Voltage-gated calcium channels in genetic diseases

AbstractVoltage-gated calcium channels (VGCCs) mediate calcium entry into excitable cells in response to membrane depolarization. During the past decade, our understanding of the gating and functions of VGCCs has been illuminated by the analysis of mutations linked to a heterogeneous group of genetic diseases called “calcium channelopathies”. Calcium channelopathies include muscular, neurological, cardiac and vision syndromes. Recent data suggest that calcium channelopathies result not only from electrophysiological defects but also from altered α1/CaV subunit protein processing, including folding, posttranslational modifications, quality control and trafficking abnormalities. Overall, functional analyses of VGCC mutations provide a more comprehensive view of the corresponding human disorders and offer important new insights into VGCC function. Ultimately, the understanding of these pathogenic channel mutations should lead to improved treatments of such hereditary diseases in humans

Endogenous cardiac Ca2+ channels do not overcome the E-C coupling defect in immortalized dysgenic muscle cells: evidence for a missing link

AbstractThe expression of subunit genes of the Ca2+ channel complex was studied in differentiating, immortalized mouse mdg cells. These cells expressedα1 andα2/δ transcripts of the skeletal muscle Ca2+ channel genes, a cardiac Ca2+ channelα1 subunit gene and several known transcript variants of skeletal, cardiac and brain β genes. The mdg mutation is retained in the 129DA3 cell line and occurs exclusively at nucleotide position 4010 in the skeletalα1 transcript in which a cytosine residue is deleted. In early stages of differentiation and fusion, Ba2+ currents were detected in dysgenic myotubes the same as the cardiac L-type Ca2+ channel. These data provide specific structural evidence [Chaudhari, N. (1992) J. Biol. Chem. 267, 25636–25639] for the major genetic defect in mouse muscular dysgenesis and show a change in the expression levels ofα1c andα1c.The upregulation of the expression ofα1c results in functional Ca2+ channel activity, however, presumably not sufficient for excitation-contraction coupling

The NALCN ion channel is activated by M3 muscarinic receptors in a pancreatic β-cell line

A previously uncharacterized putative ion channel, NALCN (sodium leak channel, non-selective), has been recently shown to be responsible for the tetrodotoxin (TTX)-resistant sodium leak current implicated in the regulation of neuronal excitability. Here, we show that NALCN encodes a current that is activated by M3 muscarinic receptors (M3R) in a pancreatic β-cell line. This current is primarily permeant to sodium ions, independent of intracellular calcium stores and G proteins but dependent on Src activation, and resistant to TTX. The current is recapitulated by co-expression of NALCN and M3R in human embryonic kidney-293 cells and in Xenopus oocytes. We also show that NALCN and M3R belong to the same protein complex, involving the intracellular I–II loop of NALCN and the intracellular i3 loop of M3R. Taken together, our data show the molecular basis of a muscarinic-activated inward sodium current that is independent of G-protein activation, and provide new insights into the properties of NALCN channels

Lysosomal and network alterations in human mucopolysaccharidosis type VII iPSC-derived neural cells

Mucopolysaccharidosis type VII (MPS VII) is a lysosomal storage disease caused by deficient β-glucuronidase (β-gluc) activity. Significantly reduced β-gluc activity leads to accumulation of glycosaminoglycans (GAGs) in many tissues, including the brain. Numerous combinations of mutations in GUSB (the gene that codes for β-gluc) cause a range of neurological features that make disease prognosis and treatment challenging. Currently, there is little understanding of the molecular basis for MPS VII brain anomalies. To identify a neuronal phenotype that could be used to complement genetic analyses, we generated two iPSC clones derived from skin fibroblasts of an MPS VII patient. We found that MPS VII neurons exhibited reduced β-gluc activity and showed previously established disease-associated phenotypes, including GAGs accumulation, expanded endocytic compartments, accumulation of lipofuscin granules, more autophagosomes, and altered lysosome function. Addition of recombinant β-gluc to MPS VII neurons, which mimics enzyme replacement therapy, restored disease-associated phenotypes to levels similar to the healthy control. MPS VII neural cells cultured as 3D neurospheroids showed upregulated GFAP gene expression, which was associated with astrocyte reactivity, and downregulation of GABAergic neuron markers. Spontaneous calcium imaging analysis of MPS VII neurospheroids showed reduced neuronal activity and altered network connectivity in patient-derived neurospheroids compared to a healthy control. These results demonstrate the interplay between reduced β-gluc activity, GAG accumulation and alterations in neuronal activity, and provide a human experimental model for elucidating the bases of MPS VII-associated cognitive defects

