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

Two sets of amino acids of the domain I of Cav2.3 Ca(2+) channels contribute to their high sensitivity to extracellular protons.

International audienceExtracellular acidification decreases Ca(2+) current amplitude and produces a depolarizing shift in the activation potential (Va) of voltage-gated Ca(2+) channels (VGCC). These effects are common to all VGCC, but differences exist between Ca(2+) channel types and the underlying molecular mechanisms remain largely unknown. We report here that the changes in current amplitude induced by extracellular acidification or alkalinisation are more important for Cav2.3 R type than for Cav2.1 P/Q-type Ca(2+) channels. This difference results from a higher shift of Va combined with a modification of channel conductance. Although involved in the sensitivity of channel conductance to extracellular protons, neither the EEEE locus nor the divalent cation selectivity locus could explain the specificity of the pH effects. We show that this specificity involves two separate sets of amino acids within domain I of the Cavα subunit. Residues of the voltage sensor domain and residues in the pore domain mediate the effects of extracellular protons on Va and on channel conductance, respectively. These new insights are important for elucidating the molecular mechanisms that control VGCC gating and conductance and for understanding the role of extracellular protons in other channels or membrane-tethered enzymes with similar pore and/or voltage sensor domains

Structure de la sous-unité β du canal calcique : la place du β-interaction domain

La structure du canal calcique, voie d’entrée privilégiée des ions calcium dans les cellules excitables, a fait l’objet de nombreuses études de biochimie et de mutagenèse associées à des approches fonctionnelles qui, même en l’absence de données cristallographiques, permettent de percevoir le mode de fonctionnement de ce pore. Au contraire, les données relatives à sa sous-unité régulatrice la plus importante, la sous-unité β, sont plutôt fragmentaires. Celle-ci joue pourtant un rôle fondamental dans l’adressage, la régulation et les propriétés biophysiques les plus intimes du canal. Ces lacunes sont maintenant partiellement comblées par la publication de plusieurs articles sur la structure tridimensionnelle de la sous-unité β. Cette structure, si elle confirme l’appartenance de la sous-unité β à la famille des guanylate kinases associées à la membrane (MAGUK), semble néanmoins remettre en cause certaines des données que l’on croyait pourtant fermement établies. Elle laisse aussi entrevoir de nouvelles fonctions dans l’assemblage et la localisation d’un canal fonctionnel.Voltage-gated calcium channels are key players in a number of fundamental physiological functions including contraction, secretion, transmitter release or gene activation. They allow a flux of calcium into the cell that constitutes a switch-on signal for most of these functions. The structures responsible for the shaping of these fluxes by the membrane voltage belong to the channel itself, but a number of associated proteins are known to more precisely tune this calcium entry and adapt it to the cellular demand. The calcium channel regulatory β subunit is undoubtedly the most important one, being influent on the expression, the kinetics, the voltage-dependence of channel opening and closing and on the pharmacology of the channel. Heterologous expression, combined to mutagenesis and electrophysiological and biochemical experiments have revealed the roles of short sequences of the β subunit, including the BID (β-interaction domain), in the physical and functional interactions with the channel pore. The resolved crystal structure of the β subunit now sheds new light on these sequences and their interactions with the rest of the protein. The presence of a type 3 src-homology (SH3) domain and a guanylate kinase (GK) domain confirms that the subunit belongs to the MAGUK protein family. Consistently, the polyproline binding site and the kinase function of the SH3 and the GK domains, respectively, are non functional, and the BID appears to be buried in the structure, preserving the SH3-GK interaction but not directly available for interactions with the channel pore subunit. Anchoring of the β subunit to the channel occurs via a hydrophobic grove in the GK domain, leaving a large surface of the subunit open to other protein-protein interactions. To what extent the intramolecular SH3-GK interaction is necessary for the stabilisation of this grove in a functional unit remains to be understood. The β subunit may thus play a key role in scaffolding multiple proteins around the channel and organizing diverse calcium-dependent signalling pathways directly linked to voltage-gated calcium entry. These findings will undoubtedly vitalize the search for new β-specific partners and functions

CANAL CALCIQUE D'ABEILLE ET UTILISATIONS

La présente invention se rapporte à un canal calcique isolé d'abeille comprenant un canal Cavα de séquence choisie dans le groupe comprenant les séquences SEQ ID n° 1 à 3. La présente invention se rapporte également à un canal calcique isolé d'abeille comprenant un canal Cavα, une protéine régulatrice Cavα2-δ de séquence choisie dans le groupe comprenant la séquence SEQ ID NO 6 à 8 et/ou une protéine régulatrice Cavβ de séquence choisie dans le groupe comprenant les séquences SEQ ID n° 4, 5 et 107. La présente invention se rapporte également à des séquences d'acides nucléiques et à des vecteurs comprenant ces séquences codantes, ainsi qu'à un procédé de fabrication de ces canaux calciques et à leurs utilisations. La présente invention trouve notamment des applications dans le domaine agricole, écologique, chimie et dans le domaine apicole. En particulier, elle trouve des applications, pour l'analyse/l'identification de la toxicité ou de l'innocuité de molécules, par exemple de molécules chimiques, vis-à-vis des abeilles

Introduction into Ca(v)2.1 of the homologous mutation of Ca(v)1.2 causing the Timothy syndrome questions the role of V421 in the phenotypic definition of P-type Ca(2+) channel.

