Structural characterization of the dihydropyridine-sensitive calcium channel alpha 2-subunit and the associated delta peptides.
Upon disulfide bond reduction, the alpha 2-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 delta peptides delta 1 (25,000 Da), delta 2 (22,000 Da), and delta 3 (17,000 Da). Densitometric scanning of Coomassie Blue-stained gels shows a stoichiometric ration of 1.0:0.31.47:0.08 for the alpha 2-subunit and the delta peptides 1, 2, and 3, respectively. Characterization of the delta peptides using antibodies, photoincorporation of a hydrophobic probe, and lectin staining shows tham to be antigenically similar hydrophobic glycoproteins. Amino-terminal sequence analysis of the delta peptides reveals three identical sequences that match the predicted amino acid sequence of the alpha 2-subunit starting at Ala935. Enzymatic deglycosylation of the reduced alpha 2.delta 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 (delta) peptide, consistent with a single transmembrane domain in the carboxyl (delta) region. The data support a model of the alpha 2-subunit in which the propeptide is processed into two chains that remain attached through disulfide linkages.
Jay
S D
SD
Howard Hughes Medical Institute, University of Iowa College of Medicine, Iowa City 52242.
Sharp
A H
AH
Kahl
S D
SD
Vedvick
T S
TS
Harpold
M M
MM
Campbell
K P
KP
D000818:Animals; D015153:Blotting, Western; D015220:Calcium Channels / Q000187:drug effects*; D004095:Dihydropyridines / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D008861:Microsomes / Q000378:metabolism; D009132:Muscles / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D011817:Rabbits
D015220:Calcium Channels; D004095:Dihydropyridines; D010455:Peptides; C038806:1,4-dihydropyridine