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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.

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

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; 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; 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.

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: 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.

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.

Introduction Phosphatidylinositol-4,5-bisphosphate (PIP2) is a cofactor necessary for the activity of KCNQ1 channels. Some Long QT mutations of KCNQ1, including R243H, R539W and R555C have been shown to decrease KCNQ1 interaction with PIP2. A previous study suggested that R539W is paradoxically less sensitive to intracellular magnesium inhibition than the WT channel, despite a decreased interaction with PIP2. In the present study, we confirm this peculiar behavior of R539W and suggest a molecular mechanism underlying it. Methods and Results COS-7 cells were transfected with WT or mutated KCNE1-KCNQ1 channel, and patch-clamp recordings were performed in giant-patch, permeabilized-patch or ruptured-patch configuration. Similar to other channels with a decreased PIP2 affinity, we observed that the R243H and R555C mutations lead to an accelerated current rundown when membrane PIP2 levels are decreasing. As opposed to R243H and R555C mutants, R539W is not more but rather less sensitive to PIP2 decrease than the WT channel. A molecular model of a fragment of the KCNQ1 C-terminus and the membrane bilayer suggested that a potential novel interaction of R539W with cholesterol stabilizes the channel opening and hence prevents rundown upon PIP2 depletion. We then carried out the same rundown experiments under cholesterol depletion and observed an accelerated R539W rundown that is consistent with this model. Conclusions We show for the first time that a mutation may shift the channel interaction with PIP2 to a preference for cholesterol. This de novo interaction wanes the sensitivity to PIP2 variations, showing that a mutated channel with a decreased affinity to PIP2 could paradoxically present a slowed current rundown compared to the WT channel. This suggests that caution is required when using measurements of current rundown as an indicator to compare WT and mutant channel PIP2 sensitivity.

Given the wealth of electrophysiological, biochemical, optical, and structural data regarding ion channels voltage-dependency, we decided to put together in this special issue, up to date reviews describing the molecular details of these complex voltage-gated channels (and in one instance voltage-dependent phosphatases: Villalba-Galea, 2012). The articles focus mostly on the molecular mechanisms underlying channels voltage-dependency, such as the electromechanical coupling governing their activation, but also on molecular mechanisms governing their regulation by lipids. We anticipate that such knowledge will help one to better understand the pathophysiology of channelopathies (Choveau et al., 2012; Delemotte et al., 2012; Jurkat-Rott et al., 2012) and lead to new pharmacological approaches. Molecular mechanisms underlying voltage-dependent activation and inactivation are complex, especially because channels are behaving in drastically different ways. Many reviews included in the present Research Topic issue describe models that rationalize these different behaviors: – In some channels, e.g., HCN, KAT, activation is promoted by hyperpolarization while in others, e.g., Kv channels, it is promoted by depolarization, despite a similar global structure and behavior of their voltage sensors. The opposite behavior may come from different kinds of S4-S5/S6 interactions, that can be transient for hyperpolarization activated channel, permanent for depolarization activated channel (Blunck and Batulan, 2012), or bimodal, with the residues implicated in the S4-S5/S6 interaction being different in the open and closed states (Choveau et al., 2012). Along the same lines, the peculiar closed state inactivation observed in Kv4 channels may also come from a transient S4-S5/S6 interaction (Bahring et al., 2012). – Forced uncoupling between the voltage sensor and the pore leads to opposite effects: this uncoupling favors channel closure of Shaker channels or, conversely, opening of the Kv-KcsA chimeric and KCNQ1 channels. This is most probably due to intrinsic properties of the pore, favoring a closed state in the former case and an open state in the latter (Blunck and Batulan, 2012; Vardanyan and Pongs, 2012). – The nature of the gating motion of S6 falls into two categories as described in details by Labro and Snyders (2012). This may due to different constraints associated with the origin of the main stimulus, which comes from either the nearby voltage sensor domain or from a distal part of the C-terminus. C-terminal domains of Kv channels are indeed critical for the modulation of channel gating by signal transduction elements (Barros et al., 2012). These two categories may also be related with the intrinsic properties of the pore mentioned above (Vardanyan and Pongs, 2012). – hERG is a very peculiar channel with slow activation gate and fast inactivation gate. Several molecular mechanisms (differences in voltage sensor dynamics, in the strength of S4-S5/S6 coupling, modulatory role of the N- and C-termini) may be at the origin of that peculiar behavior (Cheng and Claydon, 2012). Finally, in addition to the pore forming subunits, membrane lipids (Choveau et al., 2012; Moreno et al., 2012; Rodriguez Menchaca et al., 2012), intracellular ions (Goodchild and Fedida, 2012), and β-subunits (Sun et al., 2012) that can associate with multiple stoichiometry (Wrobel et al., 2012) also modulate the channel voltage-dependency. We hope that this series of reviews will bring researcher in the field (electrophysiologists, biochemists, modelers), a compendium of the knowledge gathered so far on the complex mechanisms of ion channel/enzyme voltage-dependency.

