and was easily implementable on automated platforms for future rollout in industry and ii) acquiring a reference dataset using the standardized protocol.

Methods: Data were acquired using a 'step depolarisation' protocol using manual patch-clamp at ambient temperature.

Results: Potency, kinetics and trapping characteristics of hERG block for the CiPA training panel of twelve drugs were measured. Timecourse of block and trapping characteristics could be reliably measured if the time constant for onset of block was between ~500ms and ~15s. Seven drugs, however had time courses of block faster than this cut-off.

Discussion: Here we describe the implementation of the standardized protocol for measurement of kinetics and potency of hERG block for CiPA. The results highlight the challenges in identifying a single protocol to measure hERG block over a range of kinetics. The dataset from this study is being used by the In Silico Working Group to develop models of drug binding for risk prediction and is freely available as a 'gold standard' ambient temperature dataset to evaluate variability across high throughput platforms.
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http://dx.doi.org/10.1016/j.vascn.2017.02.017DOI Listing
September 2017

Boosting the reserves: additive regulation of cardiac repolarisation.

Authors:
Adam P Hill

J Physiol 2017 04 21;595(7):2269-2270. Epub 2017 Feb 21.

Computational Cardiology, Victor Chang Cardiac Research Institute, Sydney, NSW, 2226, Australia.

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http://dx.doi.org/10.1113/JP273940DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5374104PMC
April 2017

Convergence of models of human ventricular myocyte electrophysiology after global optimization to recapitulate clinical long QT phenotypes.

J Mol Cell Cardiol 2016 Nov 20;100:25-34. Epub 2016 Sep 20.

Victor Chang Cardiac Research Institute, 405 Liverpool Street, Darlinghurst, NSW 2010, Australia; St Vincent's Clinical School, University of New South Wales, Australia. Electronic address:

In-silico models of human cardiac electrophysiology are now being considered for prediction of cardiotoxicity as part of the preclinical assessment phase of all new drugs. We ask the question whether any of the available models are actually fit for this purpose. We tested three models of the human ventricular action potential, the O'hara-Rudy (ORD11), the Grandi-Bers (GB10) and the Ten Tusscher (TT06) models. We extracted clinical QT data for LQTS1 and LQTS2 patients with nonsense mutations that would be predicted to cause 50% loss of function in I and I respectively. We also obtained clinical QT data for LQTS3 patients. We then used a global optimization approach to improve the existing in silico models so that they reproduced all three clinical data sets more closely. We also examined the effects of adrenergic stimulation in the different LQTS subsets. All models, in their original form, produce markedly different and unrealistic predictions of QT prolongation for LQTS1, 2 and 3. After global optimization of the maximum conductances for membrane channels, all models have similar current densities during the action potential, despite differences in kinetic properties of the channels in the different models, and more closely reproduce the prolongation of repolarization seen in all LQTS subtypes. In-silico models of cardiac electrophysiology have the potential to be tremendously useful in complementing traditional preclinical drug testing studies. However, our results demonstrate they should be carefully validated and optimized to clinical data before they can be used for this purpose.
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http://dx.doi.org/10.1016/j.yjmcc.2016.09.011DOI Listing
November 2016

The role of MscL amphipathic N terminus indicates a blueprint for bilayer-mediated gating of mechanosensitive channels.

Nat Commun 2016 06 22;7:11984. Epub 2016 Jun 22.

Division of Molecular Cardiology and Biophysics, Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales 2010, Australia.

The bacterial mechanosensitive channel MscL gates in response to membrane tension as a result of mechanical force transmitted directly to the channel from the lipid bilayer. MscL represents an excellent model system to study the basic biophysical principles of mechanosensory transduction. However, understanding of the essential structural components that transduce bilayer tension into channel gating remains incomplete. Here using multiple experimental and computational approaches, we demonstrate that the amphipathic N-terminal helix of MscL acts as a crucial structural element during tension-induced gating, both stabilizing the closed state and coupling the channel to the membrane. We propose that this may also represent a common principle in the gating cycle of unrelated mechanosensitive ion channels, allowing the coupling of channel conformation to membrane dynamics.
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http://dx.doi.org/10.1038/ncomms11984DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4917966PMC
June 2016

Heritability of ECG Biomarkers in the Netherlands Twin Registry Measured from Holter ECGs.

Front Physiol 2016 29;7:154. Epub 2016 Apr 29.

Department of Biological Psychology, EMGO+ Institute, VU University and VU Medical Center Amsterdam, Netherlands.