The Timothy syndrome is a multisystem disorder associated with the mutation of a Gly residue (G402 or G406) in the Ca(v)1.2 Ca(2+) channel. G406 is localized at the end of the IS6 segment and just before the intracellular I-II loop, which is important for the regulation of channel inactivation and the binding of the Ca(v)beta subunit. This Gly residue is conserved in all Ca(v)1 and Ca(v)2 channels, and the G to R exchange produces a strong decrease of inactivation not only in Ca(v)1.2 but also in Ca(v)2.3. Here, we show that the mutation into Arg or Glu of the homologous Gly residue in Ca(v)2.1 (G363) produces also a slowing of inactivation. However, the G-to-A exchange that decreases the inactivation rate in Ca(v)1.2 and Ca(v)2.3 increases inactivation in Ca(v)2.1. Each mutation affects specifically the gating properties of Ca(v)2.1 that remain nevertheless modulated by the co-expressed beta subunit as with wild-type channel. The strong decrease of inactivation produced by the G363R or G363E mutations was reminiscent to that previously described for a specific splice variant of Ca(v)2.1 that contains a single Val residue inserted in the I-II loop (V421). We unexpectedly found that the V421 insertion does not affect the inactivation rate of Ca(v)2.1 and that the effects previously attributed to this insertion, including those on G-protein regulation, can be reproduced by the G363E mutation. Altogether, our results highlight the role of G363 in gating properties, inactivation kinetics, and G-protein regulation of Ca(v)2.1 and the lack of effect of V421 insertion on inactivation

CANAL CALCIQUE D'ABEILLE ET UTILISATIONS

La présente invention se rapporte à un canal calcique isolé d'abeille comprenant un canal Cavα de séquence choisie dans le groupe comprenant les séquences SEQ ID n° 1 à 3. La présente invention se rapporte également à un canal calcique isolé d'abeille comprenant un canal Cavα, une protéine régulatrice Cavα2-δ de séquence choisie dans le groupe comprenant la séquence SEQ ID NO 6 à 8 et/ou une protéine régulatrice Cavβ de séquence choisie dans le groupe comprenant les séquences SEQ ID n° 4, 5 et 107. La présente invention se rapporte également à des séquences d'acides nucléiques et à des vecteurs comprenant ces séquences codantes, ainsi qu'à un procédé de fabrication de ces canaux calciques et à leurs utilisations. La présente invention trouve notamment des applications dans le domaine agricole, écologique, chimie et dans le domaine apicole. En particulier, elle trouve des applications, pour l'analyse/l'identification de la toxicité ou de l'innocuité de molécules, par exemple de molécules chimiques, vis-à-vis des abeilles

Sodium channel subunits of pollinator insects and uses thereof

Sodium channel subunits of pollinator insects and uses thereo

Cloning, functional expression, and pharmacological characterization of inwardly rectifying potassium channels (Kir) from Apis mellifera

Abstract Potassium channels belong to the super family of ion channels and play a fundamental role in cell excitability. Kir channels are potassium channels with an inwardly rectifying property. They play a role in setting the resting membrane potential of many excitable cells including neurons. Although putative Kir channel family genes can be found in the Apis mellifera genome, their functional expression, biophysical properties, and sensitivity to small molecules with insecticidal activity remain to be investigated. We cloned six Kir channel isoforms from Apis mellifera that derive from two Kir genes, AmKir1 and AmKir2, which are present in the Apis mellifera genome. We studied the tissue distribution, the electrophysiological and pharmacological characteristics of three isoforms that expressed functional currents (AmKir1.1, AmKir2.2, and AmKir2.3). AmKir1.1, AmKir2.2, and AmKir2.3 isoforms exhibited distinct characteristics when expressed in Xenopus oocytes. AmKir1.1 exhibited the largest potassium currents and was impermeable to cesium whereas AmKir2.2 and AmKir2.3 exhibited smaller currents but allowed cesium to permeate. AmKir1 exhibited faster opening kinetics than AmKir2. Pharmacological experiments revealed that both AmKir1.1 and AmKir2.2 are blocked by the divalent ion barium, with IC50 values of 10−5 and 10−6 M, respectively. The concentrations of VU041, a small molecule with insecticidal properties required to achieve a 50% current blockade for all three channels were higher than those needed to block Kir channels in other arthropods, such as the aphid Aphis gossypii and the mosquito Aedes aegypti. From this, we conclude that Apis mellifera AmKir channels exhibit lower sensitivity to VU041