Skeletal Muscle Na+ Channel Disorders

Five inherited human disorders affecting skeletal muscle contraction have been traced to mutations in the gene encoding the voltage-gated sodium channel Nav1.4. The main symptoms of these disorders are myotonia or periodic paralysis caused by changes in skeletal muscle fiber excitability. Symptoms of these disorders vary from mild or latent disease to incapacitating or even death in severe cases. As new human sodium channel mutations corresponding to disease states become discovered, the importance of understanding the role of the sodium channel in skeletal muscle function and disease state grows

Unexpected Gating Behaviour of an Engineered Potassium Channel Kir

In this study, we investigated the dynamics and functional characteristics of the KirBac3.1 S129R, a mutated bacterial potassium channel for which the inner pore-lining helix (TM2) was engineered so that the bundle crossing is trapped in an open conformation. The structure of this channel has been previously determined at high atomic resolution. We explored the dynamical characteristics of this open state channel using an in silico method MDeNM that combines molecular dynamics simulations and normal modes. We captured the global and local motions at the mutation level and compared these data with HDX-MS experiments. MDeNM provided also an estimation of the probability of the different opening states that are in agreement with our electrophysiological experiments. In the S129R mutant, the Arg129 mutation releases the two constriction points in the channel that existed in the wild type but interestingly creates another restriction point

Variations génétiques des canaux TREK-2, Nav1.4 et Kir2.1 et les pathologies associées

Les canaux ioniques présents à la surface ou à l intérieur des cellules sont responsables de plusieurs processus biologiques, de l excitation et la signalisation à la sécrétion et l absorption. Parmi tous les canaux ioniques, les canaux potassium (K+) sont les plus abondants, jouant un rôle important dans le maintien de la repolarisation cellulaire et dans la durée du potentiel d action. Dans le génome humain, environ 80 gènes codent pour des sous-unités de canaux K+ rendant compte d une grande diversité de cette classe de canaux. Les gènes codant pour les canaux Na+ et K+ sont porteurs de mutations naturelles associées à des pathologies comme les myotonies, les paralysies périodiques, le syndrome d Andersen, et le syndrome des QT longs. Dans cette thèse, trois types de canaux ont été étudiés : le canal TREK-2, canal de fond, le Nav1.4 du muscle squelettique associé à plusieurs pathologies musculaires, et le canal K+ à rectification entrante Kir2.1 associé au syndrome d Andersen. L objectif global de cette thèse est l étude du rôle physiologique des canaux K+ et Na+ ainsi que l impact des modifications structurales (dues à la formation des différents isoformes mutants) sur la fonction cellulaire et les états pathologiques. Cette étude commence par l analyse de la synthèse des canaux TREK-2 montrant que l initiation alternative de la traduction est responsable de la production d isoformes TREK-2 avec différents phénotypes de conductances unitaires. Ensuite, nous avons déterminé, à travers la caractérisation électrophysiologique d une mutation du canal Nav1.4, qu une activité anormalement élevée de ce canal était à l origine du phénotype clinique observé. A la fin de la thèse, nous avons évalué le rôle des canaux Kir2.1 dans la physiopathologie du muscle squelettique à travers des mutations associées au syndrome d Andersen.Ion channels present in the plasma membrane and intracellular organelles of all cells underlie a broad range of biological processes, from excitation and signaling to secretion and absorption. Among the ion channels, potassium channels are the most abundant, playing an important role in maintaining cellular repolarization and the action potential duration whereas sodium channels are responsible for the generation of the action potential. In excess of 80 genes in the human genome encode pore-forming K+ channel subunits making them the most diverse of all ion channels. Genes coding K+ and Na+ channels are subject to spontaneous mutations and are associated with disorders like myotonia, periodic paralysis, Andersen s syndrome and long QT syndrome. In the present thesis three types of channels were investigated : the TREK-2 channel which is a background leak K+ channel, the Nav1.4 skeletal muscle sodium channel associated with Andersen s syndrome. The overall goal of the work in this thesis is to study the physiological role of K+ and Na+ channels and also the impact of structural modifications (due to mutation or different isoform formation) on cellular function and in pathological states. The dissertation research commences with the analysis of TREK-2 channels synthesis showing that alternative translation initiation is responsible for producing TREK-2 isoforms with different single channel conductance phenotypes. Further, through electrophysiological characterization of a novel Nav1.4 mutation, it was determined that this mutation results in severe neonatal episodic laryngospasm by producing an overactive sodium channel. This thesis concludes with evaluation of KIR2.1 channels in skeletal muscle with regards to periodic paralysis pathophysiology in Andersen s syndrome using KIR2.1 channel mutations associated with this disorder.NICE-BU Sciences (060882101) / SudocSudocFranceF

The inward rectifier potassium channel Kir2.1 is required for osteoblastogenesis.

International audienceAndersen's syndrome (AS) is a rare and dominantly inherited pathology, linked to the inwardly rectifying potassium channel Kir2.1. AS patients exhibit a triad of symptoms that include periodic paralysis, cardiac dysrhythmia and bone malformations. Some progress has been made in understanding the contribution of the Kir2.1 channel to skeletal and cardiac muscle dysfunctions, but its role in bone morphogenesis remains unclear. We isolated myoblast precursors from muscle biopsies of healthy individuals and typical AS patients with dysmorphic features. Myoblast cultures underwent osteogenic differentiation that led to extracellular matrix mineralization. Osteoblastogenesis was monitored through the activity of alkaline phosphatase, and through the hydroxyapatite formation using Alizarin Red and Von Kossa staining techniques. Patch-clamp recordings revealed the presence of an inwardly rectifying current in healthy cells that was absent in AS osteoblasts, showing the dominant-negative effect of the Kir2.1 mutant allele in osteoblasts. We also found that while control cells actively synthesize hydroxyapatite, AS osteoblasts are unable to efficiently form any extracellular matrix. To further demonstrate the role of the Kir2.1 channels during the osteogenesis, we inhibited Kir2.1 channel activity in healthy patient cells by applying extracellular Ba(2+) or using adenoviruses carrying mutant Kir2.1 channels. In both cases, cells were no longer able to produce extracellular matrixes. Moreover, osteogenic activity of AS osteoblasts was restored by rescue experiments, via wild-type Kir2.1 channel overexpression. These observations provide a proof that normal Kir2.1 channel function is essential during osteoblastogenesis

Biological fractionation of lithium isotopes by cellular Na + /H + exchangers unravels fundamental transport mechanisms

SUMMARY Lithium (Li) has a wide range of uses in science, medicine and industry but its isotopy is underexplored, except in nuclear science and in geoscience. 6 Li and 7 Li isotopic ratio exhibits the second largest variation on Earth’s surface and constitutes a widely used tool for reconstructing past oceans and climates. As large variations have been measured in mammalian organs, plants or marine species, and as 6 Li elicits stronger effects than natural Li (~95% 7 Li) a central issue is the identification and quantification of biological influence of Li isotopes distribution. We show here that membrane ion channels and Na + -Li + /H + exchangers (NHEs), strongly fractionate Li isotopes. This systematic 6 Li enrichment is driven by membrane potential for channels, and by intracellular pH for NHEs, where it displays cooperativity, a hallmark of dimeric transport. Evidencing that transport proteins discriminate between isotopes differing by one neutron, opens new avenues for transport mechanisms, Li physiology, and paleoenvironments