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Aims Coronavirus disease of 2019 (COVID-19) has rapidly become a worldwide pandemic. Many clinical trials have been initiated to fight the disease. Among those, hydroxychloroquine and azithromycin had initially been suggested to improve clinical outcomes. Despite any demonstrated beneficial effects, they are still in use in some countries but have been reported to prolong the QT interval and induce life-threatening arrhythmia. Since a significant proportion of the world population may be treated with such COVID-19 therapies, evaluation of the arrhythmogenic risk of any candidate drug is needed. Methods and results Using the O'Hara-Rudy computer model of human ventricular wedge, we evaluate the arrhythmogenic potential of clinical factors that can further alter repolarization in COVID-19 patients in addition to hydroxychloroquine (HCQ) and azithromycin (AZM) such as tachycardia, hypokalaemia, and subclinical to mild long QT syndrome. Hydroxychloroquine and AZM drugs have little impact on QT duration and do not induce any substrate prone to arrhythmia in COVID-19 patients with normal cardiac repolarization reserve. Nevertheless, in every tested condition in which this reserve is reduced, the model predicts larger electrocardiogram impairments, as with dofetilide. In subclinical conditions, the model suggests that mexiletine limits the deleterious effects of AZM and HCQ. Conclusion By studying the HCQ and AZM co-administration case, we show that the easy-to-use O'Hara-Rudy model can be applied to assess the QT-prolongation potential of off-label drugs, beyond HCQ and AZM, in different conditions representative of COVID-19 patients and to evaluate the potential impact of additional drug used to limit the arrhythmogenic risk.

Background and aims Mutations in KCNH2 cause long or short QT syndromes (LQTS or SQTS) predisposing to life‐threatening arrhythmias. Over 1000 hERG variants have been described by clinicians, but most remain to be characterised. The objective is to standardise and accelerate the phenotyping process to contribute to clinician diagnosis and patient counselling. In silico evaluation was also included to characterise the structural impact of the variants. Methods We selected 11 variants from known LQTS patients and two variants for which diagnosis was problematic. Using the Gibson assembly strategy, we efficiently introduced mutations in hERG cDNA despite GC‐rich sequences. A pH‐sensitive fluorescent tag was fused to hERG for efficient evaluation of channel trafficking. An optimised 35‐s patch‐clamp protocol was developed to evaluate hERG channel activity in transfected cells. R software was used to speed up analyses. Results In the present work, we observed a good correlation between cell surface expression, assessed by the pH‐sensitive tag, and current densities. Also, we showed that the new biophysical protocol allows a significant gain of time in recording ion channel properties and provides extensive information on WT and variant channel biophysical parameters, that can all be recapitulated in a single parameter defined herein as the repolarisation power. The impacts of the variants on channel structure were also reported where structural information was available. These three readouts (trafficking, repolarisation power and structural impact) define three pathogenicity indexes that may help clinical diagnosis. Conclusions Fast‐track characterisation of KCNH2 genetic variants shows its relevance to discriminate mutants that affect hERG channel activity from variants with undetectable effects. It also helped the diagnosis of two new variants. This information is meant to fill a patient database, as a basis for personalised medicine. The next steps will be to further accelerate the process using an automated patch‐clamp system., We developed a method for rapid analysis of the properties of new hERG channel variants that is helpful for clinicians to diagnose an arrhythmia and help counseling the patient. This method is rapid enough to provide a complete set of channel properties and will set the stage for a more extensive characterization in the future.

Ether-a-go-go family (EAG) channels play a major role in many physiological processes in humans, including cardiac repolarization and cell proliferation. Cryo-EM structures of two of them, K(V)10.1 and human ether-a-go-go-related gene (hERG or K(V)11.1), have revealed an original nondomain-swapped structure, suggesting that the mechanism of voltage-dependent gating of these two channels is quite different from the classical mechanical-lever model. Molecular aspects of hERG voltage-gating have been extensively studied, indicating that the S4-S5 linker (S4-S5(L)) acts as a ligand binding to the S6 gate (S6 C-terminal part, S6(T)) and stabilizes it in a closed state. Moreover, the N-terminal extremity of the channel, called N-Cap, has been suggested to interact with S4-S5(L) to modulate channel voltage-dependent gating, as N-Cap deletion drastically accelerates hERG channel deactivation. In this study, using COS-7 cells, site-directed mutagenesis, electrophysiological measurements, and immunofluorescence confocal microscopy, we addressed whether these two major mechanisms of voltage-dependent gating are conserved in K(V)10.2 channels. Using cysteine bridges and S4-S5(L)–mimicking peptides, we show that the ligand/receptor model is conserved in K(V)10.2, suggesting that this model is a hallmark of EAG channels. Truncation of the N-Cap domain, Per-Arnt-Sim (PAS) domain, or both in K(V)10.2 abolished the current and altered channel trafficking to the membrane, unlike for the hERG channel in which N-Cap and PAS domain truncations mainly affected channel deactivation. Our results suggest that EAG channels function via a conserved ligand/receptor model of voltage gating, but that the N-Cap and PAS domains have different roles in these channels.