Introduction: The resting ECG is the most commonly used tool to assess cardiac electrophysiology. Previous studies have estimated heritability of ECG parameters based on these snapshots of the cardiac electrical activity. In this study we set out to determine whether analysis of heart rate specific data from Holter ECGs allows more complete assessment of the heritability of ECG parameters.

Methods And Results: Holter ECGs were recorded from 221 twin pairs and analyzed using a multi-parameter beat binning approach. Heart rate dependent estimates of heritability for QRS duration, QT interval, Tpeak-Tend and Theight were calculated using structural equation modeling. QRS duration is largely determined by environmental factors whereas repolarization is primarily genetically determined. Heritability estimates of both QT interval and Theight were significantly higher when measured from Holter compared to resting ECGs and the heritability estimate of each was heart rate dependent. Analysis of the genetic contribution to correlation between repolarization parameters demonstrated that covariance of individual ECG parameters at different heart rates overlap but at each specific heart rate there was relatively little overlap in the genetic determinants of the different repolarization parameters.

Conclusions: Here we present the first study of heritability of repolarization parameters measured from Holter ECGs. Our data demonstrate that higher heritability can be estimated from the Holter than the resting ECG and reveals rate dependence in the genetic-environmental determinants of the ECG that has not previously been tractable. Future applications include deeper dissection of the ECG of participants with inherited cardiac electrical disease.
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http://dx.doi.org/10.3389/fphys.2016.00154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4850154PMC
May 2016

Temperature Effects on Kinetics of KV11.1 Drug Block Have Important Consequences for In Silico Proarrhythmic Risk Prediction.

Mol Pharmacol 2016 07 12;90(1):1-11. Epub 2016 May 12.

Computational Cardiology, Victor Chang Cardiac Research Institute, Darlinghurst, Australia (M.J.W., S.A.M., J.I.V., A.P.H.); and St. Vincent's Clinical School, University of New South Wales, Darlinghurst, Australia (S.A.M., J.I.V., A.P.H.)

Drug block of voltage-gated potassium channel subtype 11.1 human ether-a-go-go related gene (Kv11.1) (hERG) channels, encoded by the KCNH2 gene, is associated with reduced repolarization of the cardiac action potential and is the predominant cause of acquired long QT syndrome that can lead to fatal cardiac arrhythmias. Current safety guidelines require that potency of KV11.1 block is assessed in the preclinical phase of drug development. However, not all drugs that block KV11.1 are proarrhythmic, meaning that screening on the basis of equilibrium measures of block can result in high attrition of potentially low-risk drugs. The basis of the next generation of drug-screening approaches is set to be in silico risk prediction, informed by in vitro mechanistic descriptions of drug binding, including measures of the kinetics of block. A critical issue in this regard is characterizing the temperature dependence of drug binding. Specifically, it is important to address whether kinetics relevant to physiologic temperatures can be inferred or extrapolated from in vitro data gathered at room temperature in high-throughout systems. Here we present the first complete study of the temperature-dependent kinetics of block and unblock of a proarrhythmic drug, cisapride, to KV11.1. Our data highlight a complexity to binding that manifests at higher temperatures and can be explained by accumulation of an intermediate, non-blocking encounter-complex. These results suggest that for cisapride, physiologically relevant kinetic parameters cannot be simply extrapolated from those measured at lower temperatures; rather, data gathered at physiologic temperatures should be used to constrain in silico models that may be used for proarrhythmic risk prediction.
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http://dx.doi.org/10.1124/mol.115.103127DOI Listing
July 2016

An 'alternans' way to quantify arrhythmogenic substrates.

J Physiol 2016 05;594(9):2375-6

Victor Chang Cardiac Research Institute, 405 Liverpool Street, Darlinghurst, NSW, 2010, Australia.

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http://dx.doi.org/10.1113/JP271838DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4850204PMC
May 2016

Computational cardiology and risk stratification for sudden cardiac death: one of the grand challenges for cardiology in the 21st century.

J Physiol 2016 12 9;594(23):6893-6908. Epub 2016 Jun 9.

Victor Chang Cardiac Research Institute, 405 Liverpool Street, Darlinghurst, NSW, 2010, Australia.

