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BackgroundDrug-induced QT prolongation (diLQT) is a feared side-effect as exposing susceptible individuals to fatal arrhythmias. The occurrence of diLQT is primarily attributed to unintended drug interactions with cardiac ion channels, notably the hERG channels that generate the repolarizing current (IKr) and thereby regulate the late repolarization phase. There is an important inter-individual susceptibility to develop diLQT which is of unknown origin but can be reproduced in patient-specific iPSC-derived cardiomyocytes (iPS-CMs).ObjectiveWe aimed to investigate the dynamics of hERG channels in response to sotalol and to identify regulators of the susceptibility to developing diLQT.MethodsWe measured electrophysiological activity and cellular distribution of hERG channels after hERG blocker treatment in iPS-CMs derived from patients with highest or lowest sensitivity (HS or LS) to sotalol administrationin vivo(i.e., based on the measure of the maximal change in QT interval 3 hours after administration). Specific small-interfering RNAs (siRNA) and CAVIN1-T2A-GFP adenovirus were used to manipulateCAVIN1expression.ResultsWhile HS and LS iPS-CMs showed similar electrophysiological characteristics at the baseline, the late repolarization phase was prolonged, and IKrsignificantly decreased after exposure of HS iPS-CMs to low sotalol concentrations. IKrreduction was caused by a rapid translocation of hERG channel from the plasma membrane to the cytoskeleton upon sotalol application. This phenomenon was suppressed by blocking active endocytosis using dynasore.CAVIN1, essential for caveolae biogenesis, was two-times more expressed in HS iPS-CMs and its knockdown using siRNA decreased their sensitivity to sotalol.CAVIN1overexpression in LS iPS-CMs using adenovirus showed reciprocal effects. Mechanistically, we found that treatment with sotalol promoted trafficking of the hERG channel from the plasma membrane to the cytoskeleton through caveolae and in a manner dependent on CAVIN1 expression.CAVIN1silencing reduced the number of caveolae at the membrane and abrogated the internalization of hERG channel in sotalol-treated HS iPS-CMs. CAVIN1 also controlled cardiomyocyte responses to other hERG blockers such as E4031, vandetanib, and clarithromycin.ConclusionsOur study identifies unbridled turnover of the potassium channel hERG as a mechanism supporting the inter-individual susceptibility underlying diLQT development and demonstrates how this phenomenon is finely tuned by CAVIN1.CLINICAL PERSPECTIVEWhat is new?-The inter-individual susceptibility underlying diLQT development involves unbridled turnover of cardiac ion channels from the plasma membrane.-This phenomenon is finely tuned by CAVIN1, a protein that is essential for essential for caveolae biogenesis.-Treatment with hERG blocker promoted trafficking of the hERG channel from the plasma membrane to the cytoskeleton through caveolae and in a manner dependent on CAVIN1 expression.What are the clinical implications?-While congenital long QT is primarily from a genetic origin, the development of drug-induced long QT involves differences in the trafficking machinery of cardiac ion channels.-The prediction of CAVIN1 expression levels could help preventing drug-induced cardiotoxicity.

Membrane proteins, including ion channels, became the focus of structural proteomics midway through the 20th century. Methods for studying ion channels are diverse and include structural (X-ray crystallography, cryoelectron microscopy, currently X-ray free electron lasers) and functional (e.g., patch clamp) approaches. This review highlights the evolution of approaches to study of the structure of cardiac ion channels, provides an overview of new techniques of structural biology concerning ion channels, including the use of lipo- and nanodiscs, and discusses the contribution of electrophysiological studies and molecular dynamics to obtain a complete picture of the structure and functioning of cardiac ion channels. Electrophysiological studies have become a powerful tool for deciphering the mechanisms of ion conductivity and selectivity, gating and regulation, as well as testing molecules of pharmacological interest. Obtaining the atomic structure of ion channels became possible by the active development of X-ray crystallography and cryoelectron micro-scopy, and, recently, with the use of XFEL.

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.

