Distinct regulation of expressed calcium channels 2.3 in Xenopus oocytes by direct or indirect activation of protein kinase C

Protein kinase C (PKC)-dependent regulation of voltage-gated Ca (Ca v; with α 1β1Bα2/δ subunits) channel 2.3 was investigated using phorbol 12-myristate 13-acetate (PMA), or by M 1 muscarinic receptor activation in Xenopus oocytes. The inward Ca 2+-current with Ba 2+ ( I Ba) as the charge carrier was potentiated by PMA or acetyl-β-methylcholine (MCh). The inactivating [ I (inact)] and non-inactivating [ I (noninact)] components of I Ba and the time constant of inactivation τ (inact) were all increased by MCh or PMA. This may be a PKC-dependent action since the effect of MCh and PMA was blocked by Ro-31-8425 or β-pseudosubstrate. MCh effect was blocked by atropine, guanosine-5′- O-(2-thiodiphosphate) trilithium (GDPβS) or U-73122. The effect of MCh but not PMA was blocked by the inhibition of inositol-1,4,5-trisphosphate (IP3) receptors, intracellular Ca 2+ ([Ca 2+] i) or the translocation of conventional PKC (cPKC) with heparin, BAPTA and βC2.4, respectively. While a lower concentration (25 nM) of Ro-31-8425 blocked MCh, a higher concentration (500 nM) of Ro-31-8425 was required to block PMA action. This differential susceptibility of MCh and PMA to heparin, BAPTA, βC2.4 or Ro-31-8425 is suggestive of the involvement of Ca 2+-dependent cPKC in MCh action, whereas cPKC and Ca 2+-independent novel PKC (nPKC) in PMA action. PMA led to additional increase in I Ba that was already potentiated by preadministered MCh (1 or 10 μM), leading to the suggestion that differential phosphorylation sites for cPKC and nPKC may be present in the α 12.3 subunit of Ca v 2.3 channels.