Structural characterization of the dihydropyridine-sensitive calcium channel α2- subunit and the associated δ peptides

Abstract

Upon disulfide bond reduction, the α2-subunit of the dihydropyridine-sensitive Ca2+ channel undergoes a characteristic mobility shift on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis with the concurrent appearance of the three δ peptides δ1 (25,000 Da), δ2 (22,000 Da), and δ3 (17,000 Da). Densitometric scanning of Coomassie Blue-stained gels shows a stoichiometric ratio of 1.0:0.31:0.47:0.08 for the α2-subunit and the δ peptides 1, 2, and 3, respectively. Characterization of the δ peptides using antibodies, photoincorporation of a hydrophobic probe, and lectin staining shows them to be antigenically similar hydrophobic glycoproteins. Amino-terminal sequence analysis of the δ peptides reveals three identical sequences that match the predicted amino acid sequence of the α2-subunit starting at Ala935. Enzymatic deglycosylation of the reduced α2·δ complex produces individual core peptides of 105,000 and 17,000 Da, respectively. Treatment of skeletal muscle membranes with high pH in the presence of reducing agents is able to extract the larger amino-terminal peptide but not the smaller carboxyl (δ) peptide, consistent with a single transmembrane domain in the carboxyl (δ) region. The data support a model of the α2-subunit in which the propeptide is processed into two chains that remain attached through disulfide linkages.