Activation of purified calcium channels by stoichiometric protein phosphorylation.
Purified dihydropyridine-sensitive calcium channels from rabbit skeletal muscle were reconstituted into phosphatidylcholine vesicles to evaluate the effect of phosphorylation by cyclic AMP-dependent protein kinase (PK-A) on their function. Both the rate and extent of 45Ca2+ uptake into vesicles containing reconstituted calcium channels were increased severalfold after incubation with ATP and PK-A. The degree of stimulation of 45Ca2+ uptake was linearly proportional to the extent of phosphorylation of the alpha 1 and beta subunits of the calcium channel up to a stoichiometry of approximately 1 mol of phosphate incorporated into each subunit. The calcium channels activated by phosphorylation were determined to be incorporated into the reconstituted vesicles in the inside-out orientation and were completely inhibited by low concentrations of dihydropyridines, phenylalkylamines, Cd2+, Ni2+, and Mg2+. The results demonstrate a direct relationship between PK-A-catalyzed phosphorylation of the alpha 1 and beta subunits of the purified calcium channel and activation of the ion conductance activity of the dihydropyridine-sensitive calcium channels.
Nunoki
K
K
Department of Pharmacology, University of Washington, Seattle 98195.
Florio
V
V
Catterall
W A
WA
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D002132:Calcium Radioisotopes; D002413:Cations, Divalent; D002458:Cell Fractionation; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism; D009132:Muscles / Q000502:physiology*; D010713:Phosphatidylcholines; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011817:Rabbits
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D002132:Calcium Radioisotopes; D002413:Cations, Divalent; D008565:Membrane Proteins; D010713:Phosphatidylcholines; D010761:Phosphorus Radioisotopes; D000255:Adenosine Triphosphate; D002118:Calcium