Inhibitory role of Ser-425 of the α ₁ 2.2 subunit in the enhancement of Ca _v 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-β-methylcholine (MCh). MCh-selective sites were identified in the α 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 α ₁ 2.2 subunit are homologous to the PMA-responsive sites in α ₁ 2.3 subunit was tested with Ser/Thr → Ala mutations in the α ₁ 2.2 subunit. WT α ₁ 2.2 or mutants were expressed in Xenopus oocytes in combination with β1b and α2/δ 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 α ₁ 2.2 and α 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 α ₁ 2.2 subunit.