Molecular Determinant for Specific Ca/Ba Selectivity Profiles of Low and High Threshold Ca2+ Channels

Voltage-gated Ca2+ channels (VGCC) play a key role in many physiological functions by their high selectivity for Ca2+ over other divalent and monovalent cations in physiological situations. Divalent/monovalent selection is shared by all VGCC and is satisfactorily explained by the existence, within the pore, of a set of four conserved glutamate/aspartate residues (EEEE locus) coordinating Ca2+ ions. This locus however does not explain either the choice of Ca2+ among other divalent cations or the specific conductances encountered in the different VGCC. Our systematic analysis of high- and low-threshold VGCC currents in the presence of Ca2+ and Ba2+ reveals highly specific selectivity profiles. Sequence analysis, molecular modeling, and mutational studies identify a set of nonconserved charged residues responsible for these profiles. In HVA (high voltage activated) channels, mutations of this set modify divalent cation selectivity and channel conductance without change in divalent/monovalent selection, activation, inactivation, and kinetics properties. The CaV2.1 selectivity profile is transferred to CaV2.3 when exchanging their residues at this location. Numerical simulations suggest modification in an external Ca2+ binding site in the channel pore directly involved in the choice of Ca2+, among other divalent physiological cations, as the main permeant cation for VGCC. In LVA (low voltage activated) channels, this locus (called DCS for divalent cation selectivity) also influences divalent cation selection, but our results suggest the existence of additional determinants to fully recapitulate all the differences encountered among LVA channels. These data therefore attribute to the DCS a unique role in the specific shaping of the Ca2+ influx between the different HVA channels

Cav2.1 C‐terminal fragments produced in Xenopus laevis oocytes do not modify the channel expression and functional properties

International audienceThe sequence and genomic organization of the CACNA1A gene that encodes the Cav2.1 subunit of both P and Q type Ca2+ channels are well conserved in mammals. In human, rat and mouse CACNA1A, the use of an alternative acceptor site at the exon 46‐47 boundary results in the expression of a long Cav2.1 splice variant. In transfected cells, the long isoform of human Cav2.1 produces a C‐terminal fragment, but it is not known whether this fragment affects Cav2.1 expression or functional properties. Here, we cloned the long isoform of rat Cav2.1 (Cav2.1(e47)) and identified a novel variant with a shorter C‐terminus (Cav2.1(e47s)) that differs from those previously described in the rat and mouse. When expressed in Xenopus laevis oocytes, Cav2.1(e47) and Cav2.1(e47s) displayed similar functional properties as the short isoform (Cav2.1). We show that Cav2.1 isoforms produced short (CT1) and long (CT1(e47)) C‐terminal fragments that interacted in vivo with the auxiliary Cavβ4a subunit. Overexpression of the C‐terminal fragments did not affect Cav2.1 expression and functional properties. Furthermore, the functional properties of a Cav2.1 mutant without the C‐terminal Cavβ4 binding domain (Cav2.1ΔCT2) were similar to those of Cav2.1, and were not influenced by the co‐expression of the missing fragments (CT2 or CT2(e47)). Our results exclude a functional role of the C‐terminal fragments in Cav2.1 biophysical properties in an expression system widely used to study this channel

Disruption of the IS6-AID Linker Affects Voltage-gated Calcium Channel Inactivation and Facilitation

Two processes dominate voltage-gated calcium channel (CaV) inactivation: voltage-dependent inactivation (VDI) and calcium-dependent inactivation (CDI). The CaVβ/CaVα1-I-II loop and Ca2+/calmodulin (CaM)/CaVα1–C-terminal tail complexes have been shown to modulate each, respectively. Nevertheless, how each complex couples to the pore and whether each affects inactivation independently have remained unresolved. Here, we demonstrate that the IS6–α-interaction domain (AID) linker provides a rigid connection between the pore and CaVβ/I-II loop complex by showing that IS6-AID linker polyglycine mutations accelerate CaV1.2 (L-type) and CaV2.1 (P/Q-type) VDI. Remarkably, mutations that either break the rigid IS6-AID linker connection or disrupt CaVβ/I-II association sharply decelerate CDI and reduce a second Ca2+/CaM/CaVα1–C-terminal–mediated process known as calcium-dependent facilitation. Collectively, the data strongly suggest that components traditionally associated solely with VDI, CaVβ and the IS6-AID linker, are essential for calcium-dependent modulation, and that both CaVβ-dependent and CaM-dependent components couple to the pore by a common mechanism requiring CaVβ and an intact IS6-AID linker