High-resolution laser system for the S3-Low Energy Branch

In this paper we present the first high-resolution laser spectroscopy results obtained at the GISELE laser laboratory of the GANIL-SPIRAL2 facility, in preparation for the first experiments with the S$^3$-Low Energy Branch. Studies of neutron-deficient radioactive isotopes of erbium and tin represent the first physics cases to be studied at S$^3$. The measured isotope-shift and hyperfine structure data are presented for stable isotopes of these elements. The erbium isotopes were studied using the $4f^{12}6s^2$ $^3H_6 \rightarrow 4f^{12}(^3 H)6s6p$ $J = 5$ atomic transition (415 nm) and the tin isotopes were studied by the $5s^25p^2 (^3P_0) \rightarrow 5s^25p6s (^3P_1)$ atomic transition (286.4 nm), and are used as a benchmark of the laser setup. Additionally, the tin isotopes were studied by the $5s^25p6s (^3P_1) \rightarrow 5s^25p6p (^3P_2)$ atomic transition (811.6 nm), for which new isotope-shift data was obtained and the corresponding field-shift $F_{812}$ and mass-shift $M_{812}$ factors are presented

I–II Loop Structural Determinants in the Gating and Surface Expression of Low Voltage-Activated Calcium Channels

The intracellular loops that interlink the four transmembrane domains of Ca2+- and Na+-channels (Cav, Nav) have critical roles in numerous forms of channel regulation. In particular, the intracellular loop that joins repeats I and II (I–II loop) in high voltage-activated (HVA) Ca2+ channels possesses the binding site for Cavβ subunits and plays significant roles in channel function, including trafficking the α1 subunits of HVA channels to the plasma membrane and channel gating. Although there is considerable divergence in the primary sequence of the I–II loop of Cav1/Cav2 HVA channels and Cav3 LVA/T-type channels, evidence for a regulatory role of the I–II loop in T-channel function has recently emerged for Cav3.2 channels. In order to provide a comprehensive view of the role this intracellular region may play in the gating and surface expression in Cav3 channels, we have performed a structure-function analysis of the I–II loop in Cav3.1 and Cav3.3 channels using selective deletion mutants. Here we show the first 60 amino acids of the loop (post IS6) are involved in Cav3.1 and Cav3.3 channel gating and kinetics, which establishes a conserved property of this locus for all Cav3 channels. In contrast to findings in Cav3.2, deletion of the central region of the I–II loop in Cav3.1 and Cav3.3 yielded a modest increase (+30%) and a reduction (−30%) in current density and surface expression, respectively. These experiments enrich our understanding of the structural determinants involved in Cav3 function by highlighting the unique role played by the intracellular I–II loop in Cav3.2 channel trafficking, and illustrating the prominent role of the gating brake in setting the slow and distinctive slow activation kinetics of Cav3.3

Extracorporeal Membrane Oxygenation for Severe Acute Respiratory Distress Syndrome associated with COVID-19: An Emulated Target Trial Analysis.

RATIONALE: Whether COVID patients may benefit from extracorporeal membrane oxygenation (ECMO) compared with conventional invasive mechanical ventilation (IMV) remains unknown. OBJECTIVES: To estimate the effect of ECMO on 90-Day mortality vs IMV only Methods: Among 4,244 critically ill adult patients with COVID-19 included in a multicenter cohort study, we emulated a target trial comparing the treatment strategies of initiating ECMO vs. no ECMO within 7 days of IMV in patients with severe acute respiratory distress syndrome (PaO2/FiO2 <80 or PaCO2 ≥60 mmHg). We controlled for confounding using a multivariable Cox model based on predefined variables. MAIN RESULTS: 1,235 patients met the full eligibility criteria for the emulated trial, among whom 164 patients initiated ECMO. The ECMO strategy had a higher survival probability at Day-7 from the onset of eligibility criteria (87% vs 83%, risk difference: 4%, 95% CI 0;9%) which decreased during follow-up (survival at Day-90: 63% vs 65%, risk difference: -2%, 95% CI -10;5%). However, ECMO was associated with higher survival when performed in high-volume ECMO centers or in regions where a specific ECMO network organization was set up to handle high demand, and when initiated within the first 4 days of MV and in profoundly hypoxemic patients. CONCLUSIONS: In an emulated trial based on a nationwide COVID-19 cohort, we found differential survival over time of an ECMO compared with a no-ECMO strategy. However, ECMO was consistently associated with better outcomes when performed in high-volume centers and in regions with ECMO capacities specifically organized to handle high demand. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Etude de la spécificité pharmacologique et de l'impact physiologique des canaux T