We identified a single nucleotide variation (SNV) (c.1264A > G) in the KCNQ1 gene in a 5-year-old boy who presented with a prolonged QT interval. His elder brother and mother, but not sister and father, also had this mutation. This missense mutation leads to a p.Lys422Glu (K422E) substitution in the Kv7.1 protein that has never been mentioned before. We inserted this substitution in an expression plasmid containing Kv7.1 cDNA and studied the electrophysiological characteristics of the mutated channel expressed in CHO-K1, using the whole-cell configuration of the patch-clamp technique. Expression of the mutant Kv7.1 channel in both homo- and heterozygous conditions in the presence of auxiliary subunit KCNE1 results in a significant decrease in tail current densities compared to the expression of wild-type (WT) Kv7.1 and KCNE1. This study also indicates that K422E point mutation causes a dominant negative effect. The mutation was not associated with a trafficking defect; the mutant channel protein was confirmed to localize at the cell membrane. This mutation disrupts the poly-Lys strip in the proximal part of the highly conserved cytoplasmic A–B linker of Kv7.1 that was not shown before to be crucial for channel functioning. © 2022 by the authors. Russian Science Foundation, RSF: 22-14-00088; International Association for the Evaluation of Educational Achievement, IEA: 2773 This study was funded by Russian Science Foundation (22-14-00088 to O.S.S.). Authors thank Lisa Trifonova for proof-reading the manuscript and Lisha Mai for help with . G.L. would like to acknowledge support from the CNRS International Emerging Action (IEA) PRC RUSSIE 2019 (PRC no. 2773). O.S.S. and V.N. acknowledge the support from the Interdisciplinary Scientific and Educational School of Moscow Lomonosov University «Molecular Technologies of the Living Systems and Synthetic Biology». was created with the help of Biorender.com.

International audience

IKr current, a major component of cardiac repolarization, is mediated by human Ether-&agrave, go-go-Related Gene (hERG, Kv11.1) potassium channels. The blockage of these channels by pharmacological compounds is associated to drug-induced long QT syndrome (LQTS), which is a life-threatening disorder characterized by ventricular arrhythmias and defects in cardiac repolarization that can be illustrated using cardiomyocytes derived from human-induced pluripotent stem cells (hiPS-CMs). This study was meant to assess the modification in hiPS-CMs excitability and contractile properties by BeKm-1, a natural scorpion venom peptide that selectively interacts with the extracellular face of hERG, by opposition to reference compounds that act onto the intracellular face. Using an automated patch-clamp system, we compared the affinity of BeKm-1 for hERG channels with some reference compounds. We fully assessed its effects on the electrophysiological, calcium handling, and beating properties of hiPS-CMs. By delaying cardiomyocyte repolarization, the peptide induces early afterdepolarizations and reduces spontaneous action potentials, calcium transients, and contraction frequencies, therefore recapitulating several of the critical phenotype features associated with arrhythmic risk in drug-induced LQTS. BeKm-1 exemplifies an interesting reference compound in the integrated hiPS-CMs cell model for all drugs that may block the hERG channel from the outer face. Being a peptide that is easily modifiable, it will serve as an ideal molecular platform for the design of new hERG modulators displaying additional functionalities.

