Demonstration of the phosphorylation of dihydropyridine-sensitive calcium channels in chick skeletal muscle and the resultant activation of the channels after reconstitution

MUNDIÑA - WEILENMANN C; CHANG FC; GUTIERREZ L; HOSEY M

JOURNAL OF BIOLOGICAL CHEMISTRY

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Lugar: Bethesda, Maryland; Año: 1991 vol. 266 p. 4067 - 4073

0021-9258

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 a1 subunit in the intact cells, as evidenced by a marked decrease in the ability of the a1 peptide to serve as a substrate in in vitro back phosphorylation reactions with [Y-32P]ATP and the purified catalytic subunit of CAMP-dependent protein kinase. The phosphorylation of the 52-kDa alpha subunit was not affected. The effects of isoproterenol were timeand 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 a1 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.