Influence of pH on Ca2+ current and its control of electrical and Ca2+ signaling in ventricular myocytes

Modulation of L-type Ca2+ current (ICa,L) by H+ ions in cardiac myocytes is controversial, with widely discrepant responses reported. The pH sensitivity of ICa,L was investigated (whole cell voltage clamp) while measuring intracellular Ca2+ (Ca2+i) or pHi (epifluorescence microscopy) in rabbit and guinea pig ventricular myocytes. Selectively reducing extracellular or intracellular pH (pHo 6.5 and pHi 6.7) had opposite effects on ICa,L gating, shifting the steady-state activation and inactivation curves to the right and left, respectively, along the voltage axis. At low pHo, this decreased ICa,L, whereas at low pHi, it increased ICa,L at clamp potentials negative to 0 mV, although the current decreased at more positive potentials. When Ca2+i was buffered with BAPTA, the stimulatory effect of low pHi was even more marked, with essentially no inhibition. We conclude that extracellular H+ ions inhibit whereas intracellular H+ ions can stimulate ICa,L. Low pHi and pHo effects on ICa,L were additive, tending to cancel when appropriately combined. They persisted after inhibition of calmodulin kinase II (with KN-93). Effects are consistent with H+ ion screening of fixed negative charge at the sarcolemma, with additional channel block by H+o and Ca2+i. Action potential duration (APD) was also strongly H+ sensitive, being shortened by low pHo, but lengthened by low pHi, caused mainly by H+-induced changes in late Ca2+ entry through the L-type Ca2+ channel. Kinetic analyses of pH-sensitive channel gating, when combined with whole cell modeling, successfully predicted the APD changes, plus many of the accompanying changes in Ca2+ signaling. We conclude that the pHi-versus-pHo control of ICa,L will exert a major influence on electrical and Ca2+-dependent signaling during acid–base disturbances in the heart

Calcineurin Controls Voltage-Dependent-Inactivation (VDI) of the Normal and Timothy Cardiac Channels

Ca2+-entry in the heart is tightly controlled by Cav1.2 inactivation, which involves Ca2+-dependent inactivation (CDI) and voltage-dependent inactivation (VDI) components. Timothy syndrome, a subtype-form of congenital long-QT syndrome, results from a nearly complete elimination of VDI by the G406R mutation in the α11.2 subunit of Cav1.2. Here, we show that a single (A1929P) or a double mutation (H1926A-H1927A) within the CaN-binding site at the human C-terminal tail of α11.2, accelerate the inactivation rate and enhances VDI of both wt and Timothy channels. These results identify the CaN-binding site as the long-sought VDI-regulatory motif of the cardiac channel. The substantial increase in VDI and the accelerated inactivation caused by the selective inhibitors of CaN, cyclosporine A and FK-506, which act at the same CaN-binding site, further support this conclusion. A reversal of enhanced-sympathetic tone by VDI-enhancing CaN inhibitors could be beneficial for improving Timothy syndrome complications such as long-QT and autism

Molecular endpoints of Ca2+/calmodulin- and voltage-dependent inactivation of Cav1.3 channels