Phosphatidylinositol (4,5)-bisphosphate (PIP2) is a phospholipid of the plasma membrane that has been shown to be a key regulator of several ion channels. Functional studies and more recently structural studies of Kir channels have revealed the major impact of PIP2 on the open state stabilization. A similar effect of PIP2 on the delayed rectifiers Kv7.1 and Kv11.1, two voltage-gated K+ channels, has been suggested, but the molecular mechanism remains elusive and nothing is known on PIP2 effect on other Kv such as those of the Shaker family. By combining giant-patch ionic and gating current recordings in COS-7 cells, and voltage-clamp fluorimetry in Xenopus oocytes, both heterologously expressing the voltage-dependent Shaker channel, we show that PIP2 exerts 1) a gain-of-function effect on the maximal current amplitude, consistent with a stabilization of the open state and 2) a loss-of-function effect by positive-shifting the activation voltage dependence, most likely through a direct effect on the voltage sensor movement, as illustrated by molecular dynamics simulations.

International audience; Voltage-dependent potassium (Kv) channels are tetramers of six transmembrane domain (S1-S6) proteins. Crystallographic data demonstrate that the tetrameric pore (S5-S6) is surrounded by four voltage sensor domains (S1-S4). One key question remains: how do voltage sensors (S4) regulate pore gating? Previous mutagenesis data obtained on the Kv channel KCNQ1 highlighted the critical role of specific residues in both the S4-S5 linker (S4S5(L)) and S6 C terminus (S6(T)). From these data, we hypothesized that S4S5(L) behaves like a ligand specifically interacting with S6(T) and stabilizing the closed state. To test this hypothesis, we designed plasmid-encoded peptides corresponding to portions of S4S5(L) and S6(T) of the voltage-gated potassium channel KCNQ1 and evaluated their effects on the channel activity in the presence and absence of the ancillary subunit KCNE1. We showed that S4S5(L) peptides inhibit KCNQ1, in a reversible and state-dependent manner. S4S5(L) peptides also inhibited a voltage-independent KCNQ1 mutant. This inhibition was competitively prevented by a peptide mimicking S6(T), consistent with S4S5(L) binding to S6(T). Val(254) in S4S5(L) is known to contact Leu(353) in S6(T) when the channel is closed, and mutations of these residues alter the coupling between the two regions. The same mutations introduced in peptides altered their effects, further confirming S4S5(L) binding to S6(T). Our results suggest a mechanistic model in which S4S5(L) acts as a voltage-dependent ligand bound to its receptor on S6 at rest. This interaction locks the channel in a closed state. Upon plasma membrane depolarization, S4 pulls S4S5(L) away from S6(T), allowing channel opening.

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.

Aims KCNQ1 (alias KvLQT1 or Kv7.1) and KCNE1 (alias IsK or minK) co-assemble to form the voltage-activated K+ channel responsible for I Ks—a major repolarizing current in the human heart—and their dysfunction promotes cardiac arrhythmias. The channel is a component of larger macromolecular complexes containing known and undefined regulatory proteins. Thus, identification of proteins that modulate its biosynthesis, localization, activity, and/or degradation is of great interest from both a physiological and pathological point of view. Methods and results Using a yeast two-hybrid screening, we detected a direct interaction between β-tubulin and the KCNQ1 N-terminus. The interaction was confirmed by co-immunoprecipitation of β-tubulin and KCNQ1 in transfected COS-7 cells and in guinea pig cardiomyocytes. Using immunocytochemistry, we also found that they co-localized in cardiomyocytes. We tested the effects of microtubule-disrupting and -stabilizing agents (colchicine and taxol, respectively) on the KCNQ1–KCNE1 channel activity in COS-7 cells by means of the permeabilized-patch configuration of the patch-clamp technique. None of these agents altered I Ks. In addition, colchicine did not modify the current response to osmotic challenge. On the other hand, the I Ks response to protein kinase A (PKA)-mediated stimulation depended on microtubule polymerization in COS-7 cells and in cardiomyocytes. Strikingly, KCNQ1 channel and Yotiao phosphorylation by PKA—detected by phospho-specific antibodies—was maintained, as was the association of the two partners. Conclusion We propose that the KCNQ1–KCNE1 channel directly interacts with microtubules and that this interaction plays a major role in coupling PKA-dependent phosphorylation of KCNQ1 with I Ks activation.

