Publications by authors named "Matthew D Perry"

29 Publications

  • Page 1 of 1

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

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

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

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

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

The S1 helix critically regulates the finely tuned gating of Kv11.1 channels.

J Biol Chem 2017 05 9;292(18):7688-7705. Epub 2017 Mar 9.

From the Victor Chang Cardiac Research Institute, 405 Liverpool Street, Darlinghurst, New South Wales 2010,

Congenital mutations in the cardiac Kv11.1 channel can cause long QT syndrome type 2 (LQTS2), a heart rhythm disorder associated with sudden cardiac death. Mutations act either by reducing protein expression at the membrane and/or by perturbing the intricate gating properties of Kv11.1 channels. A number of clinical LQTS2-associated mutations have been reported in the first transmembrane segment (S1) of Kv11.1 channels, but the role of this region of the channel is largely unexplored. In part, this is due to problems defining the extent of the S1 helix, as a consequence of its low sequence homology with other Kv family members. Here, we used NMR spectroscopy and electrophysiological characterization to show that the S1 of Kv11.1 channels extends seven helical turns, from Pro-405 to Phe-431, and is flanked by unstructured loops. Functional analysis suggests that pre-S1 loop residues His-402 and Tyr-403 play an important role in regulating the kinetics and voltage dependence of channel activation and deactivation. Multiple residues within the S1 helix also play an important role in fine-tuning the voltage dependence of activation, regulating slow deactivation, and modulating C-type inactivation of Kv11.1 channels. Analyses of LQTS2-associated mutations in the pre-S1 loop or S1 helix of Kv11.1 channels demonstrate perturbations to both protein expression and most gating transitions. Thus, S1 region mutations would reduce both the action potential repolarizing current passed by Kv11.1 channels in cardiac myocytes, as well as the current passed in response to premature depolarizations that normally helps protect against the formation of ectopic beats.
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http://dx.doi.org/10.1074/jbc.M117.779298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5418064PMC
May 2017

Segmental differences in upregulated apical potassium channels in mammalian colon during potassium adaptation.

Am J Physiol Gastrointest Liver Physiol 2016 11 8;311(5):G785-G793. Epub 2016 Sep 8.

Leeds Institute of Biomedical and Clinical Sciences, St James's University Hospital, Leeds, United Kingdom;

Rat proximal and distal colon are net K secretory and net K absorptive epithelia, respectively. Chronic dietary K loading increases net K secretion in the proximal colon and transforms net K absorption to net K secretion in the distal colon, but changes in apical K channel expression are unclear. We evaluated expression/activity of apical K (BK) channels in surface colonocytes in proximal and distal colon of control and K-loaded animals using patch-clamp recording, immunohistochemistry, and Western blot analyses. In controls, BK channels were more abundant in surface colonocytes from K secretory proximal colon (39% of patches) than in those from K-absorptive distal colon (12% of patches). Immunostaining demonstrated more pronounced BK channel α-subunit protein expression in surface cells and cells in the upper 25% of crypts in proximal colon, compared with distal colon. Dietary K loading had no clear-cut effects on the abundance, immunolocalization, or expression of BK channels in proximal colon. By contrast, in distal colon, K loading 1) increased BK channel abundance in patches from 12 to 41%; 2) increased density of immunostaining in surface cells, which extended along the upper 50% of crypts; and 3) increased expression of BK channel α-subunit protein when assessed by Western blotting (P < 0.001). Thus apical BK channels are normally more abundant in K secretory proximal colon than in K absorptive distal colon, and apical BK channel expression in distal (but not proximal) colon is greatly stimulated as part of the enhanced K secretory response to dietary K loading.
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http://dx.doi.org/10.1152/ajpgi.00181.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5130553PMC
November 2016

TECRL: connecting sequence to consequence for a new sudden cardiac death gene.

EMBO Mol Med 2016 12 1;8(12):1364-1365. Epub 2016 Dec 1.

Mark Cowley Lidwill Research Programme in Cardiac Electrophysiology, Victor Chang Cardiac Research Institute, Darlinghurst, NSW, Australia.

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http://dx.doi.org/10.15252/emmm.201606967DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5167129PMC
December 2016

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

Tyrosine Residues from the S4-S5 Linker of Kv11.1 Channels Are Critical for Slow Deactivation.

J Biol Chem 2016 08 17;291(33):17293-302. Epub 2016 Jun 17.