International audience; Styrene-maleic acid (SMA) copolymers are used to extract lipid-encased membrane proteins from lipid bilayers in a detergent-free manner, yielding SMA lipid particles (SMALPs). SMALPs can serve as stable water-soluble nanocontainers for structural and functional studies of membrane proteins. Here, we used SMA copolymers to study full-length pore-forming α-subunits hKCNH5 and hKCNQ1 of human neuronal and cardiac voltage-gated potassium (Kv) channels, as well as the fusion construct comprising of an α-subunit hKCNQ1 and its regulatory transmembrane KCNE1 β-subunit (hKCNE1-hKCNQ1) with added affinity tags, expressed in mammalian COS-1 cells. All these recombinant proteins were shown to be functionally active. Treatment with the SMA copolymer, followed by purification on the affinity column, enabled extraction of all three channels. A DLS experiment demonstrated that Negative stain electron microscopy and single particle image analysis revealed a four-fold symmetry within channel-containing SMALPs, which indicates that purified hKCNH5 and hKCNQ1 channels, as well as the hKCNE1-hKCNQ1 fusion construct, retained their structural integrity as tetramers.

et al., [Aims]: Polyunsaturated fatty n-3 acids (PUFAs) have been reported to exhibit antiarrhythmic properties. However, the mechanisms of action remain unclear. We studied the electrophysiological effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on IKs, and on the expression and location of Kv7.1 and KCNE1. [Methods and results]: Experiments were performed using patch-clamp, western blot, and sucrose gradient techniques in COS7 cells transfected with Kv7.1/KCNE1 channels. Acute perfusion with both PUFAs increased Kv7.1/KCNE1 current, this effect being greater for DHA than for EPA. Similar results were found in guinea pig cardiomyocytes. Acute perfusion of either PUFA slowed the activation kinetics and EPA shifted the activation curve to the left. Conversely, chronic EPA did not modify Kv7.1/KCNE1 current magnitude and shifted the activation curve to the right. Chronic PUFAs decreased the expression of Kv7.1, but not of KCNE1, and induced spatial redistribution of Kv7.1 over the cell membrane. Cholesterol depletion with methyl-β-cyclodextrin increased Kv7.1/KCNE1 current magnitude. Under these conditions, acute EPA produced similar effects than those induced in non-cholesterol-depleted cells. A ventricular action potential computational model suggested antiarrhythmic efficacy of acute PUFA application under IKr block. [Conclusions]: We provide evidence that acute application of PUFAs increases Kv7.1/KCNE1 through a probably direct effect, and shows antiarrhythmic efficacy under IKr block. Conversely, chronic EPA application modifies the channel activity through a change in the Kv7.1/KCNE1 voltage-dependence, correlated with a redistribution of Kv7.1 over the cell membrane. This loss of function may be pro-arrhythmic. This shed light on the controversial effects of PUFAs regarding arrhythmias., This work was supported by grants from CICYT (SAF2010-14916 and SAF2013-45800-R to C.V.; BFU2011-23268 and CSD2008-00005 to A.F.) and FIS (PI11/02459, RD06/0014/0006, and RD12/0042/0019 to C.V.). C.M. and M.G. hold FPI grants. N.C. and A.d.l.C. hold Juan de la Cierva and RIC contracts, respectively.

International audience; Voltage-gated Na(+) (NaV) channels are key regulators of myocardial excitability, and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII)-dependent alterations in NaV1.5 channel inactivation are emerging as a critical determinant of arrhythmias in heart failure. However, the global native phosphorylation pattern of NaV1.5 subunits associated with these arrhythmogenic disorders and the associated channel regulatory defects remain unknown. Here, we undertook phosphoproteomic analyses to identify and quantify in situ the phosphorylation sites in the NaV1.5 proteins purified from adult WT and failing CaMKIIδc-overexpressing (CaMKIIδc-Tg) mouse ventricles. Of 19 native NaV1.5 phosphorylation sites identified, two C-terminal phosphoserines at positions 1938 and 1989 showed increased phosphorylation in the CaMKIIδc-Tg compared with the WT ventricles. We then tested the hypothesis that phosphorylation at these two sites impairs fibroblast growth factor 13 (FGF13)-dependent regulation of NaV1.5 channel inactivation. Whole-cell voltage-clamp analyses in HEK293 cells demonstrated that FGF13 increases NaV1.5 channel availability and decreases late Na(+) current, two effects that were abrogated with NaV1.5 mutants mimicking phosphorylation at both sites. Additional co-immunoprecipitation experiments revealed that FGF13 potentiates the binding of calmodulin to NaV1.5 and that phosphomimetic mutations at both sites decrease the interaction of FGF13 and, consequently, of calmodulin with NaV1.5. Together, we have identified two novel native phosphorylation sites in the C terminus of NaV1.5 that impair FGF13-dependent regulation of channel inactivation and may contribute to CaMKIIδc-dependent arrhythmogenic disorders in failing hearts.