International audience; Editorial on the Research Topic Molecular Mechanisms of Voltage-Gating in Ion Channels Voltage-gated ion channels are transmembrane proteins conducting ions according to the electrochemical gradient, when opened by voltage. Hence, in these channels, at least one of the channel gates regulating the ion flux is controlled by the transmembrane potential. They are frequently ion specific and therefore selectively permeable to sodium (Na V channels), potassium (K V channels), calcium (Ca V channels) or chloride (CLC channels) ions. Depending on the channels, opening of the activation gate is triggered by membrane depolarization (e.g. K V , Na V and Ca V channels) or hyperpolarization (HCN channels for instance). In addition, in many voltage-gated channels, a so-called inactivation gate is also present. Compared to the activation gate, the latter is, when voltage-dependent, oppositely coupled to the potential: In K V , Na V and Ca V channels, upon membrane depolarization, the inactivation gate closes whereas the activation gate opens. Various voltage-dependent channels have been identified, depending on the excitable cell types in which they are expressed and their physiological role. They are characterized by their conductance, ion selectivity, pharmacology and voltage-sensitivity. These properties are mainly dictated by the amino-acid sequence and structure of the pore forming subunit(s), the presence of accessory subunit(s), the membrane composition and the intra-and extracellular ions concentrations. Many mutations have been identified in these channels, impacting their functions and provoking diseases named channelopathies. In 2012, we hosted a Research Topic on the Molecular Mechanisms of Voltage Dependency (Loussouarn and Tarek, 2012), bringing together scientists to collaborate and showcase the latest developments in the field. Since this Frontiers Research Topic, the development of new approaches, such as the use of cryo-electron microscopy (cryo-EM) at the atomic scale and the original approach of split channels, to name a few, has led to a more precise understanding of the mechanisms of voltage-gating, their targeting by toxins, and also their physio-pathological implications. Given the wealth of recent electrophysiological, biochemical, optical, and structural data regarding ion channel voltage-dependence, we felt there was clearly a need for putting together a new Research Topic that would include up to date Reviews and Original Research describing molecular details of the functioning of these complex voltage-gated channels. The review of Brewer et al. underlines how ion channel structures and models reveal critical differences in the atomic details of KCNQ1, hERG, and Na V 1.5 structures associated with their distinct voltage-gating and implication in Long QT syndrome, and also their pharmacological profiles. Such structural data may help defining the pathogenicity of the hundreds of variants in absence of functional data. It also mentions an important point: Molecular Dynamics represents a useful tool in refining cryo-EM structures, which are often of lower resolution in the periphery of protein core structures.

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.

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The patch-clamp technique and more recently the high throughput patch-clamp technique have contributed to major advances in the characterization of ion channels. However, the whole-cell voltage-clamp technique presents certain limits that need to be considered for robust data generation. One major caveat is that increasing current amplitude profoundly impacts the accuracy of the biophysical analyses of macroscopic ion currents under study. Using mathematical kinetic models of a cardiac voltage-gated sodium channel and a cardiac voltage-gated potassium channel, we demonstrated how large current amplitude and series resistance artefacts induce an undetected alteration in the actual membrane potential and affect the characterization of voltage-dependent activation and inactivation processes. We also computed how dose–response curves are hindered by high current amplitudes. This is of high interest since stable cell lines frequently demonstrating high current amplitudes are used for safety pharmacology using the high throughput patch-clamp technique. It is therefore critical to set experimental limits for current amplitude recordings to prevent inaccuracy in the characterization of channel properties or drug activity, such limits being different from one channel type to another. Based on the predictions generated by the kinetic models, we draw simple guidelines for good practice of whole-cell voltage-clamp recordings.

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.