From the Victor Chang Cardiac Research Institute, 405 Liverpool Street, Darlinghurst and the St. Vincent's Clinical School, University of New South Wales, Victoria Street, Darlinghurst, New South Wales 2010, Australia and

Slow deactivation of Kv11.1 channels is critical for its function in the heart. The S4-S5 linker, which joins the voltage sensor and pore domains, plays a critical role in this slow deactivation gating. Here, we use NMR spectroscopy to identify the membrane-bound surface of the S4S5 linker, and we show that two highly conserved tyrosine residues within the KCNH subfamily of channels are membrane-associated. Site-directed mutagenesis and electrophysiological analysis indicates that Tyr-542 interacts with both the pore domain and voltage sensor residues to stabilize activated conformations of the channel, whereas Tyr-545 contributes to the slow kinetics of deactivation by primarily stabilizing the transition state between the activated and closed states. Thus, the two tyrosine residues in the Kv11.1 S4S5 linker play critical but distinct roles in the slow deactivation phenotype, which is a hallmark of Kv11.1 channels.
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http://dx.doi.org/10.1074/jbc.M116.729392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5016128PMC
August 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

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

Steady-state modulation of voltage-gated K+ channels in rat arterial smooth muscle by cyclic AMP-dependent protein kinase and protein phosphatase 2B.

PLoS One 2015 20;10(3):e0121285. Epub 2015 Mar 20.

Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, United Kingdom.

Voltage-gated potassium channels (Kv) are important regulators of membrane potential in vascular smooth muscle cells, which is integral to controlling intracellular Ca2+ concentration and regulating vascular tone. Previous work indicates that Kv channels can be modulated by receptor-driven alterations of cyclic AMP-dependent protein kinase (PKA) activity. Here, we demonstrate that Kv channel activity is maintained by tonic activity of PKA. Whole-cell recording was used to assess the effect of manipulating PKA signalling on Kv and ATP-dependent K+ channels of rat mesenteric artery smooth muscle cells. Application of PKA inhibitors, KT5720 or H89, caused a significant inhibition of Kv currents. Tonic PKA-mediated activation of Kv appears maximal as application of isoprenaline (a β-adrenoceptor agonist) or dibutyryl-cAMP failed to enhance Kv currents. We also show that this modulation of Kv by PKA can be reversed by protein phosphatase 2B/calcineurin (PP2B). PKA-dependent inhibition of Kv by KT5720 can be abrogated by pre-treatment with the PP2B inhibitor cyclosporin A, or inclusion of a PP2B auto-inhibitory peptide in the pipette solution. Finally, we demonstrate that tonic PKA-mediated modulation of Kv requires intact caveolae. Pre-treatment of the cells with methyl-β-cyclodextrin to deplete cellular cholesterol, or adding caveolin-scaffolding domain peptide to the pipette solution to disrupt caveolae-dependent signalling each attenuated PKA-mediated modulation of the Kv current. These findings highlight a novel, caveolae-dependent, tonic modulatory role of PKA on Kv channels providing new insight into mechanisms and the potential for pharmacological manipulation of vascular tone.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0121285PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4368632PMC
February 2016

Can many subunits make light work of ion channel inactivation?

J Physiol 2014 Oct;592(20):4411-2

Victor Chang Cardiac Research Institute, Level 6, 405 Liverpool Street, Darlinghurst, NSW 2010, Australia St Vincent's Clinical School, University of New South Wales, Victoria Street, Darlinghurst, NSW 2010, Australia.

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http://dx.doi.org/10.1113/jphysiol.2014.281568DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4287733PMC
October 2014

Multiscale cardiac modelling reveals the origins of notched T waves in long QT syndrome type 2.

Nat Commun 2014 Sep 25;5:5069. Epub 2014 Sep 25.

1] Division of Molecular Cardiology and Biophysics, Victor Chang Cardiac Research Institute, 405 Liverpool Street, Darlinghurst, New South Wales 2010, Australia [2] St Vincent's Clinical School, University of New South Wales, Sydney, New South Wales 2052, Australia.