International audience; Human immunodeficiency virus (HIV)-infected cells actively release the transcriptional activator (Tat) viral protein that is required for efficient HIV gene transcription. Extracellular Tat is able to enter uninfected cells. We recently reported that internalized Tat escapes endosomes to reach the cytosol and is then recruited to the plasma membrane by phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). As a consequence, Tat strongly impairs different critical cellular functions in several cell types. Here we will review recent evidences showing that Tat, by affecting the interaction of key cellular effectors with PtdIns(4,5)P2, blocks exocytosis from neuroendocrine cells, perturbs the synaptic vesicle exo-endocytosis cycle, prevents efficient phagocytosis by macrophages, and alters potassium channel activity in cardiac cells. Potential mechanistic aspects of Tat effects on these cellular processes will be discussed.

Voltage-gated Na+ (NaV) channels are key regulators of myocardial excitability, and Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent alterations in NaV1.5 channel inactivation are emerging as a critical determinant of arrhythmias in heart failure. However, the global native phosphorylation pattern of NaV1.5 subunits associated with these arrhythmogenic disorders and the associated channel regulatory defects remain unknown. Here, we undertook phosphoproteomic analyses to identify and quantify in situ the phosphorylation sites in the NaV1.5 proteins purified from adult WT and failing CaMKIIδc-overexpressing (CaMKIIδc-Tg) mouse ventricles. Of 19 native NaV1.5 phosphorylation sites identified, two C-terminal phosphoserines at positions 1938 and 1989 showed increased phosphorylation in the CaMKIIδc-Tg compared with the WT ventricles. We then tested the hypothesis that phosphorylation at these two sites impairs fibroblast growth factor 13 (FGF13)-dependent regulation of NaV1.5 channel inactivation. Whole-cell voltage-clamp analyses in HEK293 cells demonstrated that FGF13 increases NaV1.5 channel availability and decreases late Na+ current, two effects that were abrogated with NaV1.5 mutants mimicking phosphorylation at both sites. Additional co-immunoprecipitation experiments revealed that FGF13 potentiates the binding of calmodulin to NaV1.5 and that phosphomimetic mutations at both sites decrease the interaction of FGF13 and, consequently, of calmodulin with NaV1.5. Together, we have identified two novel native phosphorylation sites in the C terminus of NaV1.5 that impair FGF13-dependent regulation of channel inactivation and may contribute to CaMKIIδc-dependent arrhythmogenic disorders in failing hearts.

Background The Brugada syndrome is an inherited cardiac arrhythmia associated with high risk of sudden death. Although 20% of patients with Brugada syndrome carry mutations in SCN 5A , the molecular mechanisms underlying this condition are still largely unknown. Methods and Results We combined whole‐exome sequencing and linkage analysis to identify the genetic variant likely causing Brugada syndrome in a pedigree for which SCN 5A mutations had been excluded. This approach identified 6 genetic variants cosegregating with the Brugada electrocardiographic pattern within the pedigree. In silico gene prioritization pointed to 1 variant residing in KCNAB 2 , which encodes the voltage‐gated K + channel β2‐subunit (Kvβ2‐R12Q). Kvβ2 is widely expressed in the human heart and has been shown to interact with the fast transient outward K + channel subunit Kv4.3, increasing its current density. By targeted sequencing of the KCNAB 2 gene in 167 unrelated patients with Brugada syndrome, we found 2 additional rare missense variants (L13F and V114I). We then investigated the physiological effects of the 3 KCNAB 2 variants by using cellular electrophysiology and biochemistry. Patch‐clamp experiments performed in COS ‐7 cells expressing both Kv4.3 and Kvβ2 revealed a significant increase in the current density in presence of the R12Q and L13F Kvβ2 mutants. Although biotinylation assays showed no differences in the expression of Kv4.3, the total and submembrane expression of Kvβ2‐R12Q were significantly increased in comparison with wild‐type Kvβ2. Conclusions Altogether, our results indicate that Kvβ2 dysfunction can contribute to the Brugada electrocardiographic pattern.

The two components of the cardiac delayed rectifier current have been the subject of numerous studies since firstly described. This current controls the action potential duration and is highly regulated. After identification of the channel subunits underlying IKs, KCNQ1 associated with KCNE1, and IKr, HERG, their involvement in human cardiac channelopathies have provided various models allowing the description of the molecular mechanisms of the KCNQ1 and HERG channels trafficking, activity and regulation. More recently, studies have been focusing on the unveiling of different partners of the pore-forming proteins that contribute to their maturation, trafficking, activity and/or degradation, on one side, and on their respective expression in the heterogeneous cardiac tissue, on the other side. The aim of this review is to report and discuss the major works on IKs and IKr and the most recent ones that help to understand the precise function of these currents in the heart.