Aims: several inherited arrhythmic diseases have been linked to single gene mutations in cardiac ion channels and interacting proteins. However, the mechanisms underlying most arrhythmias, are thought to involve altered regulation of the expression of multiple effectors. In this study, we aimed to examine the role of a transcription factor (TF) belonging to the Iroquois homeobox family, IRX5, in cardiac electrical function. Methods and results: using human cardiac tissues, transcriptomic correlative analyses between IRX5 and genes involved in cardiac electrical activity showed that in human ventricular compartment, IRX5 expression strongly correlated to the expression of major actors of cardiac conduction, including the sodium channel, Nav1.5, and Connexin 40 (Cx40). We then generated human-induced pluripotent stem cells (hiPSCs) derived from two Hamamy syndrome-affected patients carrying distinct homozygous loss-of-function mutations in IRX5 gene. Cardiomyocytes derived from these hiPSCs showed impaired cardiac gene expression programme, including misregulation in the control of Nav1.5 and Cx40 expression. In accordance with the prolonged QRS interval observed in Hamamy syndrome patients, a slower ventricular action potential depolarization due to sodium current reduction was observed on electrophysiological analyses performed on patient-derived cardiomyocytes, confirming the functional role of IRX5 in electrical conduction. Finally, a cardiac TF complex was newly identified, composed by IRX5 and GATA4, in which IRX5 potentiated GATA4-induction of SCN5A expression. Conclusion: altogether, this work unveils a key role for IRX5 in the regulation of human ventricular depolarization and cardiac electrical conduction, providing therefore new insights into our understanding of cardiac diseases., National Research Agency; European Union (EU); Horizon 2020; Marie Curie Actions International Incoming Fellowship FP7-PEOPLE-2012-IIF; La Fédération Française de Cardiologie; Fondation LefoulonDelalande; Eiffel Scholarship Programme of Excellence (Campus France), Doctoral School of Science and Technology-Lebanese University and The Fondation Genavie

Aim Deletion of QKP1507-1509 amino-acids in SCN5A gene product, the voltage-gated Na+ channel Nav1.5, has been associated with a large phenotypic spectrum of type 3 long QT syndrome, conduction disorder, dilated cardiomyopathy and high incidence of sudden death. The aim of this study was to develop and characterize a novel model of type 3 long QT syndrome to study the consequences of the QKP1507–1509 deletion. Methods and results We generated a knock-in mouse presenting the delQKP1510–1512 mutation (Scn5a+/ΔQKP) equivalent to human deletion. Scn5a+/ΔQKP mice showed prolonged QT interval, conduction defects and ventricular arrhythmias at the age of 2 weeks, and, subsequently, structural defects and premature mortality. The mutation increased Na+ window current and generated a late Na+ current. Ventricular action potentials from Scn5a+/ΔQKP mice were prolonged. At the age of 4 weeks, Scn5a+/ΔQKP mice exhibited a remodeling leading to [Ca2+]i transients with higher amplitude and slower kinetics, combined with enhanced SR Ca2+ load. SERCA2 expression was not altered. However, total phospholamban expression was higher whereas the amount of Ca2+-calmodulin-dependent kinase II (CaMKII)-dependent T17-phosphorylated form was lower, in hearts from 4-week-old mice only. This was associated with a lower activity of CaMKII and lower calmodulin expression. In addition, Scn5a+/ΔQKP cardiomyocytes showed larger Ca2+ waves, correlated with the presence of afterdepolarizations during action potential recording. Ranolazine partially prevented action potential and QT interval prolongation in 4-week-old Scn5a+/ΔQKP mice and suppressed arrhythmias. Conclusion The Scn5a+/ΔQKP mouse model recapitulates the clinical phenotype of mutation carriers and provides new and unexpected insights into the pathological development of the disease in patients carrying the QKP1507–1509 deletion.

Introduction Mutations in KCNH2 (coding for hERG) cause long or short QT syndromes (LQTS or SQTS), predisposing to life-threatening arrhythmias. More than 1000 variations in hERG sequences have been described, most of them are to be characterized. Objective The objective is to standardize and accelerate the entire process necessary to phenotype hERG variants. An in silico evaluation was also included to characterize the structural impact of the variants. Methods We selected 12 variants from patients with LQTS, and 1 with SQTS. We optimized the protocol to efficiently introduce mutations in hERG cDNA despite GC-rich sequences, using the Gibson assembly strategy. A pH-sensitive fluorescent tag was fused to hERG for fast-track evaluation of hERG cell trafficking. An optimized patch-clamp protocol of 35 sec 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 biophycical parameters, that can all be recapitulated in a single parameter defined herein as the repolarization power. The impact of the variants on channel structure were also reported where structural information was available. These three readouts (trafficking, repolarization power and structural impact) define 3 pathogenicity indexes that may help clinical diagnosis. Conclusion Fast track characterization of KCNH2 genetic variants shows its relevance to discriminate mutants that affect hERG channel activity from variants with undetectable effects. This information is meant to fill a patient database, as a basis for personalized medicine. The next steps will be to further accelerate the process using an automated patch-clamp system.