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.

Point mutation of nearby residues in ion channels can be associated with diametrically opposed clinical phenotypes despite the mutant channels exhibit similar biophysical characteristics. Hence a characterization of the channel structure/function at the amino-acid scale is required for better understanding of channel genotype/phenotype relationship. R555C and R539W KCNQ1 mutant channels are a good illustration of this idea: R555C mutation is associated with a fruste form of type 1 long QT syndrome, whereas R539W mutation is associated with sudden death. Puzzling enough, the genotype/phenotype relationship is difficult to understand because the mutated residues are in the same helix C module, they both concern arginine residues, both channels have the same biophysical properties, and the same sensitivity to short chain phosphatidylinositol-4,5-bisphosphate (PIP2).To better understand the genotype/phenotype relationship in the context of these mutations, we performed several tests in COS-7 cells expressing the WT or mutant channels and used tail-currents amplitudes as readout. We show that R539W is very peculiar: As opposed to WT and R555C channels, the R539W channel current is barely running down when available PIP2 is decreased, either by wortmannin application in whole-cell, or by magnesium application in inside-out configuration. Consistent with that, the R539W channel is also insensitive to extracellular osmolarity, known to modulate the channel activity via PIP2. These results suggest that KCNQ1-R539W mutation shortcuts PIP2 in the channel open pore stabilization. Both structural model prediction and functional analysis implicate membrane cholesterol in this effect. Indeed, structural model prediction suggests that the introduced tryptophan in R539W interacts with cholesterol. Both cyclodextrin application on R539W and substitution of R539 by residues other than tryptophan restore channel rundown, consistent with the supposed tryptophan-cholesterol interaction. We conclude that the R539W/cholesterol interaction substitutes for R539/PIP2 interaction in the channel open pore stabilization.

Voltage-gated calcium channels (CaVs) provide the primary source of calcium influx in excitable cells and couple electrical signals to chemical signaling cascades. Due to CaV size and the difficulty of expressing CaVs at quantities sufficient for high-resolution determination, detailed structural information is limited to isolated cytoplasmic domains. However, CaVs are homologous to voltage-gated sodium channels (NaVs) and NaV structure can serve as a template for CaV structure. We determined the structure of the closed conformation of NaVAe1p, a pore only bacterial NaV derived from NaVAe1, an Alkalilimnicola ehrlichei bacterial NaV. This structure reveals the site of a putative calcium ion at the extracellular mouth of the selectivity filter liganded by four serines. At the equivalent site in mammalian calcium channel selectivity filters, there is a conserved aspartate in one of the calcium channel domains. Our functional studies show that this aspartate is a previously unknown determinant of CaV high affinity calcium binding in the mammalian calcium channel CaV1.2. These findings show the extent of similarities between bacterial sodium channels and eukaryotic voltage gated channels and shed new light on the selectivity filter in mammalian calcium channels.

Using a candidate-gene approach, we detected a variant of SCN5A, encoding the cardiac Na+ channel Nav1.5, by screening a family with cardiac arrhythmia resulting in frequent premature ventricular contractions (PVCs) and non-sustained ventricular tachycardia. Arrhythmia mechanism involved ectopic foci originating from the His-Purkinje system. The same mutation, leading to the R222Q substitution, was present in two additional unrelated families with the same associated cardiac phenotype. Exercise or hydroquinidine dramatically decreased the number of PVCs. To evaluate the functional incidence of this substitution, whole-cell patch-clamp experiments were performed on transfected COS-7 cells. The activation and inactivation curves were negatively shifted in the presence of the mutation (V1/2act, WT: −30.6±2.1 mV, n=9; heterozygous: −37.2±1.6 mV, n=9; p

Phosphatidylinositol-(4,5)-bisphosphate (PIP2) is a phospholipid of the plasma membrane that has been shown to be a key regulator of several ion channels. Functional studies and more recently structural studies of Kir channels have revealed the major impact of PIP2 on the open state stabilization. A similar effect of PIP2 on the delayed rectifiers Kv7.1 and Kv11.1, two voltage-gated K+ channels, has been suggested, but the molecular mechanism remains elusive. By combining giant-patch ionic and gating current recordings in COS-7 cells, and voltage-clamp fluorimetry in Xenopus Oocytes, both heterologously expressing the voltage-dependent Shaker channel, we show that PIP2 exerts (1) a gain-of-function effect on the maximal current amplitude, consistent with a stabilization of the open state and (2) a loss-of-function effect by positive-shifting the activation voltage dependence, most likely through a direct effect on the voltage sensor movement, as illustrated by Molecular Dynamics simulations.