Identification of a disulfide bridge essential for structure and function of the voltage-gated Ca2+ channel α2δ-1 auxiliary subunit

Abstract

Voltage-gated calcium (CaV) channels are transmembrane proteins that form Ca2+-selective pores gated by depolarization and are essential regulators of the intracellular Ca2+ concentration. By providing a pathway for rapid Ca2+ influx, CaV channels couple membrane depolarization to a wide array of cellular responses including neurotransmission, muscle contraction and gene expression. CaV channels fall into two major classes, low voltage-activated (LVA) and high voltage-activated (HVA). The ion-conducting pathway of HVA channels is the α1 subunit, which typically contains associated β and α2δ ancillary subunits that regulate the properties of the channel. Although it is widely acknowledged that α2δ-1 is post-translationally cleaved into an extracellular α2 polypeptide and a membrane-anchored δ protein that remain covalently linked by disulfide bonds, to date the contribution of different cysteine (Cys) residues to the formation of disulfide bridges between these proteins has not been investigated. In the present report, by predicting disulfide connectivity with bioinformatics, molecular modeling and protein biochemistry experiments we have identified two Cys residues involved in the formation of an intermolecular disulfide bond of critical importance for the structure and function of the α2δ-1 subunit. Site directed-mutagenesis of Cys404 (located in the von Willebrand factor-A region of α2) and Cys1047 (in the extracellular domain of δ) prevented the association of the α2 and δ peptides upon proteolysis, suggesting that the mature protein is linked by a single intermolecular disulfide bridge. Furthermore, co-expression of mutant forms of α2δ-1 Cys404Ser and Cys1047Ser with recombinant neuronal N-type (CaV2.2α1/β3) channels, showed decreased whole-cell patch-clamp currents indicating that the disulfide bond between these residues is required for α2δ-1 function.

Transcriptional regulation of voltage‐gated Ca2+ channels

“…[78][79][80] Four separate genes code for the a 2 d subunits which display distinct tissue distribution and strong expression in the developing and mature CNS. [74][75][76] The a 2 d proteins consist of post-translationally cleaved a 2 and d peptides which remain associated with each other by a single disulphide bond, 81 and glycosylation of a 2 d seems to be required for the functional membrane expression of Ca V channels. [82][83][84] Molecular topology studies show that the a 2 d subunits may be linked to the plasma membrane via a GPI-anchor or by the d subunit that might constitute a single-pass membrane protein.…”

“…[82][83][84] Molecular topology studies show that the a 2 d subunits may be linked to the plasma membrane via a GPI-anchor or by the d subunit that might constitute a single-pass membrane protein. 81,[85][86][87] The majority of the a 2 d subunit is extracellular ( Fig. 1) and ideally located to interact with constituents of the extracellular matrix or extracellularly exposed proteins and serve also as a membrane receptor for binding of anti-epileptic and anti-allodynic drugs.…”

Regulation of high‐voltage‐activated Ca2+ channel function, trafficking, and membrane stability by auxiliary subunits

“…As mentioned earlier, during biosynthesis the Ca V α 2 δ subunits are generated as precursor proteins which undergo post‐translational cleavage, oxidation, and glycosylation to yield mature proteins comprised of disulphide‐linked α 2 and δ glycopolypeptides . Although the precise functional role of the post‐translational modifications of the Ca V α 2 δ subunits has not yet been fully defined, it has been suggested that they could play a role in channel trafficking.…”

“…Disulfide bond formation seems to be also important for channel cell surface expression. Recent studies have shown that a pair of conserved cysteine residues at positions 404 and 1047, located in the vWFA region of α 2 and the extracellular domain of δ, respectively, form a single intermolecular disulfide bridge required for normal α 2 δ‐1 subunit function . In these studies, it was shown that whereas there was a significant increase in current density through Ca V 2.2α 1 /β 3 channels upon cotransfection with wild‐type Ca V α 2 δ‐1, Cys404Ser, and Cys1047Ser mutations had little influence in current density, implying that formation of an intra‐subunit disulfide bond between these residues is essential for Ca V α 2 δ‐1 functional enhancement of currents, and perhaps for Ca V channel trafficking to the plasma membrane.…”

“…As mentioned earlier, during biosynthesis the Ca V α 2 δ subunits are generated as precursor proteins which undergo post-translational cleavage, oxidation, and glycosylation to yield mature proteins comprised of disulphide-linked α 2 and δ glycopolypeptides. [86][87][88] Although the precise functional role of the posttranslational modifications of the Ca V α 2 δ subunits has not yet been fully defined, it has been suggested that they could play a role in channel trafficking. Initial experiments by Gurnett et al 75 showed that N-linked glycosylation play a critical role for surface expression of Ca V 2.2 channels since the Ca 2+ current stimulation normally observed after Ca V α 2 δ coexpression was virtually abolished by deglycosylation.…”