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.

International audience; Patients with HIV present with a higher prevalence of QT prolongation, of which molecularbases are still not clear. Among HIV proteins, Tat serves as a transactivator that stimulatesviral genes expression and is required for efficient HIV replication. Tat is actively secretedinto the blood by infected T-cells and affects organs such as the heart. Tat has been shown toalter cardiac repolarization in animal models but how this is mediated and whether this is alsothe case in human cells is unknown. In the present study, we show that Tat transfection inheterologous expression systems led to a decrease in hERG (underlying cardiac IKr) andhuman KCNE1-KCNQ1 (underlying cardiac IKs) currents and to an acceleration of theirdeactivation. This is consistent with a decrease in available phosphatidylinositol-(4,5)-bisphosphate (PIP2). A mutant Tat, unable to bind PIP2, did not reproduce the observedeffects. In addition, WT-Tat had no effect on a mutant KCNQ1 which is PIP2-insensitive,further confirming the hypothesis. Twenty four-hour incubation of human induced pluripotentstem cells-derived cardiomyocytes with Wild-type Tat reduced IKr and accelerated itsdeactivation. Concordantly, this Tat incubation led to a prolongation of the action potential(AP) duration. Events of AP alternans were also recorded in the presence of Tat, and wereexacerbated at a low pacing cycle length. Altogether, these data obtained on human K+channels both in heterologous expression systems and in human cardiomyocytes stronglysuggest that Tat sequesters PIP2, leading to a reduction of IKr and IKs, and provide a molecularmechanism for QT prolongation in HIV-infected patients.Key

Introduction Kv11.1 channel (hERG) and the corresponding IKr current is a major component of cardiac repolarization and sinoatrial pacemaking. Dysfunctions in hERG channels are associated with cardiac arrhythmias and remains a driving force to study the structure, biophysics and pharmacology of this channel. Several compounds that block hERG channels have been developed. In this study, we focused on BeKm-1, a toxin which is highly selective for the hERG channel. Objective To study hERG channels at high temporal and spatial resolution, we developed a photo-protected analog of BeKm-1 that enables control of hERG using light. Method We developed a photo-protected BeKm-1 analog (BeKm-1-Lys18Nvoc) which is cleaved by UV-light at 365 nm. Using a hERG stable cell line and high-throughput automated patch-clamp (Syncropatch 384, Nanion) we validated several properties of the BeKm-1 analog blockage. To demonstrate spatial control of cardiomyocyte function, hiPS-derived cardiomyocytes were plated on CardioExcyte96 to record extrafield potentials. Results We first validated physico-chemical properties of BeKm-1-Nvoc compared to BeKm-1 using HPLC analyses. Patch-clamp experiments displayed no inhibition of hERG current by BeKm-1-Nvoc at concentrations for which wild-type Bekm-1 is maximally inhibiting. Illumination of BeKm-1-Nvoc (100 nM) at 365 nm allows cleavage of the protecting group and strong inhibition of hERG current. The block of hERG is reversible after washes. Illumination of BeKm-1-Nvoc on hiPS-derived cardiomyocytes induces prolongation of extrafied potential which reflects an increase in repolarization time. Study of effects of photoactivation of BeKm-1 on mouse and zebrafish heart rates are ongoing. Conclusion We developed for the first time a photo-sensitive blocker of endogenous hERG channel which allows control of its activity with the spatiotemporal precision of light. Studies of implication of hERG on arrhythmias using this compound are ongoing.

