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

Abstract: Voltage-gated calcium (Ca_V) 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, Ca_V channels couple membrane depolarization to a wide array of cellular responses including neurotransmission, muscle contraction and gene expression. Ca_V 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 (Ca_V2.2α₁/β₃) channels, showed decreased whole-cell patch-clamp currents indicating that the disulfide bond between these residues is required for α₂δ-1 function. [Copyright &y& Elsevier]