Les canaux calciques à bas seuil d'activation ou de type T (canaux T) sont impliqués dans plusieurs fonctions physiologiques telles que l'activité pacemaker cardiaque, l'excitabilité neuronale, la sécrétion, la fécondation... Le courant calcique correspondant est caractérisé par une activation et une inactivation transitoire (T), une déactivation lente et une faible conductance. Trois sous-unités codant pour les canaux T ont été récemment clonées : a1G, a1H et a1I. Ces canaux n'ont malheureusement pas une pharmacologie spécifique. L'objectif de ma thèse a été de caractériser le rôle de canaux T cardiaques et d'étudier la pharmacologie de ces canaux. La première partie des résultats correspond à l'étude du rôle de la sous-unité a1G cardiaque grâce à une souris knock-out pour a1G. Nous avons montré que ces souris présentaient une bradycardie et un ralentissement de la conduction atrio-ventriculaire par un approche d'électrophysiologie cellulaire (cellules isolées du sinus et de l'oreillette), d'électrophysiologie in vivo (ECG sur animaux anesthésiés et enregistrements télémétriques) et moléculaire. Ces résultats démontrent l'implication de la sous-unité a1G dans la genèse et la conduction du rythme cardiaque. La deuxième partie de la thèse concerne l'étude pharmacologique des canaux T. La fluoxétine (Prozac ®) inhibe les 3 sous-unités de canaux T avec la même affinité. Elle se fixe et maintient le canal T dans l'état inactivé. Le zinc est un bloqueur plus spécifique de la sous-unité a1H. Il se fixe préférentiellement sur l'état inactivé du canal. L'efonidipine (isomère R -), qui appartient à la famille des dihydropyridines, inhibe plus spécifiquement les canaux T que les canaux L. Dans son ensemble, ces travaux contribuent à une meilleure compréhension du rôle physiologique et des propriétés pharmacologiques des canaux T.MONTPELLIER-BU Pharmacie (341722105) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

New Challenges Resulting From the Loss of Function of Nav1.4 in Neuromuscular Diseases

International audienceThe voltage-gated sodium channel Na v 1.4 is a major actor in the excitability of skeletal myofibers, driving the muscle force in response to nerve stimulation. Supporting further this key role, mutations in SCN4A , the gene encoding the pore-forming α subunit of Na v 1.4, are responsible for a clinical spectrum of human diseases ranging from muscle stiffness (sodium channel myotonia, SCM) to muscle weakness. For years, only dominantly-inherited diseases resulting from Na v 1.4 gain of function (GoF) were known, i.e. , non-dystrophic myotonia (delayed muscle relaxation due to myofiber hyperexcitability), paramyotonia congenita and hyperkalemic or hypokalemic periodic paralyses (episodic flaccid muscle weakness due to transient myofiber hypoexcitability). These last 5 years, SCN4A mutations inducing Na v 1.4 loss of function (LoF) were identified as the cause of dominantly and recessively-inherited disorders with muscle weakness: periodic paralyses with hypokalemic attacks, congenital myasthenic syndromes and congenital myopathies. We propose to name this clinical spectrum sodium channel weakness (SCW) as the mirror of SCM. Na v 1.4 LoF as a cause of permanent muscle weakness was quite unexpected as the Na + current density in the sarcolemma is large, securing the ability to generate and propagate muscle action potentials. The properties of SCN4A LoF mutations are well documented at the channel level in cellular electrophysiological studies However, much less is known about the functional consequences of Na v 1.4 LoF in skeletal myofibers with no available pertinent cell or animal models. Regarding the therapeutic issues for Na v 1.4 channelopathies, former efforts were aimed at developing subtype-selective Na v channel antagonists to block myofiber hyperexcitability. Non-selective, Na v channel blockers are clinically efficient in SCM and paramyotonia congenita , whereas patient education and carbonic anhydrase inhibitors are helpful to prevent attacks in periodic paralyses. Developing therapeutic tools able to counteract Na v 1.4 LoF in skeletal muscles is then a new challenge in the field of Na v channelopathies. Here, we review the current knowledge regarding Na v 1.4 LoF and discuss the possible therapeutic strategies to be developed in order to improve muscle force in SCW