Phosphorylation of an alpha 1-like subunit of an omega-conotoxin-sensitive brain calcium channel by cAMP-dependent protein kinase and protein kinase.
Antibodies that recognize the alpha 2 delta and alpha 1 subunits of skeletal muscle L-type calcium channels have been used to investigate the subunit components and phosphorylation of omega-conotoxin (omega-CgTx)-sensitive N-type calcium channels from rabbit brain. Photolabeling of the N-type channel with a photoreactive derivative of 125I-omega-CgTx results in the identification of a single polypeptide of 240 kDa. MANC-1, a monoclonal antibody recognizing alpha 2 delta subunits of L-type calcium channels from skeletal muscle, immunoprecipitates the omega-CgTx-labeled 240-kDa polypeptide and approximately 6% of the digitonin-solubilized 125I-omega-CgTx-labeled N-type channels. MANC-1 also immunoprecipitates a phosphoprotein of 240 kDa that comigrates with 125I-omega-CgTx-labeled N-type calcium channels, but not with L-type calcium channels, in sucrose gradients. Both cAMP-dependent protein kinase and protein kinase C are effective in the phosphorylation of this polypeptide. Similar to the alpha 1 subunits of skeletal muscle L-type calcium channels, the immunoprecipitation of the 240-kDa phosphoprotein by MANC-1 is prevented by the detergent Triton X-100. Anti-CP-(1382-1400), an antipeptide antibody against a highly conserved segment of the alpha 1 subunits of calcium channels, immunoprecipitates the 240-kDa phosphopeptide in Triton X-100. The 240-kDa protein is phosphorylated to a stoichiometry of approximately 1 mol of phosphate/mol of omega-CgTx-binding N-type calcium channels by both cAMP-dependent protein kinase and protein kinase C. Our results show that the 240-kDa polypeptide is an alpha 1-like subunit of an omega-CgTx-sensitive N-type calcium channel. The N-type calcium channels containing this subunit are phosphorylated by cAMP-dependent protein kinase and protein kinase C and contain noncovalently associated alpha 1-like and alpha 2 delta-like subunits as part of their oligomeric structure.
Ahlijanian
M K
MK
Department of Pharmacology, University of Washington, Seattle 98195.
Striessnig
J
J
Catterall
W A
WA
D000818:Animals; D001345:Autoradiography; D001923:Brain Chemistry; D015220:Calcium Channels / Q000187:drug effects*; D004591:Electrophoresis, Polyacrylamide Gel; D010456:Peptides, Cyclic / Q000378:metabolism* / Q000494:pharmacology; D010766:Phosphorylation; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D011817:Rabbits; D020866:omega-Conotoxin GVIA
D015220:Calcium Channels; D010456:Peptides, Cyclic; D020866:omega-Conotoxin GVIA; D011494:Protein Kinases; D011493:Protein Kinase C