Site-specific regulation of CA(V)2.2 channels by protein kinase C isozymes betaII and epsilon.
Site-specific regulation of CA(V)2.2 channels by protein kinase C isozymes betaII and epsilon.
复制标题
蛋白激酶 C 同工酶 betaII 和 epsilon 对 CA(V)2.2 通道的位点特异性调节。
DOI：
10.1016/j.neuroscience.2008.12.047
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发表时间：
2009
期刊：
影响因子：
3.3
通讯作者：
Kamatchi,GL
中科院分区：
文献类型：
--
作者：
Rajagopal,S;Fang,H;Oronce,CIA;Jhaveri,S;Taneja,S;Dehlin,EM;Snyder,SL;Sando,JJ;Kamatchi,GL
佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）通过α12.2亚基的I-II接头和C端的丝氨酸/苏氨酸蛋白激酶C（PKC）磷酸化位点调节Cav2.2高压门控钙通道。我们发现PMA对爪蟾卵母细胞表达的Cav2.2电流的增强作用可以被PKC βII或PKC ε同工酶的抑制剂阻断，如先前对Cav2.3电流所示，并且在表达WT Cav2.2通道的卵母细胞中显微注射PKC βII或PKC ε同工酶增加了基础钡电流（IBa）。I-V曲线显示，PKC β Ⅱ和PKC ε同工酶的电流幅度大幅增加，而在超极化方向上，I B峰仅发生微小位移。用GDPβS抑制G蛋白不改变PKC β Ⅱ和PKC ε同工酶对Cav2.2电流的增强作用。同工酶特异性抑制剂与先前产生的α12.2亚基的Ser/Thr至Ala突变体的组合显示，PKC βII或PKC ε同工酶（但不是PKC α或δ）可以通过I-II接头中的刺激位点（Thr-422）提供完全增强，但PKC ε更擅长通过抑制位点Ser-425降低通道活性。PKC βII或ε对Thr-422的增强作用优于对Ser-425的抑制作用。当C端存在任何潜在的刺激位点（Ser-1757、Ser-2108和Ser-2132）时，注射PKC βII也可增强Cav2.2电流。PKC ε对C末端位点的增强作用较小，仅对Ser-2108和Ser-2132的增强作用较小。位点Ser-1757和Ser-2132（但不是Ser-2108）对抑制位点Ser-425具有优势。总的来说，这些结果揭示了Cav2.2通道中的调节位点的层次结构。不同的PKC同工酶的位点特异性调节可能允许分级水平的通道激活和对随后的刺激事件的敏感性或抗性。
Cav2.2 high voltage-gated calcium channels are regulated by phorbol-12-myristae, 13-acetate (PMA) via Ser/Thr protein kinase C (PKC) phosphorylation sites in the I–II linker and C-terminus of the α12.2 subunit. Here we show that PMA enhancement of Cav2.2 currents expressed in Xenopus oocytes can be blocked by inhibitors of PKC βII or PKC ε isozymes, as shown previously for Cav2.3 currents, and that microinjection of PKC βII or PKC ε isozymes in the oocytes expressing the WT Cav2.2 channels increases the basal barium current (IBa). The I–V plot shows a large increase in current amplitude with PKC βII and PKC ε isozymes with only a small shift in the peak IBain the hyperpolarizing direction. The potentiation of Cav2.2 currents by microinjection of PKC βII and PKC ε isozymes was not altered by the inhibition of G proteins with GDPβS. The combination of isozyme specific inhibitors with previously generated Ser/Thr to Ala mutants of α12.2 subunit revealed that PKC βII or PKC ε isozymes (but not PKC α or δ) can provide full enhancement through the stimulatory site (Thr-422) in the I–II linker but that PKC ε is better at decreasing channel activity through the inhibitory site Ser-425. The enhancing effect of PKC βII or ε at Thr-422 is dominant over the inhibitory effect at Ser-425. Injected PKC βII also enhances Cav2.2 current when any of the potential stimulatory sites (Ser-1757, Ser-2108 and Ser-2132) are available in the C-terminus. PKC ε provides lesser enhancement with C-terminal sites and only with Ser-2108 and Ser-2132. Sites Ser-1757 and Ser-2132, but not Ser-2108, are dominant over the inhibitory site Ser-425. Collectively, these results reveal a hierarchy of regulatory sites in Cav2.2 channels. Site-specific regulation by different PKC isozymes may allow graded levels of channel activation and susceptibility or resistance to subsequent stimulatory events.