Demonstration of the phosphorylation of dihydropyridine-sensitive calcium channels in chick skeletal muscle and the resultant activation of the channels after reconstitution.
We have examined the effects of cAMP elevating agents on the phosphorylation of dihydropyridine-sensitive Ca2+ channels in intact newborn chick skeletal muscle. In situ treatment with the beta-adrenergic receptor agonist isoproterenol resulted in the phosphorylation of the 170-kDa alpha 1 subunit in the intact cells, as evidenced by a marked decrease in the ability of the alpha 1 peptide to serve as a substrate in in vitro back phosphorylation reactions with [gamma-32P]ATP and the purified catalytic subunit of cAMP-dependent protein kinase. The phosphorylation of the 52-kDa beta subunit was not affected. The effects of isoproterenol were time- and concentration-dependent and were mimicked by other cAMP elevating agents but not by the Ca2+ ionophore A23187 or a protein kinase C activator. To test for functional effects of the observed phosphorylation, purified channels were reconstituted into liposomes containing entrapped fluo-3, and depolarization-sensitive and dihydropyridine-sensitive Ca2+ influx was measured. Channels from isoproterenol-treated muscle exhibited an increased rate and extent of Ca2+ influx compared to control preparations. The effects of isoproterenol pretreatment could be mimicked by phosphorylating the channels with cAMP-dependent protein kinase in vitro. These results demonstrate that the alpha 1 subunit of the dihydropyridine-sensitive Ca2(+)-channels is the primary target of cAMP-dependent phosphorylation in intact muscle and that the phosphorylation of this protein leads to activation of channel activity.
Mundiña-Weilenmann
C
C
Department of Pharmacology, Northwestern University Medical School, Chicago, Illinois 60611.
Chang
C F
CF
Gutierrez
L M
LM
Hosey
M M
MM
D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism*; D002478:Cells, Cultured; D002645:Chickens; D000242:Cyclic AMP / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D066298:In Vitro Techniques; D007545:Isoproterenol / Q000494:pharmacology; D008081:Liposomes; D009132:Muscles / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000502:physiology*; D013329:Structure-Activity Relationship; D013997:Time Factors
D015220:Calcium Channels; D008081:Liposomes; D010750:Phosphoproteins; D000242:Cyclic AMP; D011494:Protein Kinases; D007545:Isoproterenol; D002118:Calcium