Fang Hongyu, Patanavanich Saharat, Rajagopal Senthilkumar, Yi Xiaobin, Gill Monica S, Sando Julianne J, Kamatchi Ganesan L
Department of Anesthesiology, University of Virginia Health Sciences Systems, Charlottesville, Virginia 22908, USA.
J Biol Chem. 2006 Jul 21;281(29):20011-7. doi: 10.1074/jbc.M601776200. Epub 2006 May 16.
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.
电压门控钙通道（Ca(v)）2.2电流可被佛波醇-12-肉豆蔻酸酯13-乙酸酯（PMA）增强，而Ca(v) 2.3电流可被PMA和乙酰-β-甲基胆碱（MCh）增强。在α(1) 2.3亚基中鉴定出了MCh选择性位点，而鉴定出的PMA位点对PMA和MCh均有反应（卡马奇，G.L.，弗兰克，R.，林奇，C.三世，以及桑多，J.J.（2004年）《生物化学杂志》279，4102 - 4109；方，H.，弗兰克，R.，帕塔纳瓦尼奇，S.，拉尔瓦尼，A.，鲍威尔，N.K.，桑多，J.J.，以及卡马奇，G.L.（2005年）《生物化学杂志》280，23559 - 23565）。通过在α(1) 2.2亚基中进行丝氨酸/苏氨酸→丙氨酸突变，对α(1) 2.2亚基中的PMA位点与α(1) 2.3亚基中对PMA有反应的位点同源这一假说进行了测试。野生型α(1) 2.2或突变体与β1b和α2/δ亚基一起在非洲爪蟾卵母细胞中表达。使用Ba(2+)作为电荷载体记录内向电流（I(Ba)）。T422A、S1757A、S2108A或S2132A降低了PMA反应。相反，S425A增强了对PMA的反应，因此，它被认为是一个抑制位点。将每个鉴定出的刺激性丝氨酸/苏氨酸位点替换为天冬氨酸会增加基础电流并降低PMA诱导的增强作用，这与这些位点的磷酸化调节一致。多个突变体组合显示：（i）比单个丙氨酸突变引起的抑制作用更强；（ii）当苏氨酸-422和丝氨酸-2108存在时观察到的增强作用可能会被丝氨酸-425的存在所抑制；（iii）当丝氨酸-425被丙氨酸取代时，苏氨酸-422、丝氨酸-2108与丝氨酸-1757或丝氨酸-2132的组合对PMA可产生更大的反应。α(1) 2.2和α(1) 2.3亚基中的同源位点在功能上似乎有所不同。I-II连接区中抑制性磷酸化位点的存在似乎是α(1) 2.2亚基所特有的。
