Functional characterization of CaVα2δ mutations associated with sudden cardiac death

L-type Ca(2+) channels play a critical role in cardiac rhythmicity. These ion channels are oligomeric complexes formed by the pore-forming CaVα1 with the auxiliary CaVβ and CaVα2δ subunits. CaVα2δ increases the peak current density and improves the voltage-dependent activation gating of CaV1.2 channels without increasing the surface expression of the CaVα1 subunit. The functional impact of genetic variants of CACNA2D1 (the gene encoding for CaVα2δ), associated with shorter repolarization QT intervals (the time interval between the Q and the T waves on the cardiac electrocardiogram), was investigated after recombinant expression of the full complement of L-type CaV1.2 subunits in human embryonic kidney 293 cells. By performing side-by-side high resolution flow cytometry assays and whole-cell patch clamp recordings, we revealed that the surface density of the CaVα2δ wild-type protein correlates with the peak current density. Furthermore, the cell surface density of CaVα2δ mutants S755T, Q917H, and S956T was not significantly different from the cell surface density of the CaVα2δ wild-type protein expressed under the same conditions. In contrast, the cell surface expression of CaVα2δ D550Y, CaVα2δ S709N, and the double mutant D550Y/Q917H was reduced, respectively, by ≈30-33% for the single mutants and by 60% for the latter. The cell surface density of D550Y/Q917H was more significantly impaired than protein stability, suggesting that surface trafficking of CaVα2δ was disrupted by the double mutation. Co-expression with D550Y/Q917H significantly decreased CaV1.2 currents as compared with results obtained with CaVα2δ wild type. It is concluded that D550Y/Q917H reduced inward Ca(2+) currents through a defect in the cell surface trafficking of CaVα2δ. Altogether, our results provide novel insight in the molecular mechanism underlying the modulation of CaV1.2 currents by CaVα2δ

Structural Determinants of the Closed KCa3.1 Channel Pore in Relation to Channel Gating: Results from a Substituted Cysteine Accessibility Analysis

In this work we address the question of the KCa3.1 channel pore structure in the closed configuration in relation to the contribution of the C-terminal end of the S6 segments to the Ca2+-dependent gating process. Our results based on SCAM (substituted cysteine accessibility method) experiments first demonstrate that the S6 transmembrane segment of the open KCa3.1 channel contains two distinct functional domains delimited by V282 with MTSEA and MTSET binding leading to a total channel inhibition at positions V275, T278, and V282 and to a steep channel activation at positions A283 and A286. The rates of modification by MTSEA (diameter 4.6 Å) of the 275C (central cavity) and 286C residues (S6 C-terminal end) for the closed channel configuration were found to differ by less than sevenfold, whereas experiments performed with the larger MTSET reagent (diameter 5.8 Å) resulted in modification rates 103–104 faster for cysteines at 286 compared with 275. Consistent with these results, the modification rates of the cavity lining 275C residue by MTSEA, Et-Hg+, and Ag+ appeared poorly state dependent, whereas modification rates by MTSET were 103 faster for the open than the closed configuration. A SCAM analysis of the channel inner vestibule in the closed state revealed in addition that cysteine residues at 286 were accessible to MTS reagents as large as MTS-PtrEA, a result supported by the observation that binding of MTSET to cysteines at positions 283 or 286 could neither sterically nor electrostatically block the access of MTSEA to the closed channel cavity (275C). It follows that the closed KCa3.1 structure can hardly be accountable by an inverted teepee-like structure as described for KcsA, but is better represented by a narrow passage centered at V282 (equivalent to V474 in Shaker) connecting the channel central cavity to the cytosolic medium. This passage would not be however restrictive to the diffusion of small reagents such as MTSEA, Et-Hg+, and Ag+, arguing against the C-terminal end of S6 forming an obstructive barrier to the diffusion of K+ ions for the closed channel configuration

Cysteine Mutagenesis and Computer Modeling of the S6 Region of an Intermediate Conductance IKCa Channel

Cysteine-scanning mutagenesis (SCAM) and computer-based modeling were used to investigate key structural features of the S6 transmembrane segment of the calcium-activated K+ channel of intermediate conductance IKCa. Our SCAM results show that the interaction of [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET) with cysteines engineered at positions 275, 278, and 282 leads to current inhibition. This effect was state dependent as MTSET appeared less effective at inhibiting IKCa in the closed (zero Ca2+ conditions) than open state configuration. Our results also indicate that the last four residues in S6, from A283 to A286, are entirely exposed to water in open IKCa channels, whereas MTSET can still reach the 283C and 286C residues with IKCa maintained in a closed state configuration. Notably, the internal application of MTSET or sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES) caused a strong Ca2+-dependent stimulation of the A283C, V285C, and A286C currents. However, in contrast to the wild-type IKCa, the MTSET-stimulated A283C and A286C currents appeared to be TEA insensitive, indicating that the MTSET binding at positions 283 and 286 impaired the access of TEA to the channel pore. Three-dimensional structural data were next generated through homology modeling using the KcsA structure as template. In accordance with the SCAM results, the three-dimensional models predict that the V275, T278, and V282 residues should be lining the channel pore. However, the pore dimensions derived for the A283–A286 region cannot account for the MTSET effect on the closed A283C and A286 mutants. Our results suggest that the S6 domain extending from V275 to V282 possesses features corresponding to the inner cavity region of KcsA, and that the COOH terminus end of S6, from A283 to A286, is more flexible than predicted on the basis of the closed KcsA crystallographic structure alone. According to this model, closure by the gate should occur at a point located between the T278 and V282 residues

Prix Nobel de Chimie 2003 : Canaux hydriques et ioniques

Ces prix Nobel récompensent, au travers de la distinction accordée à Peter Agre et Roderick MacKinnon, des avancées décisives dans la connaissance de la structure et du fonctionnement de protéines membranaires, les canaux hydriques et ioniques, essentiels à la communication entre les cellules et leur environnement

A bas les barrières… d’énergie dans les canaux potassiques !

Un des grands défis de l’électrophysiologie cellulaire des 50 dernières années fut de comprendre comment la très grande sélectivité des canaux ioniques peut être compatible avec le flux rapide des ions (106 à 108 ions par seconde). Trois articles récents jettent un éclairage nouveau sur ce dilemme en décrivant, au niveau atomique, le passage des ions dans le canal KcsA, un canal potassique initialement isolé de la bactérie Streptomyces lividans. Le pore du canal est constitué de 7 sites de liaison principaux pour les ions potassiques répartis entre la cavité interne (1 site), le filtre de sélectivité (4 sites) et le vestibule externe du pore (2 sites). Seuls deux ions peuvent être présents de façon simultanée dans le filtre de sélectivité et la diffusion des ions dans le filtre procède dès lors par un mécanisme de knock-on où le passage d’un ion de la cavité centrale dans le site voisin du filtre entraîne la sortie d’un des ions déjà présent au niveau des sites externes. Les calculs d’énergie libre révèlent que le passage d’un ion d’un site à un site voisin se produit avec un minimum de changement dans l’énergie potentielle de l’ion. Cette conclusion est d’autant plus inattendue qu’elle remet en cause un des dogmes les plus importants de la théorie de la diffusion dans les canaux ioniques, selon lequel la déshydratation des ions constitue la plus haute barrière d’énergie que doit surmonter un ion pour diffuser dans un canal ionique