Cyclic AMP-Dependent Protein Kinase but Not Protein Kinase C Regulates the Cardiac Ca²⁺ Channel through Phosphorylation of Its α₁ Subunit

Abstract

The voltage-dependent L-type Ca²⁺ channel in the heart is regulated by cAMP-dependent protein kinase (PKA) and possibly by protein kinase C (PKC). We have investigated the channel modulation through phosphorylation by these protein kinases, using liposomes into which Ca²⁺ channels from bovine heart were reconstituted. Phosphorylation of the proteoliposomes with PKA increased the dihydropyridine-sensitive Ca²⁺ efflux from them by about 70%. PKA rapidly phosphorylated membrane proteins of 210 and 170 kDa. A dihydropyridine-class Ca²⁺ channel blocker, [³H] azidopine, specifically photo-labeled a protein of 210 kDa, suggesting that the 210-kDa phosphoprotein might be the α₁ subunit of the Ca²⁺ channel. In contrast, phosphorylation of the proteoliposomes with PKC failed to modulate the Ca²⁺ efflux. Although PKC catalyzed the phosphorylation of membrane proteins of 150, 130, 95, 67, and 62 kDa, the 210- and 170-kDa proteins were not phosphorylated by this kinase. These results suggest that phosphorylation of the 210-kDa protein in the cardiac sarcolemma by PKA may be responsible for modulation of the channel function, whereas modulation of the channel by PKC, if it occurs, must be the result of an indirect mechanism, e. g. phosphorylation of a cytoplasmic protein or an associated channel polypeptide, that cannot function in the reconstituted system.