| Abstrakt: |
We have expressed the pore-forming alpha1S (skeletal muscle isoform) and alpha1C (cardiac/brain isoform) subunits, as well as the accessory beta2a (cardiac/brain isoform) and alpha2/delta subunits of the L-type, dihydropyridine-sensitive calcium (Ca) channels in Spodoptera frugiperda insect cells (Sf9 cells) by infection with recombinant baculoviruses in order to facilitate biochemical studies of these rare, heteromultimeric membrane proteins. Since the L-type channels are believed to be regulated by protein phosphorylation, this expression system allowed us to investigate which subunits could act as substrates for protein kinase A and C (PKA and PKC) and to determine the potential role of subunit interactions in phosphorylation of the channel proteins. Using purified protein kinases in vitro, the membrane-associated alpha1S, alpha1C, and beta2a subunits were demonstrated to be phosphorylated 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 alpha1C and beta2a subunits were phosphorylated stoichiometrically by PKC when expressed individually. In contrast, the alpha1S subunit, when expressed alone, was a poor substrate for PKC, despite the fact that this subunit has been shown to be an excellent substrate for PKC in native skeletal muscle membranes. Interestingly, co-expression of alpha1S with the beta2a subunit restored the ability of the alpha1S subunit to serve as a substrate for PKC. These results strongly suggests that subunit interactions play an important and potentially differential role in channel regulation by PKC, whereas phosphorylation of the same subunit by PKA occurs independent of subunit interaction. Furthermore, our results provide biochemical evidence that, when co-expressed, the alpha1C, alpha1S, and beta2a 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. |