Mutations in the potassium channel KCNQ1 that determine retention of the mutated proteins in the endoplasmic reticulum (ER) are associated with the autosomal dominant negative Romano-Ward LQT1 cardiac syndrome. In the present study, we have analyzed the consequences and the potential molecular mechanisms involved in the ER retention of three Romano-Ward mutations located in KCNQ1 N terminus (Y111C, L114P, and P117L). We showed that the mutant KCNQ1 proteins exhibited reduced expression levels with respect to wild-type (WT)-KCNQ1. Radiolabeling pulse-chase experiments revealed that the lower expression levels did not result from reduced rate of synthesis. Instead, using a combination of Western blot and pulse-chase experiments, we showed that the mutant channel Y111C-KCNQ1, used as a model, was ubiquitinated and degraded in the proteasome more rapidly (t((1/2)) = 82 min) than WT-KCNQ1 channel (t((1/2)) = 113 min). On the other hand, KCNQ1 degradation did not appear to involve the GTP-dependent pathway. We also showed that KCNE1 stabilized both wild-type and Y111C proteins. To identify potential actors involved in KCNQ1 degradation, we studied the implication of the ER-resident protein Derlin-1 in KCNQ1 degradation. We showed that although KCNQ1 and Derlin-1 share the same molecular complex and co-immunoprecipitate when co-expressed in HEK293FT cells, Derlin-1 did not affect KCNQ1 steady state expression and degradation. These data were confirmed in T84 cells that express endogenous KCNQ1 and Derlin-1. Small interfering RNA knock-down of Derlin-1 did not modify KCNQ1 expression level, and no interaction between endogenous KCNQ1 and Derlin-1 could be detected.

Background: Early repolarization in the inferolateral leads has been recently recognized as a frequent syndrome associated with idiopathic ventricular fibrillation (VF). We report the case of a patient presenting dramatic changes in the ECG in association with recurrent VF in whom a novel genetic variant has been identified. Case Report: This young female (14 years) was resuscitated in 2001 following an episode of sudden death due to VF. All examinations including coronary angiogram with ergonovine injection, MRI, and flecainide or isoproterenol infusion were normal. The patient had multiple (>100) recurrences of VF unresponsive to beta-blockers, lidocaine/mexiletine, verapamil, and amiodarone. Recurrences of VF were associated with massive accentuation of the early repolarization pattern at times mimicking acute myocardial ischemia. Coronary angiography during an episode with 1.2 mV J/ST elevation was normal. Isoproterenol infusion acutely suppressed electrical storms, while quinidine eliminated all recurrences of VF and restored a normal ECG over a follow-up of 65 months. Genomic DNA sequencing of KATP channel genes showed missense variant in exon 3 (NC_000012) of the KCNJ8 gene, a subunit of the KATP channel, conferring predisposition to dramatic repolarization changes and ventricular vulnerability.

Patients with HIV present with a higher prevalence of QT prolongation, of which molecular bases are still not clear. Among HIV proteins, Tat serves as a transactivator that stimulates viral genes expression and is required for efficient HIV replication. Tat is actively secreted into the blood by infected T-cells and affects organs such as the heart. Tat has been shown to alter cardiac repolarization in animal models but how this is mediated and whether this is also the case in human cells is unknown. In the present study, we show that Tat transfection in heterologous expression systems led to a decrease in hERG (underlying cardiac I

Background Human genetically inherited cardiac diseases have been studied mainly in heterologous systems or animal models, independent of patients' genetic backgrounds. Because sources of human cardiomyocytes ( CM s) are extremely limited, the use of urine samples to generate induced pluripotent stem cell–derived CM s would be a noninvasive method to identify cardiac dysfunctions that lead to pathologies within patients' specific genetic backgrounds. The objective was to validate the use of CMs differentiated from urine‐derived human induced pluripotent stem (UhiPS) cells as a new cellular model for studying patients' specific arrhythmia mechanisms. Methods and Results Cells obtained from urine samples of a patient with long QT syndrome who harbored the HERG A561P gene mutation and his asymptomatic noncarrier mother were reprogrammed using the episomal‐based method. Uhi PS cells were then differentiated into CM s using the matrix sandwich method. Uhi PS ‐ CM s showed proper expression of atrial and ventricular myofilament proteins and ion channels. They were electrically functional, with nodal‐, atrial‐ and ventricular‐like action potentials recorded using high‐throughput optical and patch‐clamp techniques. Comparison of HERG expression from the patient's Uhi PS ‐ CM s to the mother's Uhi PS ‐ CM s showed that the mutation led to a trafficking defect that resulted in reduced delayed rectifier K + current (I Kr ). This phenotype gave rise to action potential prolongation and arrhythmias. Conclusions UhiPS cells from patients carrying ion channel mutations can be used as novel tools to differentiate functional CMs that recapitulate cardiac arrhythmia phenotypes.