Risk stratification in the context of sudden cardiac death has been acknowledged as one of the major challenges facing cardiology for the past four decades. In recent years, the advent of high performance computing has facilitated organ-level simulation of the heart, meaning we can now examine the causes, mechanisms and impact of cardiac dysfunction in silico. As a result, computational cardiology, largely driven by the Physiome project, now stands at the threshold of clinical utility in regards to risk stratification and treatment of patients at risk of sudden cardiac death. In this white paper, we outline a roadmap of what needs to be done to make this translational step, using the relatively well-developed case of acquired or drug-induced long QT syndrome as an exemplar case.
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http://dx.doi.org/10.1113/JP272015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5134408PMC
December 2016

Rescue of protein expression defects may not be enough to abolish the pro-arrhythmic phenotype of long QT type 2 mutations.

J Physiol 2016 07 27;594(14):4031-49. Epub 2016 May 27.

Victor Chang Cardiac Research Institute, Molecular Cardiology and Biophysics Division, Darlinghurst, NSW, 2010, Australia.

Key Points: Most missense long QT syndrome type 2 (LQTS2) mutations result in Kv11.1 channels that show reduced levels of membrane expression. Pharmacological chaperones that rescue mutant channel expression could have therapeutic potential to reduce the risk of LQTS2-associated arrhythmias and sudden cardiac death, but only if the mutant Kv11.1 channels function normally (i.e. like WT channels) after membrane expression is restored. Fewer than half of mutant channels exhibit relatively normal function after rescue by low temperature. The remaining rescued missense mutant Kv11.1 channels have perturbed gating and/or ion selectivity characteristics. Co-expression of WT subunits with gating defective missense mutations ameliorates but does not eliminate the functional abnormalities observed for most mutant channels. For patients with mutations that affect gating in addition to expression, it may be necessary to use a combination therapy to restore both normal function and normal expression of the channel protein.

Abstract: In the heart, Kv11.1 channels pass the rapid delayed rectifier current (IKr ) which plays critical roles in repolarization of the cardiac action potential and in the suppression of arrhythmias caused by premature stimuli. Over 500 inherited mutations in Kv11.1 are known to cause long QT syndrome type 2 (LQTS2), a cardiac electrical disorder associated with an increased risk of life threatening arrhythmias. Most missense mutations in Kv11.1 reduce the amount of channel protein expressed at the membrane and, as a consequence, there has been considerable interest in developing pharmacological agents to rescue the expression of these channels. However, pharmacological chaperones will only have clinical utility if the mutant Kv11.1 channels function normally after membrane expression is restored. The aim of this study was to characterize the gating phenotype for a subset of LQTS2 mutations to assess what proportion of mutations may be suitable for rescue. As an initial screen we used reduced temperature to rescue expression defects of mutant channels expressed in Xenopus laevis oocytes. Over half (∼56%) of Kv11.1 mutants exhibited functional gating defects that either dramatically reduced the amount of current contributing to cardiac action potential repolarization and/or reduced the amount of protective current elicited in response to premature depolarizations. Our data demonstrate that if pharmacological rescue of protein expression defects is going to have clinical utility in the treatment of LQTS2 then it will be important to assess the gating phenotype of LQTS2 mutations before attempting rescue.
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http://dx.doi.org/10.1113/JP271805DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4945714PMC
July 2016

In silico assessment of kinetics and state dependent binding properties of drugs causing acquired LQTS.

Prog Biophys Mol Biol 2016 Jan 20;120(1-3):89-99. Epub 2015 Dec 20.

Victor Chang Cardiac Research Institute, 405 Liverpool Street, Darlinghurst, NSW 2010, Australia; St Vincent's Clinical School, University of New South Wales, NSW 2052, Australia. Electronic address:

The Kv11.1 or hERG potassium channel is responsible for one of the major repolarising currents (IKr) in cardiac myocytes. Drug binding to hERG can result in reduction in IKr, action potential prolongation, acquired long QT syndrome and fatal cardiac arrhythmias. The current guidelines for pre-clinical assessment of drugs in development is based on the measurement of the drug concentration that causes 50% current block, i.e., IC50. However, drugs with the same apparent IC50 may have very different kinetics of binding and unbinding, as well as different affinities for the open and inactivated states of Kv11.1. Therefore, IC50 measurements may not reflect the true risk of drug induced arrhythmias. Here we have used an in silico approach to test the hypothesis that drug binding kinetics and differences in state-dependent affinity will influence the extent of cardiac action potential prolongation independent of apparent IC50 values. We found, in general that drugs with faster overall kinetics and drugs with higher affinity for the open state relative to the inactivated state cause more action potential prolongation. These characteristics of drug-hERG interaction are likely to be more arrhythmogenic but cannot be predicted by IC50 measurement alone. Our results suggest that the pre-clinical assessment of Kv11.1-drug interactions should include descriptions of the kinetics and state dependence of drug binding. Further, incorporation of this information into sophisticated in silico models should be able to better predict arrhythmia risk and therefore more accurately assess safety of new drugs in development.
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http://dx.doi.org/10.1016/j.pbiomolbio.2015.12.005DOI Listing
January 2016

PhosphOrtholog: a web-based tool for cross-species mapping of orthologous protein post-translational modifications.