Ca2+/calmodulin- and voltage-dependent inactivation (CDI and VDI) comprise vital prototypes of Ca2+ channel modulation, rich with biological consequences. Although the events initiating CDI and VDI are known, their downstream mechanisms have eluded consensus. Competing proposals include hinged-lid occlusion of channels, selectivity filter collapse, and allosteric inhibition of the activation gate. Here, novel theory predicts that perturbations of channel activation should alter inactivation in distinctive ways, depending on which hypothesis holds true. Thus, we systematically mutate the activation gate, formed by all S6 segments within CaV1.3. These channels feature robust baseline CDI, and the resulting mutant library exhibits significant diversity of activation, CDI, and VDI. For CDI, a clear and previously unreported pattern emerges: activation-enhancing mutations proportionately weaken inactivation. This outcome substantiates an allosteric CDI mechanism. For VDI, the data implicate a “hinged lid–shield” mechanism, similar to a hinged-lid process, with a previously unrecognized feature. Namely, we detect a “shield” in CaV1.3 channels that is specialized to repel lid closure. These findings reveal long-sought downstream mechanisms of inactivation and may furnish a framework for the understanding of Ca2+ channelopathies involving S6 mutations

Modeling CICR in rat ventricular myocytes: voltage clamp studies

<p>Abstract</p> <p>Background</p> <p>The past thirty-five years have seen an intense search for the molecular mechanisms underlying calcium-induced calcium-release (CICR) in cardiac myocytes, with voltage clamp (VC) studies being the leading tool employed. Several VC protocols including lowering of extracellular calcium to affect <it>Ca</it>²⁺loading of the sarcoplasmic reticulum (SR), and administration of blockers caffeine and thapsigargin have been utilized to probe the phenomena surrounding SR <it>Ca</it>²⁺release. Here, we develop a deterministic mathematical model of a rat ventricular myocyte under VC conditions, to better understand mechanisms underlying the response of an isolated cell to calcium perturbation. Motivation for the study was to pinpoint key control variables influencing CICR and examine the role of CICR in the context of a physiological control system regulating cytosolic <it>Ca</it>²⁺concentration ([<it>Ca</it>²⁺]<it>_myo</it>).</p> <p>Methods</p> <p>The cell model consists of an electrical-equivalent model for the cell membrane and a fluid-compartment model describing the flux of ionic species between the extracellular and several intracellular compartments (cell cytosol, SR and the dyadic coupling unit (DCU), in which resides the mechanistic basis of CICR). The DCU is described as a controller-actuator mechanism, internally stabilized by negative feedback control of the unit's two diametrically-opposed <it>Ca</it>²⁺channels (trigger-channel and release-channel). It releases <it>Ca</it>²⁺flux into the cyto-plasm and is in turn enclosed within a negative feedback loop involving the SERCA pump, regulating[<it>Ca</it>²⁺]<it>_myo</it>.</p> <p>Results</p> <p>Our model reproduces measured VC data published by several laboratories, and generates graded <it>Ca</it>²⁺release at high <it>Ca</it>²⁺gain in a homeostatically-controlled environment where [<it>Ca</it>²⁺]<it>_myo</it>is precisely regulated. We elucidate the importance of the DCU elements in this process, particularly the role of the ryanodine receptor in controlling SR <it>Ca</it>²⁺release, its activation by trigger <it>Ca</it>²⁺, and its refractory characteristics mediated by the luminal SR <it>Ca</it>²⁺sensor. Proper functioning of the DCU, sodium-calcium exchangers and SERCA pump are important in achieving negative feedback control and hence <it>Ca</it>²⁺homeostasis.</p> <p>Conclusions</p> <p>We examine the role of the above <it>Ca</it>²⁺regulating mechanisms in handling various types of induced disturbances in <it>Ca</it>²⁺levels by quantifying cellular <it>Ca</it>²⁺balance. Our model provides biophysically-based explanations of phenomena associated with CICR generating useful and testable hypotheses.</p

Quelques données sur l'épidémie de Covid-19

Par Tristan Poullaouec Face au flot d’informations et à l’incertitude de la situation, un sociologue spécialiste de l’école et des investigations quantitatives cherche à comprendre l’épidémie de Covid-19 en se familiarisant avec les concepts de base de l’épidémiologie, discipline médicale qui étudie les maladies épidémiques, leurs modes de contagion et les moyens de les combattre et qui a beaucoup d’affinités avec la statistique. Les coronavirus sont une grande famille de virus découverts..