Objective: The K + channel encoded by the human ether-a`-go-go-related gene (HERG) is crucial for repolarization in the human heart. In order to investigate the impact of HERG current (IKr) on the incidence of cardiac arrhythmias, we generated a transgenic mouse expressing HERG specifically in the heart. Methods and results: ECG recordings at baseline showed no obvious difference between transgenic and wild-type (WT) mice with the exception of the T wave, which was more negative in transgenic mice than in WT mice. E4031 (20 mg/kg) prolonged the QTc interval and flattened the Twave in transgenic mice, but not in WT mice. Injection of BaCl2 (25 mg/kg) induced short runs of ventricular tachycardia in 9/ 10 WT mice, but not in transgenic animals. Atrial pacing reproducibly induced atrial tachyarrhythmias in 11/15 WT mice. In contrast, atrial arrhythmia was inducible in only 2/11 transgenic mice. When pretreated with dofetilide (10 mg/kg), transgenic mice were as sensitive to experimental arrhythmias as WT mice. Microelectrode studies showed that atrial action potentials have a steeper slope of duration-rate adaptation in WT than in transgenic mice. Transgenic mice were also characterized by a post-repolarization refractoriness, which could result from the substantial amount of IKr subsisting after repolarization as assessed with action potential-clamp experiments and simulations with a model of the transgenic mouse action potential. Conclusion: HERG expression in the mouse heart can protect against experimental induction of arrhythmias. This is the first report of such a protective effect of HERG in vivo.

The last 10 years have seen rapid advances in the understanding of potassium channel function. Since the first inward rectifying (Kir) channels were cloned in 1994, the structural basis of channel function has been significantly elucidated, and determination of the crystal structure of a bacterial K channel (KcsA) in 1998 provided an atomic resolution of the permeation pathway. This review considers recent experimental studies aimed at uncovering the structural basis of Kir channel activity, and the applicability of comparative models based on KcsA to illuminate Kir channel pore structure and opening and closing processes.

Brugada syndrome (BrS) is characterized by ST-segment elevation in the right precordial leads and is associated with increased risk of sudden cardiac death. We have recently reported families with BrS and SCN5A mutations where some affected members do not carry the familial mutation. We evaluated the involvement of additional genetic determinants for BrS in an affected family. We identified three distinct gene variants within a family presenting BrS (5 individuals), cardiac conduction defects (CCD, 3 individuals) and shortened QT interval (4 individuals). The first mutation is nonsense, p.Q1695*, lying within the SCN5A gene, which encodes for NaV1.5, the α-subunit of the cardiac Na(+) channel. The second mutation is missense, p.N300D, and alters the CACNA1C gene, which encodes the α-subunit CaV1.2 of the L-type cardiac Ca(2+) channel. The SCN5A mutation strictly segregates with CCD. Four out of the 5 BrS patients carry the CACNA1C variant, and three of them present shortened QT interval. One of the BrS patients carries none of these mutations but a rare variant located in the ABCC9 gene as well as his asymptomatic mother. Patch-clamp studies identified a loss-of-function of the mutated CaV1.2 channel. Western-blot experiments showed a global expression defect while increased mobility of CaV1.2 channels on cell surface was revealed by FRAP experiments. Finally, computer simulations of the two mutations recapitulated patient phenotypes. We report a rare CACNA1C mutation as causing BrS and/or shortened QT interval in a family also carrying a SCN5A stop mutation, but which does not segregate with BrS. This study underlies the complexity of BrS inheritance and its pre-symptomatic genetic screening interpretation.