The heart rhythm disorder long QT syndrome (LQTS) can result in sudden death in the young or remain asymptomatic into adulthood. The features of the surface electrocardiogram (ECG), a measure of the electrical activity of the heart, can be equally variable in LQTS patients, posing well-described diagnostic dilemmas. Here we report a correlation between QT interval prolongation and T-wave notching in LQTS2 patients and use a novel computational framework to investigate how individual ionic currents, as well as cellular and tissue level factors, contribute to notched T waves. Furthermore, we show that variable expressivity of ECG features observed in LQTS2 patients can be explained by as little as 20% variation in the levels of ionic conductances that contribute to repolarization reserve. This has significant implications for interpretation of whole-genome sequencing data and underlies the importance of interpreting the entire molecular signature of disease in any given individual.
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http://dx.doi.org/10.1038/ncomms6069DOI Listing
September 2014

Multiple interactions between cytoplasmic domains regulate slow deactivation of Kv11.1 channels.

J Biol Chem 2014 Sep 29;289(37):25822-32. Epub 2014 Jul 29.

From the Victor Chang Cardiac Research Institute and St. Vincent's Clinical School, University of New South Wales, Darlinghurst, New South Wales 2010, Australia.

The intracellular domains of many ion channels are important for fine-tuning their gating kinetics. In Kv11.1 channels, the slow kinetics of channel deactivation, which are critical for their function in the heart, are largely regulated by the N-terminal N-Cap and Per-Arnt-Sim (PAS) domains, as well as the C-terminal cyclic nucleotide-binding homology (cNBH) domain. Here, we use mutant cycle analysis to probe for functional interactions between the N-Cap/PAS domains and the cNBH domain. We identified a specific and stable charge-charge interaction between Arg(56) of the PAS domain and Asp(803) of the cNBH domain, as well an additional interaction between the cNBH domain and the N-Cap, both of which are critical for maintaining slow deactivation kinetics. Furthermore, we found that positively charged arginine residues within the disordered region of the N-Cap interact with negatively charged residues of the C-linker domain. Although this interaction is likely more transient than the PAS-cNBD interaction, it is strong enough to stabilize the open conformation of the channel and thus slow deactivation. These findings provide novel insights into the slow deactivation mechanism of Kv11.1 channels.
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http://dx.doi.org/10.1074/jbc.M114.558379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4162183PMC
September 2014

Role of the cytoplasmic N-terminal Cap and Per-Arnt-Sim (PAS) domain in trafficking and stabilization of Kv11.1 channels.

J Biol Chem 2014 May 2;289(20):13782-91. Epub 2014 Apr 2.

From the Mark Cowley Lidwill Research Program in Cardiac Electrophysiology, Victor Chang Cardiac Research Institute, 405 Liverpool Street and St. Vincent's Clinical School, University of New South Wales, Victoria Street, Darlinghurst, New South Wales 2010, Australia

The N-terminal cytoplasmic region of the Kv11.1a potassium channel contains a Per-Arnt-Sim (PAS) domain that is essential for the unique slow deactivation gating kinetics of the channel. The PAS domain has also been implicated in the assembly and stabilization of the assembled tetrameric channel, with many clinical mutants in the PAS domain resulting in reduced stability of the domain and reduced trafficking. Here, we use quantitative Western blotting to show that the PAS domain is not required for normal channel trafficking nor for subunit-subunit interactions, and it is not necessary for stabilizing assembled channels. However, when the PAS domain is present, the N-Cap amphipathic helix must also be present for channels to traffic to the cell membrane. Serine scan mutagenesis of the N-Cap amphipathic helix identified Leu-15, Ile-18, and Ile-19 as residues critical for the stabilization of full-length proteins when the PAS domain is present. Furthermore, mutant cycle analysis experiments support recent crystallography studies, indicating that the hydrophobic face of the N-Cap amphipathic helix interacts with a surface-exposed hydrophobic patch on the core of the PAS domain to stabilize the structure of this critical gating domain. Our data demonstrate that the N-Cap amphipathic helix is critical for channel stability and trafficking.
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http://dx.doi.org/10.1074/jbc.M113.531277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4022852PMC
May 2014

C-terminal β9-strand of the cyclic nucleotide-binding homology domain stabilizes activated states of Kv11.1 channels.

PLoS One 2013 25;8(10):e77032. Epub 2013 Oct 25.

Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales, Australia ; St Vincent's Clinical School, University of New South Wales, New South Wales, Australia.