Introduction hERG potassium channels are essential for normal cardiac electrical activity. Mutations in the hERG gene (KCNH2) cause long QT syndrome, a disorder that predisposes individuals to life-threatening arrhythmias. More than 1000 mutations in hERG sequence are described in databases and most of them remains to be characterized. Objective The objective of the work is to determine the consequences of the variants identified in LQT2 patients on the expression and function of the hERG channel. Methods An optimized sequence hERG fused to pHluorin tag was synthesized. By amplifying mutated overlapping fragments, and using the Gibson assembly strategy, we constructed hERG-pHluorin plasmids with missense variations. CHO cells were transfected by electroporation with the Maxcyte system or Fugene method, and studied after 40 hours. Confocal images and flow cytometry analysis were used to evaluate the percentage of transfected cells and to quantify the membrane channel expression, through pH sensitive pHluorin tag. Conventional or automated patch clamp were used to evaluate the hERG activity. Results In the present work, we constructed hERG plasmids carrying 48 variations identified in patients in France. Higher efficiency of transfection was observed with electroporation as compared to the Fugene method. The activity of hERG-pHluorin was validated in manual patch-clamp, the pHluorin tag does not interfere with channel activity. Characterization of several mutations in conventional patch clamp showed difference in current amplitude. Automated patch-clamp data was compared to manual patch-clamp, as an initial validation for high throughput screening of all constructed mutations. Conclusion High-throughput characterization of KCNH2 genetic variants is relevant to discriminate mutants that affect hERG channel activity from variants with undetectable effects. This information will be indicated in a patient database, as a basis of personalized medicine.

Deletion of QKP1507-1509 amino-acids in SCN5A gene product, the voltage-gated NaWe generated a knock-in mouse presenting the delQKP1510-1512 mutation (Scn5aThe Scn5a

International audience; In recent years, molecular modeling techniques, combined with MD simulations, provided significant insights on voltage-gated (Kv) potassium channels intrinsic properties. Among the success stories are the highlight of molecular level details of the effects of mutations, the unraveling of several metastable intermediate states, and the influence of a particular lipid, PIP2, in the stability and the modulation of Kv channel function. These computational studies offered a detailed view that could not have been reached through experimental studies alone. With the increase of cross disciplinary studies, numerous experiments provided validation of these computational results, which endows an increase in the reliability of molecular modeling for the study of Kv channels. This chapter offers a description of the main techniques used to model Kv channels at the atomistic level.

International audience; Voltage-gated potassium channels play pivotal roles in excitable and non-excitable cells. For many decades, structural properties and molecular mechanisms of these channels were inferred from functional observations. At the turn of the twenty-first century, structural biology revealed major aspects in the structural basis of ion channel organization, permeation, and gating. Among the available tools, homology modeling associated with low resolution microscopy helps in delineating the different structural elements of voltage-gated channels. Here, we describe in detail the methodology of homology modeling, using the 3D structure of the Kv2.1ΔCTA ion channel as a reference.

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.

There has been considerable debate as to whether adenosine triphosphate (ATP) is compartmentalized within cells and, in particular, whether the ATP concentration directly beneath the plasma membrane, experienced by membrane proteins, is the same as that of the bulk cytoplasm. This issue has been difficult to address because there is no indicator of cytosolic ATP, such as those available for Ca(2+), capable of resolving the submembrane ATP concentration ([ATP](sm)) in real time within a single cell. We show here that mutant ATP-sensitive K(+) channels can be used to measure [ATP](sm) by comparing the increase in current amplitude on patch excision with the ATP dose-response curve. In Xenopus oocytes, [ATP](sm) was 4.6 +/- 0.3 mm (n = 29) under resting conditions, slightly higher than that measured for the bulk cytoplasm (2.3 mm). In mammalian (COSm6) cells, [ATP](sm) was slightly lower and averaged 1.4 +/- 0.1 mm (n = 66). Metabolic poisoning (10 min of 3 mm azide) produced a significant fall in [ATP](sm) in both types of cells: to 1.2 +/- 0.1 mm (n = 24) in oocytes and 0.8 +/- 0.11 mm for COSm6 cells. We conclude that [ATP](sm) lies in the low millimolar range and that there is no gradient between bulk cytosolic and submembrane [ATP].

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.