The perforated-patch configuration of the patch-clamp technique was used to record whole cell currents from human epithelial CFPAC-1 cells defective for functional cystic fibrosis transmembrane conductance regulator (CFTR). In CFPAC-1 cells, adenosine 3',5'-cyclic monophosphate (cAMP) stimulation with forskolin (10 microM) plus 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (400 microM) activated neither Cl- nor K+ currents. In the same cells transfected with wild-type CFTR gene, cAMP stimulation produced activation of both Cl- and K+ currents. In Cl(-)-depleted medium (gluconate as a substitute), cAMP stimulation evoked a K+ current in CFTR-transfected but not in untransfected CFPAC-1 cells. This cAMP-evoked K+ current was the sum of two components: 1) a time-independent inwardly rectifying component, and 2) a slowly relaxing component activated at positive voltages. Increasing intracellular Ca2+ with ionomycin (1 microM) activated K+ currents in either transfected or untransfected cells. In transfected cells, blocking the CFTR conductance with high-concentration glibenclamide (100 microM) reduced the K+ current when activated by cAMP but not when activated by Ca2+. Pretreating CFTR-transfected cells for 48 h with interferon-gamma downregulated CFTR gene expression and reduced cAMP but not Ca2+ activation of the whole cell K+ current. From these results, we conclude that functional membrane CFTR protein influences activation by cAMP of epithelial K+ currents.

In vivo, KCNQ1 α-subunits associate with the β-subunit KCNE1 to generate the slowly activating cardiac potassium current (I(Ks)). Structurally, they share their topology with other Kv channels and consist out of six transmembrane helices (S1-S6) with the S1-S4 segments forming the voltage-sensing domain (VSD). The opening or closure of the intracellular channel gate, which localizes at the bottom of the S6 segment, is directly controlled by the movement of the VSD via an electromechanical coupling. In other Kv channels, this electromechanical coupling is realized by an interaction between the S4-S5 linker (S4S5(L)) and the C-terminal end of S6 (S6(T)). Previously we reported that substitutions for Leu(353) in S6(T) resulted in channels that failed to close completely. Closure could be incomplete because Leu(353) itself is the pore-occluding residue of the channel gate or because of a distorted electromechanical coupling. To resolve this and to address the role of S4S5(L) in KCNQ1 channel gating, we performed an alanine/tryptophan substitution scan of S4S5(L). The residues with a "high impact" on channel gating (when mutated) clustered on one side of the S4S5(L) α-helix. Hence, this side of S4S5(L) most likely contributes to the electromechanical coupling and finds its residue counterparts in S6(T). Accordingly, substitutions for Val(254) resulted in channels that were partially constitutively open and the ability to close completely was rescued by combination with substitutions for Leu(353) in S6(T). Double mutant cycle analysis supported this cross-talk indicating that both residues come in close contact and stabilize the closed state of the channel.

International audience; Phosphatidylinositol-4,5-bisphosphate (PIP(2)) is a phospholipid that has been shown to modulate several ion channels, including some voltage-gated channels like Kv11.1 (hERG). From a biophysical perspective, the mechanisms underlying this regulation are not well characterized. From a physiological perspective, it is critical to establish whether the PIP(2) effect is within the physiological concentration range. Using the giant-patch configuration of the patch-clamp technique on COS-7 cells expressing hERG, we confirmed the activating effect of PIP(2). PIP(2) increased the hERG maximal current and concomitantly slowed deactivation. Regarding the molecular mechanism, these increased amplitude and slowed deactivation suggest that PIP(2) stabilizes the channel open state, as it does in KCNE1-KCNQ1. We used kinetic models of hERG to simulate the effects of the phosphoinositide. Simulations strengthened the hypothesis that PIP(2) is more likely stabilizing the channel open state than affecting the voltage sensors. From the physiological aspect, we established that the sensitivity of hERG to PIP(2) comes close to that of KCNE1-KCNQ1 channels, which lies in the range of physiological PIP(2) variations.

KCNQ1 is a voltage-activated potassium channel alpha-subunit expressed in various cell types, including cardiac myocytes and epithelial cells. KCNQ1 associates with different beta-subunits of the KCNE protein family. In the human heart, KCNQ1 associates with KCNE1 to generate the IKs current characterized by its slow activation and deactivation kinetics. Mutations in either KCNQ1 or KCNE1 are responsible for at least four channelopathies that lead to cardiac dysfunction and one that leads to congenital deafness: the Romano-Ward syndrome, the short QT syndrome, atrial fibrillation, and the Jervell and Lange-Nielsen syndrome (cardioauditory syndrome). To date, nearly 100 different KCNQ1 mutations have been reported as responsible for the cardiac long QT syndrome, characterized by prolonged QT interval, syncopes, and sudden death. Patch clamp and immunofluorescence techniques are instrumental for characterization of the molecular mechanisms responsible for the altered function of KCNQ1 and its partners.

