DIFFERENTIAL-EFFECTS OF SUBUNIT INTERACTIONS ON PROTEIN-KINASE-A-MEDIATED AND PROTEIN-KINASE-C-MEDIATED PHOSPHORYLATION OF L-TYPE CALCIUM CHANNELS

We have expressed the pore-forming als (skeletal muscle isoform) and a lpha(1C) (cardiac/brain isoform) subunits, as well as the accessory be ta(2a) (cardiac/brain isoform) and alpha(2)/delta subunits of the L-ty pe, dihydropyridine-sensitive calcium (Ca) channels in Spodoptera frug iperda insect cells (Sf9 cells) by infection with recombinant baculovi ruses in order to facilitate biochemical studies of these rare, hetero multimeric membrane proteins. Since the L-type channels are believed t o be regulated by protein phosphorylation, this expression system allo wed us to investigate which subunits could act as substrates for prote in kinase A and C (PKA and PKC) and to determine the potential role of subunit interactions in phosphorylation of the channel proteins. Usin g purified protein kinases in vitro, the membrane-associated alpha(1S) , alpha(1C), and beta(2a) subunits were demonstrated to be phosphoryla ted stoichiometrically by PKA. The extent of phosphorylation of these subunits by PKA was similar whether the subunits were expressed alone or in combination. In addition, the alpha(1C) and beta(2a) subunits we re phosphorylated stoichiometrically by PKC when expressed individuall y. In contrast, the als subunit, when expressed alone, was a poor subs trate for PKC, despite the fact that this subunit has been shown to be an excellent substrate for PKC in native skeletal muscle membranes. I nterestingly, co-expression of alpha(1S) with the beta(2a) subunit res tored the ability of the alpha(1S) subunit to serve as a substrate for PKC. These results strongly suggests that subunit interactions play a n important and potentially differential role in channel regulation by PKC, whereas phosphorylation of the same subunit by PKA occurs indepe ndent of subunit interaction. Furthermore, our results provide biochem ical evidence that, when co-expressed, the alpha(1C), alpha(1S), and b eta(2a) subunits of L-type Ca2+ channels are excellent substrates for PKA and PKC and support the hypothesis that phosphorylation of each of these subunits may participate in channel regulation by these kinases .