Kv (voltage-gated potassium) channels are formed by a tetrameric pore (S5–S6) surrounded by four voltage sensor domains (S1–S4). Molecular mechanisms underlying gating remain poorly understood.Previously, we showed that peptides mimicking S4-S5L inhibit a cardiac Kv channel, KCNQ1 (Kv7.1). On the other hand, peptides mimicking S6T upregulate the channel activity by competing with the endogenous S6T for the endogenous S4-S5L (Choveau et al, 2010). Those observations suggested that S4-S5L acts as a voltage controled ligand that binds S6T and locks the channel in a closed state. However, given the low affinity between the peptide and the KCNQ1 channel, effects were moderate. In the present study, we asked whether covalently locking s4s5l onto s6t would lead to a permanent closure of the channel. To this end, we selected the human ether-a-go-go-related gene (hERG or Kv11.1) channel as a model since in this channel, a couple of cysteine in S4-S5L and S6T was introduced and led to channel closure (Ferrer et al, 2006). We used the whole-cell configuration of the patch-clamp technique in transfected COS-7 cells. We observed a complete inhibition of hERG in oxidative condition when coexpressing the S4-S5L peptide carrying an introduced cysteine (S4S5L-Cys-peptide) and the channel with a cysteine introduced in S6T (S6T-Cys-channel). Reciprocally, coexpressing S6TCyspeptide and the S4-S5L-Cys-channel led to a virtually voltage independent channel in oxidative condition. These results: 1) validate a model of gating in which S4-S5 acts as a voltage-dependent ligand binding the activation gate and locking it in a closed state and 2) suggest a generalization of this model.Choveau et al, J Biol Chem. 2011 286:707-16Ferrer et al, J Biol Chem. 2006 281:12858-64

OBJECTIVES: The aim of this study was to describe a new familial cardiac phenotype and to elucidate the electrophysiological mechanism responsible for the disease. BACKGROUND: Mutations in several genes encoding ion channels, especially SCN5A, have emerged as the basis for a variety of inherited cardiac arrhythmias. METHODS: Three unrelated families comprising 21 individuals affected by multifocal ectopic Purkinje-related premature contractions (MEPPC) characterized by narrow junctional and rare sinus beats competing with numerous premature ventricular contractions with right and/or left bundle branch block patterns were identified. RESULTS: Dilated cardiomyopathy was identified in 6 patients, atrial arrhythmias were detected in 9 patients, and sudden death was reported in 5 individuals. Invasive electrophysiological studies demonstrated that premature ventricular complexes originated from the Purkinje tissue. Hydroquinidine treatment dramatically decreased the number of premature ventricular complexes. It normalized the contractile function in 2 patients. All the affected subjects carried the c.665G>A transition in the SCN5A gene. Patch-clamp studies of resulting p.Arg222Gln (R222Q) Nav1.5 revealed a net gain of function of the sodium channel, leading, in silico, to incomplete repolarization in Purkinje cells responsible for premature ventricular action potentials. In vitro and in silico studies recapitulated the normalization of the ventricular action potentials in the presence of quinidine. CONCLUSIONS: A new SCN5A-related cardiac syndrome, MEPPC, was identified. The SCN5A mutation leads to a gain of function of the sodium channel responsible for hyperexcitability of the fascicular-Purkinje system. The MEPPC syndrome is responsive to hydroquinidine.

KCNQ1 osmosensitivity is of physiological and pathophysiological relevance in epithelial and cardiac cells, but the mechanism involved remains elusive. In COS-7 cells expressing the KCNE1–KCNQ1 fusion protein, extracellular hypoosmolarity and hyperosmolarity modify the channel biophysical parameters. These changes are consistent with hypoosmolarity increasing the level of membrane phosphatidylinositol-4,5-bisphosphate (PIP2), which in turn upregulates KCNE1–KCNQ1 channels. We showed that increasing PIP2 levels with a water-soluble PIP2 analogue prevented channel upregulation in hypoosmotic condition, suggesting a variation of the channel–PIP2 interaction during channel osmoregulation. Furthermore, we showed that polyamines and Mg2+, already known to tonically inhibit KCNQ channels by screening PIP2 negative charges, are involved in the osmoregulatory process. Indeed, intracellular Mg2+ removal and polyamines chelation inhibited the channel osmoregulation. Thus, the dilution of those cations during cell swelling might decrease channel inhibition and explain the channel upregulation by hypoosmolarity. To support this idea, we quantified the role of Mg2+ in the osmodependent channel activity. Direct measurement of intracellular [Mg2+] variations during osmotic changes and characterization of the channel Mg2+ sensitivity showed that Mg2+ participates significantly to the osmoregulation. Using intracellular solutions that mimic the variation of Mg2+ and polyamines, we were able to recapitulate the current amplitude variations in response to extracellular osmolarity changes. Altogether, these results support the idea of a modulation of the channel–PIP2 interactions by Mg2+ and polyamines during cell volume changes. It is likely that this mechanism applies to other channels that are sensitive to both osmolarity and PIP2.