Nearly a hundred different KCNQ1 mutations have been reported as leading to the cardiac long QT syndrome, characterized by prolonged QT interval, syncopes, and sudden death. We have previously shown that phosphatidylinositol-4,5-bisphosphate (PIP 2 ) regulates the KCNQ1–KCNE1 complex. In the present study, we show that PIP 2 affinity is reduced in three KCNQ1 mutant channels (R243H, R539W, and R555C) associated with the long QT syndrome. In giant excised patches, direct application of PIP 2 on the cytoplasmic face of the three mutant channels counterbalances the loss of function. Reintroduction of a positive charge by application of methanethiosulfonate ethylammonium on the cytoplasmic face of R555C mutant channels also restores channel activity. The channel affinity for a soluble analog of PIP 2 is decreased in the three mutant channels. By using a model that describes the KCNQ1–KCNE1 channel behavior and by fitting the relationship between the kinetics of deactivation and the current amplitude obtained in whole-cell experiments, we estimated the PIP 2 binding and dissociation rates on wild-type and mutant channels. The dissociation rate of the three mutants was higher than for the wild-type channel, suggesting a decreased affinity for PIP 2 . PIP 2 binding was magnesium-dependent, and the PIP 2 -dependent equilibrium constant in the absence of magnesium was higher with the wild-type than with the mutant channels. Altogether, our data suggest that a reduced PIP 2 affinity of KCNQ1 mutants can lead to the long QT syndrome.

International audience; The β-galactosidase reporter gene, either free or complexed with various cationic vectors, was microinjected into mammalian cells. Cationic lipids but not polyethylenimine or polylysine prevent transgene expression when complexes are injected in the nucleus. Polyethylenimine and to a lesser extent polylysine, but not cationic lipids, enhance transgene expression when complexes are injected into the cytoplasm. This latter effect was independent of the polymer vector/cDNA ionic charge ratio, suggesting that nucleic acid compaction rather than surface charge was critical for efficient nuclear trafficking. Cell division was not required for nuclear entry. Finally, comparative transfection and microinjection experiments with various cell lines confirm that barriers to gene transfer vary with cell type. We conclude that polymers but not cationic lipids promote gene delivery from the cytoplasm to the nucleus and that transgene expression in the nucleus is prevented by complexation with cationic lipids but not with cationic polymers.

Studies in Shaker, a voltage-dependent potassium channel, suggest a coupling between activation and inactivation. This coupling is controversial in hERG, a fast-inactivating voltage-dependent potassium channel. To address this question, we transferred to hERG the S3-S4 linker of the voltage-independent channel, rolf, to selectively disrupt the activation process. This chimera shows an intact voltage-dependent inactivation process consistent with a weak coupling, if any, between both processes. Kinetic models suggest that the chimera presents only an open and an inactivated states, with identical transition rates as in hERG. The lower sensitivity of the chimera to BeKm-1, a hERG preferential closed-state inhibitor, also suggests that the chimera presents mainly open and inactivated conformations. This chimera allows determining the mechanism of action of hERG blockers, as exemplified by the test on ketoconazole.

We investigated whether high levels of expression of the cystic fibrosis transmembrane conductance regulator (CFTR) would alter the functional properties of newly synthesized recombinant proteins. COS-7, CFPAC-1, and A549 cells were intranuclearly injected with a Simian virus 40-driven pECE-CFTR plasmid and assayed for halide permeability using the 6-methoxy- N-(3-sulfopropyl)quinolinium fluorescent probe. With increasing numbers of microinjected pECE-CFTR copies, the baseline permeability to halide dose dependently increased, and the response to adenosine 3′,5′-cyclic monophosphate (cAMP) stimulation decreased. In cells hyperexpressing CFTR, the high level of halide permeability was reduced when a cell metabolism poisoning cocktail was applied to decrease intracellular ATP and, inversely, was increased by orthovanadate. In CFPAC-1 cells investigated with the patch-clamp technique, CFTR hyperexpression led to a time-independent nonrectifying chloride current that was not sensitive to cAMP stimulation. CFPAC-1 cells hyperexpressing CFTR exhibited no outward rectifying chloride current nor inward rectifying potassium current either spontaneously or under cAMP stimulation. We conclude that hyperexpression of recombinant CFTR proteins modifies their properties inasmuch as 1) CFTR channels are permanently activated and not susceptible to cAMP regulation and 2) they lose their capacity to regulate heterologous ionic channels.