Protein kinase C isozyme-specific potentiation of expressed Ca v 2.3 currents by acetyl-beta-methylcholine and phorbol-12-myristate, 13-acetate.

Protein kinase C (PKC) is implicated in the potentiation of Ca v 2.3 currents by acetyl-beta-methylcholine (MCh), a muscarinic M1 receptor agonist or phorbol-12-myristate, 13-acetate (PMA). The PKC isozymes responsible for the action of MCh and PMA were investigated using translocation as a measure of activation and with isozyme-selective antagonists and siRNA. Ca v channels were expressed with alpha1 2.3, beta1b and alpha2delta subunits and muscarinic M1 receptors in the Xenopus oocytes and the expressed currents (I Ba) were studied using Ba2+ as the charge carrier. Translocation of PKC isozymes to the membrane studied by Western blot revealed that all eleven known PKC isozymes are present in the Xenopus oocytes. Exposure of the oocytes to MCh led to the translocation of PKC alpha whereas PMA activated PKC betaII and epsilon isozymes. The action of MCh was inhibited by Go 6976, an inhibitor of cPKC isozymes or PKC alpha siRNA. PMA-induced potentiation of Ca v 2.3 currents was inhibited by CG533 53, a PKC betaII antagonist, betaIIV5.3, a peptide translocation inhibitor of PKC betaII or PKC betaII siRNA. Similarly, epsilonV1.2, a peptide translocation inhibitor of PKC epsilon or PKC epsilon siRNA inhibited PMA action. The inhibitors of PKC increased the basal I Ba slightly. It is possible that some PKC isozymes have negative control over the I Ba. Our results implicate PKC alpha in the potentiation of Ca v 2.3 currents by MCh and PKC betaII and epsilon in the potentiation of Ca v 2.3 currents by PMA.

Inhibitory role of Ser-425 of the alpha1 2.2 subunit in the enhancement of Cav 2.2 currents by phorbol-12-myristate, 13-acetate.

Voltage-gated calcium channels (Ca(v)) 2.2 currents are potentiated by phorbol-12-myristate, 13-acetate (PMA), whereas Ca(v) 2.3 currents are increased by both PMA and acetyl-beta-methylcholine (MCh). MCh-selective sites were identified in the alpha(1) 2.3 subunit, whereas the identified PMA sites responded to both PMA and MCh (Kamatchi, G. L., Franke, R., Lynch, C., III, and Sando, J. J. (2004) J. Biol. Chem. 279, 4102-4109; Fang, H., Franke, R., Patanavanich, S., Lalvani, A., Powell, N. K., Sando, J. J., and Kamatchi, G. L. (2005) J. Biol. Chem. 280, 23559-23565). The hypothesis that PMA sites in the alpha(1) 2.2 subunit are homologous to the PMA-responsive sites in alpha(1) 2.3 subunit was tested with Ser/Thr --> Ala mutations in the alpha(1) 2.2 subunit. WT alpha(1) 2.2 or mutants were expressed in Xenopus oocytes in combination with beta1b and alpha2/delta subunits. Inward current (I(Ba)) was recorded using Ba(2+) as the charge carrier. T422A, S1757A, S2108A, or S2132A decreased the PMA response. In contrast, S425A increased the response to PMA, and thus, it was considered an inhibitory site. Replacement of each of the identified stimulatory Ser/Thr sites with Asp increased the basal current and decreased the PMA-induced enhancement, consistent with regulation by phosphorylation at these sites. Multiple mutant combinations showed (i) greater inhibition than that caused by the single Ala mutations; (ii) that enhancement observed when Thr-422 and Ser-2108 are available may be inhibited by the presence of Ser-425; and (iii) that the combination of Thr-422, Ser-2108, and either Ser-1757 or Ser-2132 can provide a greater response to PMA when Ser-425 is replaced with Ala. The homologous sites in alpha(1) 2.2 and alpha(1) 2.3 subunits seem to be functionally different. The existence of an inhibitory phosphorylation site in the I-II linker seems to be unique to the alpha(1) 2.2 subunit.

Role of alpha1 2.3 subunit I-II linker sites in the enhancement of Ca(v) 2.3 current by phorbol 12-myristate 13-acetate and acetyl-beta-methylcholine.

Potentiation of Ca(v) 2.3 currents by phorbol 12-myristate 13-acetate (PMA) or acetyl-beta-methylcholine (MCh) may be due to protein kinase C (PKC)-mediated phosphorylation of the alpha1 2.3 subunit. Mutational analysis of potential PKC sites unique to the alpha1 2.3 subunit revealed several sites in the II-III linker that are specific to MCh (Kamatchi, G., Franke, R., Lynch, C., III, and Sando, J. (2004) J. Biol. Chem. 279, 4102-4109). To identify sites responsive to PMA, Ser/Thr --> Ala mutations were made in potential PKC sites homologous to the alpha1 2.3 and 2.2 subunits, both of which respond to PMA. Wild type alpha1 2.3 or mutants were expressed in Xenopus oocytes in combination with beta1b and alpha2/delta subunits and muscarinic M1 receptors. Inward current (I(Ba)) was recorded using Ba2+ as the charge carrier. Thr-365 of the I-II linker was identified as the primary site of PMA action, and this site also was required, along with the previously identified MCh-selective sites, for the MCh response. Ser-369 and Ser-1995 contributed to current enhancement only if Thr-365 also was available. Mutation of the essential sites to Asp increased the basal I(Ba) and caused a corresponding decrease in the PMA or MCh responses, consistent with possible regulation of these sites by phosphorylation. These results suggest that PMA and MCh both activate a pathway that can regulate the common PMA-sensitive sites in the I-II linker but that MCh also activates an additional pathway required for regulation of the MCh-unique sites, especially in the II-III linker.