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.

KCNQ1 is the pore-forming subunit of a channel complex whose expression and function have been rather well characterized in the heart. Almost 300 mutations of KCNQ1 have been identified in patients and a vast majority of the described mutations are linked to the long QT syndrome. Only a few mutations are linked to other pathologies such as atrial fibrillation and the short QT syndrome. However, a considerable amount of work remains to be done to get a clear picture of the molecular mechanisms responsible for the pathogenesis related to each mutation. The present review gives three examples of recent studies towards this goal and illustrates the diversity of the molecular mechanisms involved.

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.

Rationale Brugada syndrome is a human hereditary cardiac disease known to cause ventricular tachyarrhythmias (torsade de pointes) that can lead to sudden death. In about 20% of the Brugada affected patients, mutations have been identified in the gene encoding the Na+ ion channel, SCN5A. Up to now, genotype-phenotype studies have been performed using heterologous expression systems that lack the correct cellular context of a cardiomyocyte. Human Induced Pluripotent stem cells (hiPSCs) offer a now new paradigm for gene mutation characterization. Objective In this study, using cardiomyocytes differentiated from hiPSCs, we have electrophysiologically characterized a previously undescribed mutation in SCN5A gene, carried by a Brugada affected patient. Methods and results hiPSCs from a Brugada affected patient carrying the N1722D mutation in SCN5A have been generated and validated. hiPSCs from a healthy subject were used as control. Using patch clamp techniques, the biophysical properties of the Na+ channel and action potential characteristics were evaluated in both cardiomyocytes differentiated from these hiPSCs and in a mammalian expression system expressing the mutant channel. Preliminary data from both cellular models suggest a three-times reduction in Na+ current. The hiPSCs-derived cardiomyocytes revealed a specific action potential phenotype which is still under investigation Conclusion Brugada syndrome modeling using hiPSCs-derived cardiomyocytes suggests that this cellular model recapitulates the characteristics of a loss-of-function Na+ channel mutation and that hiPSCs-derived cardiomyocytes can be used as an accurate model for cardiac Na+ channel disease.

Polyamines cause inward rectification of Kir K(+) channels by blocking deep within the channel pore. We investigated structural constraints of polyamine block of strongly rectifying mutant K(ATP) channels (Kir6.2[L164C,N160D,C166S] + SUR1). We studied three groups of polyamine analogs: 1) conformationally restricted linear tetra-amines with a cycloalkyl or alkene group between the second and third amines (CGC-11047, CGC-11093, CGC-11099, and CGC-11098), 2) conformationally restricted linear deca-amines with a cycloalkyl or alkene group between the fifth and sixth amines (CGC-11150, CGC-11179, and CGC-11241), and 3) cyclic tetra-amines (CGC-11174, CGC-11197, CGC-11199, and CGC-11254). All linear analogs cause a voltage-dependent block similar to that of spermine, but slightly weaker (at 1 microM, V(1/2) for spermine block = -10 +/- 1 mV, Z = 2.9 +/- 0.1, n = 19; V(1/2) for analogs varies from polyamine -7 to +10 mV, Z = 2.6-3.9). These data indicate tolerance for conformational restriction and an upper limit to the voltage dependence of the blocking process. There was no voltage-dependent block by the cyclic compounds; instead, they induce irreversible rundown of the current. Structural models of Kir channels suggest that a narrow entry at the top of the cytoplasmic pore may exclude cyclic analogs from the inner cavity, thereby explaining the structure-activity relationship that we observe.