Kv11.1 potassium channels are important for regulation of the normal rhythm of the heartbeat. Reduced activity of Kv11.1 channels causes long QT syndrome type 2, a disorder that increases the risk of cardiac arrhythmias and sudden cardiac arrest. Kv11.1 channels are members of the KCNH subfamily of voltage-gated K(+) channels. However, they also share many similarities with the cyclic nucleotide gated ion channel family, including having a cyclic nucleotide-binding homology (cNBH) domain. Kv11.1 channels, however, are not directly regulated by cyclic nucleotides. Recently, crystal structures of the cNBH domain from mEAG and zELK channels, both members of the KCNH family of voltage-gated potassium channels, revealed that a C-terminal β9-strand in the cNBH domain occupied the putative cyclic nucleotide-binding site thereby precluding binding of cyclic nucleotides. Here we show that mutations to residues in the β9-strand affect the stability of the open state relative to the closed state of Kv11.1 channels. We also show that disrupting the structure of the β9-strand reduces the stability of the inactivated state relative to the open state. Clinical mutations located in this β9-strand result in reduced trafficking efficiency, which suggests that binding of the C-terminal β9-strand to the putative cyclic nucleotide-binding pocket is also important for assembly and trafficking of Kv11.1 channels.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0077032PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3808384PMC
August 2014

Hydrophobic interactions between the voltage sensor and pore mediate inactivation in Kv11.1 channels.

J Gen Physiol 2013 Sep;142(3):275-88

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

Kv11.1 channels are critical for the maintenance of a normal heart rhythm. The flow of potassium ions through these channels is controlled by two voltage-regulated gates, termed "activation" and "inactivation," located at opposite ends of the pore. Crucially in Kv11.1 channels, inactivation gating occurs much more rapidly, and over a distinct range of voltages, compared with activation gating. Although it is clear that the fourth transmembrane segments (S4), within each subunit of the tetrameric channel, are important for controlling the opening and closing of the activation gate, their role during inactivation gating is much less clear. Here, we use rate equilibrium free energy relationship (REFER) analysis to probe the contribution of the S4 "voltage-sensor" helix during inactivation of Kv11.1 channels. Contrary to the important role that charged residues play during activation gating, it is the hydrophobic residues (Leu529, Leu530, Leu532, and Val535) that are the key molecular determinants of inactivation gating. Within the context of an interconnected multi-domain model of Kv11.1 inactivation gating, our REFER analysis indicates that the S4 helix and the S4-S5 linker undergo a conformational rearrangement shortly after that of the S5 helix and S5P linker, but before the S6 helix. Combining REFER analysis with double mutant cycle analysis, we provide evidence for a hydrophobic interaction between residues on the S4 and S5 helices. Based on a Kv11.1 channel homology model, we propose that this hydrophobic interaction forms the basis of an intersubunit coupling between the voltage sensor and pore domain that is an important mediator of inactivation gating.
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http://dx.doi.org/10.1085/jgp.201310975DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3753607PMC
September 2013

Pore helices play a dynamic role as integrators of domain motion during Kv11.1 channel inactivation gating.

J Biol Chem 2013 Apr 7;288(16):11482-91. Epub 2013 Mar 7.

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

Proteins that form ion-selective pores in the membrane of cells are integral to many rapid signaling processes, including regulating the rhythm of the heartbeat. In potassium channels, the selectivity filter is critical for both endowing an exquisite selectivity for potassium ions, as well as for controlling the flow of ions through the pore. Subtle rearrangements in the complex hydrogen-bond network that link the selectivity filter to the surrounding pore helices differentiate conducting (open) from nonconducting (inactivated) conformations of the channel. Recent studies suggest that beyond the selectivity filter, inactivation involves widespread rearrangements of the channel protein. Here, we use rate equilibrium free energy relationship analysis to probe the structural changes that occur during selectivity filter gating in Kv11.1 channels, at near atomic resolution. We show that the pore helix plays a crucial dynamic role as a bidirectional interface during selectivity filter gating. We also define the molecular bases of the energetic coupling between the pore helix and outer helix of the pore domain that occurs early in the transition from open to inactivated states, as well as the coupling between the pore helix and inner helix late in the transition. Our data demonstrate that the pore helices are more than just static structural elements supporting the integrity of the selectivity filter; instead they play a crucial dynamic role during selectivity filter gating.
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http://dx.doi.org/10.1074/jbc.M113.461442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3630859PMC
April 2013

hERG K(+) channels: structure, function, and clinical significance.

