Rajagopal S, Fang H, Oronce C I A, Jhaveri S, Taneja S, Dehlin E M, Snyder S L, Sando J J, Kamatchi G L
Department of Anesthesiology, University of Virginia, Charlottesville, VA 22903, USA.
Neuroscience. 2009 Mar 17;159(2):618-28. doi: 10.1016/j.neuroscience.2008.12.047. Epub 2009 Jan 3.
Ca(v)2.2 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 alpha(1) 2.2 subunit. Here we show that PMA enhancement of Ca(v)2.2 currents expressed in Xenopus oocytes can be blocked by inhibitors of PKC betaII or PKC epsilon isozymes, as shown previously for Ca(v)2.3 currents, and that microinjection of PKC betaII or PKC epsilon isozymes in the oocytes expressing the WT Ca(v)2.2 channels increases the basal barium current (I(Ba)). The I-V plot shows a large increase in current amplitude with PKC betaII and PKC epsilon isozymes with only a small shift in the peak I(Ba) in the hyperpolarizing direction. The potentiation of Ca(v)2.2 currents by microinjection of PKC betaII and PKC epsilon isozymes was not altered by the inhibition of G proteins with GDPbetaS. The combination of isozyme specific inhibitors with previously generated Ser/Thr to Ala mutants of alpha(1) 2.2 subunit revealed that PKC betaII or PKC epsilon isozymes (but not PKC alpha or delta) can provide full enhancement through the stimulatory site (Thr-422) in the I-II linker but that PKC epsilon is better at decreasing channel activity through the inhibitory site Ser-425. The enhancing effect of PKC betaII or epsilon at Thr-422 is dominant over the inhibitory effect at Ser-425. Injected PKC betaII also enhances Ca(v)2.2 current when any of the potential stimulatory sites (Ser-1757, Ser-2108 and Ser-2132) are available in the C-terminus. PKC epsilon 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 Ca(v)2.2 channels. Site-specific regulation by different PKC isozymes may allow graded levels of channel activation and susceptibility or resistance to subsequent stimulatory events.
Ca(v)2.2 高电压门控钙通道受佛波醇-12-肉豆蔻酸酯-13-乙酸酯（PMA）通过α(1) 2.2亚基I-II连接区和C末端的丝氨酸/苏氨酸蛋白激酶C（PKC）磷酸化位点进行调节。在此我们表明，PMA对非洲爪蟾卵母细胞中表达的Ca(v)2.2电流的增强作用可被PKC βII或PKC ε同工酶的抑制剂阻断，这与之前对Ca(v)2.3电流的研究结果一致，并且在表达野生型Ca(v)2.2通道的卵母细胞中显微注射PKC βII或PKC ε同工酶会增加基础钡电流（I(Ba)）。I-V曲线显示，PKC βII和PKC ε同工酶使电流幅度大幅增加，而峰值I(Ba)仅在超极化方向有小的偏移。用GDPβS抑制G蛋白并不会改变显微注射PKC βII和PKC ε同工酶对Ca(v)2.2电流的增强作用。将同工酶特异性抑制剂与先前构建的α(1) 2.2亚基丝氨酸/苏氨酸到丙氨酸的突变体相结合的研究表明，PKC βII或PKC ε同工酶（而非PKC α或δ）可通过I-II连接区的刺激位点（苏氨酸-422）实现完全增强作用，但PKC ε通过抑制位点丝氨酸-425降低通道活性的效果更好。PKC βII或ε在苏氨酸-422处的增强作用强于在丝氨酸-425处的抑制作用。当C末端存在任何潜在的刺激位点（丝氨酸-1757、丝氨酸-2108和丝氨酸-2132）时，注射的PKC βII也会增强Ca(v)2.2电流。PKC ε对C末端位点的增强作用较小，且仅对丝氨酸-2108和丝氨酸-2132有增强作用。丝氨酸-1757和丝氨酸-2132位点（而非丝氨酸-2108）的作用强于抑制位点丝氨酸-425。总体而言，这些结果揭示了Ca(v)2.2通道中调节位点的层级关系。不同PKC同工酶的位点特异性调节可能允许通道激活以及对后续刺激事件的敏感性或抗性达到不同程度。
