Abstract 1505: Protein kinase C Activation Inhibits α1D L-type Calcium Channel at N-terminal Serine 81 Phosphorylation Site
Introduction: α1D L-type Ca channel plays a vital role in the function of the neuroendocrine and cardiovascular systems. Here, we report on the molecular and functional basis of α1D Ca channel modulation by protein kinase C (PKC).
Methods: Wild type (WT) and α1D mutants lacking one or more putative phosphorylation sites at N-terminal region were expressed in tsA201cells and studied using the patch-clamp and biochemical techniques.
Results: The general PKC activator, PMA consistently inhibited α1D Ca current, α1D ICaL, recorded from WT and from four other mutants (ΔN(1–59), S98A/S100A, S91A and S121A) of the α1D channel but not from α1D/S81A mutant (see graph). Introduction of a negatively charged aspartate at position S81 mimicked PKC phosphorylation effect on α1D Ca channel. The modulation of α1D Ca channel by PKC was prevented by dialyzing cells with a 35 amino acid peptide corresponding to α1D N-terminal region comprising S81. Using PKC isozyme-specific antagonist peptides, we showed that inhibition of α1D ICaL by PMA was partially prevented by βII- and ϵPKC inhibitor peptides when used separately but not by βIPKC, δPKC, ηPKC and αPKC (see graph). The combination of βII- and ϵPKC inhibitor peptides completely antagonized PMA-inhibition of α1D ICaL. Biochemical experiments showed that S81 was readily phophorylated in vitro by PKC.
Conclusion: These data establish that PKC modulates α1D Ca channel at Serine 81 of the N-terminal. Moreover, ϵPKC and βIIPKC, are specifically implicated in the modulation of α1D Ca channel in tsA201 cells. These findings have significant implications in the pathophysiology of α1D Ca channel and in the development of PKC isozyme targeted therapeutics.