Site-specific regulation of CAV2.2 channels by protein kinase C isozymes βII and ε

Cav2.2 high voltage-gated calcium channels are regulated by phorbol-12-myristae, 13-acetate (PMA) via Ser/Thr protein kinase C (PKC) phosphorylation sites in the I–II linker and C-terminus of the α1 2.2 subunit. Here we show that PMA enhancement of Cav2.2 currents expressed in Xenopus oocytes can be blocked by inhibitors of PKC βII or PKC ε isozymes, as shown previously for Cav2.3 currents, and that microinjection of PKC βII or PKC ε isozymes in the oocytes expressing the WT Cav2.2 channels increases the basal barium current (IBa). The I–V plot shows a large increase in current amplitude with PKC βII and PKC ε isozymes with only a small shift in the peak IBa in the hyperpolarizing direction. The potentiation of Cav2.2 currents by microinjection of PKC βII and PKC ε isozymes was not altered by the inhibition of G proteins with GDPβS. The combination of isozyme specific inhibitors with previously generated Ser/Thr to Ala mutants of α1 2.2 subunit revealed that PKC βII or PKC ε isozymes (but not PKC α or δ) can provide full enhancement through the stimulatory site (Thr-422) in the I–II linker but that PKC ε is better at decreasing channel activity through the inhibitory site Ser-425. The enhancing effect of PKC βII or ε at Thr-422 is dominant over the inhibitory effect at Ser-425. Injected PKC βII also enhances Cav2.2 current when any of the potential stimulatory sites (Ser-1757, Ser-2108 and Ser-2132) are available in the C-terminus. PKC ε provides lesser enhancement with C-terminal sites and only with Ser-2108 and Ser-2132. Sites Ser-1757 and Ser-2132, but not Ser-2108, are dominant over the inhibitory site Ser-425. Collectively, these results reveal a hierarchy of regulatory sites in Cav2.2 channels. Site-specific regulation by different PKC isozymes may allow graded levels of channel activation and susceptibility or resistance to subsequent stimulatory events.