Physiol Rev 2012 Jul;92(3):1393-478

Mark Cowley Lidwill Research Programme in Cardiac Electrophysiology, Victor Chang Cardiac Research Institute, Sydney, New South Wales, Australia.

The human ether-a-go-go related gene (hERG) encodes the pore-forming subunit of the rapid component of the delayed rectifier K(+) channel, Kv11.1, which are expressed in the heart, various brain regions, smooth muscle cells, endocrine cells, and a wide range of tumor cell lines. However, it is the role that Kv11.1 channels play in the heart that has been best characterized, for two main reasons. First, it is the gene product involved in chromosome 7-associated long QT syndrome (LQTS), an inherited disorder associated with a markedly increased risk of ventricular arrhythmias and sudden cardiac death. Second, blockade of Kv11.1, by a wide range of prescription medications, causes drug-induced QT prolongation with an increase in risk of sudden cardiac arrest. In the first part of this review, the properties of Kv11.1 channels, including biogenesis, trafficking, gating, and pharmacology are discussed, while the second part focuses on the pathophysiology of Kv11.1 channels.
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http://dx.doi.org/10.1152/physrev.00036.2011DOI Listing
July 2012

Voltage-sensing domain mode shift is coupled to the activation gate by the N-terminal tail of hERG channels.

J Gen Physiol 2012 Sep 13;140(3):293-306. Epub 2012 Aug 13.

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

Human ether-a-go-go-related gene (hERG) potassium channels exhibit unique gating kinetics characterized by unusually slow activation and deactivation. The N terminus of the channel, which contains an amphipathic helix and an unstructured tail, has been shown to be involved in regulation of this slow deactivation. However, the mechanism of how this occurs and the connection between voltage-sensing domain (VSD) return and closing of the gate are unclear. To examine this relationship, we have used voltage-clamp fluorometry to simultaneously measure VSD motion and gate closure in N-terminally truncated constructs. We report that mode shifting of the hERG VSD results in a corresponding shift in the voltage-dependent equilibrium of channel closing and that at negative potentials, coupling of the mode-shifted VSD to the gate defines the rate of channel closure. Deletion of the first 25 aa from the N terminus of hERG does not alter mode shifting of the VSD but uncouples the shift from closure of the cytoplasmic gate. Based on these observations, we propose the N-terminal tail as an adaptor that couples voltage sensor return to gate closure to define slow deactivation gating in hERG channels. Furthermore, because the mode shift occurs on a time scale relevant to the cardiac action potential, we suggest a physiological role for this phenomenon in maximizing current flow through hERG channels during repolarization.
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http://dx.doi.org/10.1085/jgp.201110761DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3434099PMC
September 2012

The S4-S5 linker acts as a signal integrator for HERG K+ channel activation and deactivation gating.

PLoS One 2012 16;7(2):e31640. Epub 2012 Feb 16.

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

Human ether-à-go-go-related gene (hERG) K(+) channels have unusual gating kinetics. Characterised by slow activation/deactivation but rapid inactivation/recovery from inactivation, the unique gating kinetics underlie the central role hERG channels play in cardiac repolarisation. The slow activation and deactivation kinetics are regulated in part by the S4-S5 linker, which couples movement of the voltage sensor domain to opening of the activation gate at the distal end of the inner helix of the pore domain. It has also been suggested that cytosolic domains may interact with the S4-S5 linker to regulate activation and deactivation kinetics. Here, we show that the solution structure of a peptide corresponding to the S4-S5 linker of hERG contains an amphipathic helix. The effects of mutations at the majority of residues in the S4-S5 linker of hERG were consistent with the previously identified role in coupling voltage sensor movement to the activation gate. However, mutations to Ser543, Tyr545, Gly546 and Ala548 had more complex phenotypes indicating that these residues are involved in additional interactions. We propose a model in which the S4-S5 linker, in addition to coupling VSD movement to the activation gate, also contributes to interactions that stabilise the closed state and a separate set of interactions that stabilise the open state. The S4-S5 linker therefore acts as a signal integrator and plays a crucial role in the slow deactivation kinetics of the channel.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0031640PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3280985PMC
August 2012

Principal role of adenylyl cyclase 6 in K⁺ channel regulation and vasodilator signalling in vascular smooth muscle cells.

