Abstract
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.
Highlights
Subunits are collectively called the auxiliary subunits, and they dramatically influence the surface expression of the channels and the kinetics of the current (5)
It is possible that protein kinase C (PKC) phosphorylation sites (Ser/Thr) with varying selectivity to PKC isozymes exist in the ␣1 subunit
Since Cav 2.3 currents are potentiated by MCh or PMA and Cav 2.2 currents are increased by PMA only, we hypothesized that (i) the ␣1 2.3 subunit has both MCh-selective (Ser-888, Ser-892, Ser-894, and Ser-1987) and PMA-selective (Thr-365, Ser-369, Thr-879, Ser-1995, and Ser-2011) potential PKC phosphorylation sites; (ii) the ␣1 2.2 subunit has PMA-selective sites (Thr-422, Ser-425, Ser-926, Ser-2108, and Ser-2132) only; and (iii) the PMA-selective sites of ␣1 2.2 and 2.3 subunits are homologous
Summary
Construction of Mutants—We used the splice variant 37b of the ␣1 2.2 subunit from the rat superior cervical ganglion in this study (11). The oocytes were washed twice in calcium-free OR2 solution (in mM: 82.5 NaCl, 2 KCl, 1.8 MgCl2, 5 HEPES, pH 7.5) and transferred to OR2 solution containing 1 mg/ml collagenase (type 1A; Sigma). The oocytes were returned to Barth’s solution and incubated at 16 °C for 6 – 8 days before the recording of current. The current-voltage (I-V) relationship in oocytes expressing the wild type Cav 2.2 channel or the mutant was determined wherever necessary. Following the recording of the control IBa, PMA was perfused for 60 s, and the current was recorded after another 60 s, exposing the oocyte to the agonist for a period of 2 min. To block endogenous ClϪ currents, niflumic acid (Sigma) was added to the recording solution, which was stirred overnight in order for it to dissolve. Statistical significance was determined using Student’s t test or paired t test, and p Ͻ 0.05 was considered significant
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