title: Identification of a disulfide bridge essential for structure and function of the voltage-gated Ca(2+) channel α(2)δ-1 auxiliary subunit.
abstract: Voltage-gated calcium (Ca(V)) channels are transmembrane proteins that form Ca(2+)-selective pores gated by depolarization and are essential regulators of the intracellular Ca(2+) concentration. By providing a pathway for rapid Ca(2+) 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 α(1) subunit, which typically contains associated β and α(2)δ ancillary subunits that regulate the properties of the channel. Although it is widely acknowledged that α(2)δ-1 is post-translationally cleaved into an extracellular α(2) 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 α(2)δ-1 subunit. Site directed-mutagenesis of Cys404 (located in the von Willebrand factor-A region of α(2)) and Cys1047 (in the extracellular domain of δ) prevented the association of the α(2) 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 α(2)δ-1 Cys404Ser and Cys1047Ser with recombinant neuronal N-type (Ca(V)2.2α(1)/β(3)) channels, showed decreased whole-cell patch-clamp currents indicating that the disulfide bond between these residues is required for α(2)δ-1 function.
authors: Department of Cell Biology, Center for Research and Advanced Studies of the National Polytechnic Institute (Cinvestav-IPN), Mexico City, Mexico.
authors: Aida
authors: A
authors: Calderón-Rivera
authors: Arturo
authors: A
authors: Andrade
authors: Oscar
authors: O
authors: Hernández-Hernández
authors: Ricardo
authors: R
authors: González-Ramírez
authors: Alejandro
authors: A
authors: Sandoval
authors: Manuel
authors: M
authors: Rivera
authors: Juan Carlos
authors: JC
authors: Gomora
authors: Ricardo
authors: R
authors: Felix
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D015220:Calcium Channels / Q000737:chemistry* / Q000378:metabolism*; D020746:Calcium Channels, L-Type; D017124:Conserved Sequence / Q000235:genetics; D003545:Cysteine / Q000235:genetics; D004220:Disulfides / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry* / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D013329:Structure-Activity Relationship
chemical_list: C486060:Cacna2d1 protein, rat; D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D004220:Disulfides; D050505:Mutant Proteins; D021122:Protein Subunits; D003545:Cysteine