Cardiovasc Res 2011 Sep 23;91(4):694-702. Epub 2011 May 23.

Department of Cell Physiology and Pharmacology, University of Leicester, Henry Wellcome Building, Lancaster Road, Leicester LE1 9HN, UK.

Aims: Membrane potential is a key determinant of vascular tone and many vasodilators act through the modulation of ion channel currents [e.g. the ATP-sensitive potassium channel (K(ATP))] involved in setting the membrane potential. Adenylyl cyclase (AC) isoenzymes are potentially important intermediaries in such vasodilator signalling pathways. Vascular smooth muscle cells (VSMCs) express multiple AC isoenzymes, but the reason for such redundancy is unknown. We investigated which of these isoenzymes are involved in vasodilator signalling and regulation of vascular ion channels important in modulating membrane potential.

Methods And Results: AC isoenzymes were selectively depleted (by >75%) by transfection of cultured VSMCs with selective short interfering RNA sequences. AC6 was the predominant isoenzyme involved in vasodilator-mediated cAMP accumulation in VSMCs, accounting for ∼60% of the total response to β-adrenoceptor (β-AR) stimulation. AC3 played a minor role in β-AR signalling, whereas AC5 made no significant contribution. AC6 was also the principal isoenzyme involved in β-AR-mediated protein kinase A (PKA) signalling (determined using the fluorescent biosensor for PKA activity, AKAR3) and the substantial β-AR/PKA-dependent enhancement of K(ATP) current. K(ATP) current was shown to play a vital role in setting the resting membrane potential and in mediating the hyperpolarization observed upon β-AR stimulation.

Conclusion: AC6, but not the closely related AC5, plays a principal role in vasodilator signalling and regulation of the membrane potential in VSMCs. These findings identify AC6 as a vital component in the vasodilatory apparatus central to the control of blood pressure.
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http://dx.doi.org/10.1093/cvr/cvr137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3156907PMC
September 2011

The N-terminal tail of hERG contains an amphipathic α-helix that regulates channel deactivation.

PLoS One 2011 Jan 13;6(1):e16191. Epub 2011 Jan 13.

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

The cytoplasmic N-terminal domain of the human ether-a-go-go related gene (hERG) K+ channel is critical for the slow deactivation kinetics of the channel. However, the mechanism(s) by which the N-terminal domain regulates deactivation remains to be determined. Here we show that the solution NMR structure of the N-terminal 135 residues of hERG contains a previously described Per-Arnt-Sim (PAS) domain (residues 26-135) as well as an amphipathic α-helix (residues 13-23) and an initial unstructured segment (residues 2-9). Deletion of residues 2-25, only the unstructured segment (residues 2-9) or replacement of the α-helix with a flexible linker all result in enhanced rates of deactivation. Thus, both the initial flexible segment and the α-helix are required but neither is sufficient to confer slow deactivation kinetics. Alanine scanning mutagenesis identified R5 and G6 in the initial flexible segment as critical for slow deactivation. Alanine mutants in the helical region had less dramatic phenotypes. We propose that the PAS domain is bound close to the central core of the channel and that the N-terminal α-helix ensures that the flexible tail is correctly orientated for interaction with the activation gating machinery to stabilize the open state of the channel.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0016191PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3020963PMC
January 2011

Molecular determinants of high-affinity drug binding to HERG channels.

Curr Opin Drug Discov Devel 2003 Sep;6(5):667-74

University of Leicester, Department of Cell Physiology and Pharmacology, Maurice Shock Medical Sciences Building, University Road, Leicester, LE1 9HN, UK.

Human ether-a-go-go-related gene (HERG) subunits mediate a K+ current that is required for normal repolarization of the cardiac action potential. The unintentional inhibition of HERG currents by numerous medications results in prolongation of the QT interval, as measured on the electrocardiogram, and is associated with increased risk of patients suffering from life-threatening cardiac arrhythmias. QT interval prolongation is considered a major safety concern by worldwide drug regulatory bodies, and early detection of new compounds with this undesirable side effect has become an important objective for pharmaceutical companies. New studies are shedding light on the structural basis of drug binding and the gating-dependent repositioning of key residues in the inner cavity of HERG, which are responsible for the unusual sensitivity of HERG to pharmacological agents. Insights from these studies may help develop novel strategies to reduce the proarrhythmic potential of the next generation of drugs.
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September 2003
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