SITE-SPECIFIC REGULATION OF CAV2.2 CHANNELS BY PROTEIN KINASE C ISOZYMES βII AND ε

Ca(v)2.2 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 alpha(1) 2.2 subunit. Here we show that PMA enhancement of Ca(v)2.2 currents expressed in Xenopus oocytes can be blocked by inhibitors of PKC beta II or PKC epsilon isozymes, as shown previously for Ca(v)2.3 currents, and that microinjection of PKC beta II or PKC epsilon isozymes in the oocytes expressing the WT Ca(v)2.2 channels increases the basal barium current (I-Ba). The I-V plot shows a large increase in current amplitude with PKC beta II and PKC epsilon isozymes with only a small shift in the peak I-Ba in the hyperpolarizing direction. The potentiation of Ca(v)2.2 currents by microinjection of PKC beta II and PKC epsilon isozymes was not altered by the inhibition of G proteins with GDP beta S. The combination of isozyme specific inhibitors with previously generated Ser/Thr to Ala mutants of alpha(1) 2.2 subunit revealed that PKC beta II or PKC epsilon isozymes (but not PKC alpha or delta) can provide full enhancement through the stimulatory site (Thr-422) in the I-II linker but that PKC epsilon is better at decreasing channel activity through the inhibitory site Ser-425. The enhancing effect of PKC beta II or epsilon at Thr-422 is dominant over the inhibitory effect at Ser-425. Injected PKC beta II also enhances Ca(v)2.2 current when any of the potential stimulatory sites (Ser-1757, Ser-2108 and Ser-2132) are available in the C-terminus. PKC epsilon 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 Ca(v)2.2 channels. Site-specific regulation by different PKC isozymes may allow graded levels of channel activation and susceptibility or resistance to subsequent stimulatory events. (C) 2009 IBRO. Published by Elsevier Ltd. All rights reserved.