BMC Genomics 2015 Aug 19;16:617. Epub 2015 Aug 19.

School of Mathematics and Statistics, University of Sydney, Camperdown, NSW, 2006, Australia.

Background: Most biological processes are influenced by protein post-translational modifications (PTMs). Identifying novel PTM sites in different organisms, including humans and model organisms, has expedited our understanding of key signal transduction mechanisms. However, with increasing availability of deep, quantitative datasets in diverse species, there is a growing need for tools to facilitate cross-species comparison of PTM data. This is particularly important because functionally important modification sites are more likely to be evolutionarily conserved; yet cross-species comparison of PTMs is difficult since they often lie in structurally disordered protein domains. Current tools that address this can only map known PTMs between species based on known orthologous phosphosites, and do not enable the cross-species mapping of newly identified modification sites. Here, we addressed this by developing a web-based software tool, PhosphOrtholog ( www.phosphortholog.com ) that accurately maps protein modification sites between different species. This facilitates the comparison of datasets derived from multiple species, and should be a valuable tool for the proteomics community.

Results: Here we describe PhosphOrtholog, a web-based application for mapping known and novel orthologous PTM sites from experimental data obtained from different species. PhosphOrtholog is the only generic and automated tool that enables cross-species comparison of large-scale PTM datasets without relying on existing PTM databases. This is achieved through pairwise sequence alignment of orthologous protein residues. To demonstrate its utility we apply it to two sets of human and rat muscle phosphoproteomes generated following insulin and exercise stimulation, respectively, and one publicly available mouse phosphoproteome following cellular stress revealing high mapping and coverage efficiency. Although coverage statistics are dataset dependent, PhosphOrtholog increased the number of cross-species mapped sites in all our example data sets by more than double when compared to those recovered using existing resources such as PhosphoSitePlus.

Conclusions: PhosphOrtholog is the first tool that enables mapping of thousands of novel and known protein phosphorylation sites across species, accessible through an easy-to-use web interface. Identification of conserved PTMs across species from large-scale experimental data increases our knowledgebase of functional PTM sites. Moreover, PhosphOrtholog is generic being applicable to other PTM datasets such as acetylation, ubiquitination and methylation.
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http://dx.doi.org/10.1186/s12864-015-1820-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4539857PMC
August 2015

Getting to the heart of hERG K(+) channel gating.

J Physiol 2015 Jun;593(12):2575-85

Victor Chang Cardiac Research Institute, 405 Liverpool Street, Darlinghurst, NSW 2010, Australia.

Potassium ion channels encoded by the human ether-a-go-go related gene (hERG) form the ion-conducting subunit of the rapid delayed rectifier potassium current (IKr ). Although hERG channels exhibit a widespread tissue distribution they play a particularly important role in the heart. There has been considerable interest in hERG K(+) channels for three main reasons. First, they have very unusual gating kinetics, most notably rapid and voltage-dependent inactivation coupled to slow deactivation, which has led to the suggestion that they may play a specific role in the suppression of arrhythmias. Second, mutations in hERG are the cause of 30-40% of cases of congenital long QT syndrome (LQTS), the commonest inherited primary arrhythmia syndrome. Third, hERG is the molecular target for the vast majority of drugs that cause drug-induced LQTS, the commonest cause of drug-induced arrhythmias and cardiac death. Drug-induced LQTS has now been reported for a large range of both cardiac and non-cardiac drugs, in which this side effect is entirely undesired. In recent years there have been comprehensive reviews published on hERG K(+) channels (Vandenberg et al. 2012) and we will not re-cover this ground. Rather, we focus on more recent work on the structural basis and dynamics of hERG gating with an emphasis on how the latest developments may facilitate translational research in the area of stratifying risk of arrhythmias.
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http://dx.doi.org/10.1113/JP270095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4500344PMC
June 2015
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