Phosphorylation of protein kinase C and cAMP-dependent protein kinase involves the modulation of nACh receptor channel and the regulation of calcium permeability

已知，Torpedo nACh受体（AChR）分别被α和δ亚基上Ser^<333>和Ser^<377>的蛋白激酶C （PKC）磷酸化，以及γ和δ亚基上Ser^<353,354>和Ser^<361,362>的camp依赖性蛋白激酶（PKA）磷酸化。利用双电极电压钳和单通道膜片钳技术，研究了这些激酶磷酸化对表达天然和突变AChR的非洲爪蟾卵母细胞AChR通道特性的影响。应用10^<-6> M ACh引起的单通道电流的斜率电导为31 pS，内源性PKC激活增加了电导（41 pS），而带负电荷的氨基酸（malpha+PKC/NA333mdelta+PKC/NA377）替代PKC磷酸化位点模仿了这一效应（41 pS）。值得注意的是，高浓度ACh （10^<-4> M）预处理也将电导提高到相同水平（43 pS），这被PKC抑制剂阻断。这些结果表明，更多的AChR通过ACh自身激活的新的PKC途径被磷酸化。随后，通过分析从切除斑块中获得的整体单通道电流，研究了PKC磷酸化对AChR脱敏的影响。缺失δ亚基PKC磷酸化位点（mdeltaDELTAPKC/Ser377）的突变体AChR显著延缓了脱敏率，而缺失α亚基（malphaDELTAPKC/Ser333）或缺失α +PKC/NA333mdelta+PKC/NA377的突变体AChR则没有影响。这提供了另一个证据，证明AChR脱敏是由PKC自磷酸化调节的。此外，通过AChR通道的Ca^<2+>内流也受到PKC自磷酸化的调节。共表达5 -羟色胺受体介导的PKC活化降低了Ca^<2+>的通透性。相比之下，PKC抑制剂预处理使Ca^<2+>通透性增加2倍。mdelta+PKC/NA377模拟了PKC磷酸化的作用，而mdeltaDELTAPKC/Ser377增强了Ca^<2+>的通透性，就像在去磷酸化状态下一样。malpha+PKC/NA333或malphaDELTAPKC/Ser333对其无影响，表明delta亚基上的Ser^<377>由于PKC自磷酸化而负责调节Ca^<2+>的通透性。否则，PKA磷酸化加速了脱敏的速度，也加速了PKC磷酸化的速度。除了PKC磷酸化外，PKA磷酸化还增强了通过AChR通道的Ca^<2+>内流，突变体AChR检测到其负责位点为γ亚基上两个PKA磷酸化位点的Ser^<353>。本研究结果表明，PKC和PKA磷酸化对AChR的信号转导至关重要。少

Torpedo nACh receptor (AChR) is known to be phosphorylated by protein kinase C (PKC) at Ser^<333> and Ser^<377> on the alpha and delta subunit, respectively, and by cAMP-dependent protein kinase (PKA) at Ser^<353,354> and Ser^<361,362> on the gamma and delta subunit, respectively. The effects of phosphorylation by these kinases on AChR channel properties were examined in Xenopus oocytes expressing native and mutant AChRs using two-electrode voltage clamp and single channel patch clamp techniques.The slope conductance of single channel currents elicited by the application of 10^<-6> M ACh was 31 pS.Endogenous PKC activation increased the conductance (41 pS), and replacement of PKC phosphorylation sites with negatively charged amino acid (malpha+PKC/NA333mdelta+PKC/NA377) mimicked this effect (41 pS). Notably pretreatment with higher concentration of ACh (10^<-4> M) also enhanced the conductance to a same level (43 pS), and this was blocked by a PKC inhibitor. These results suggest that … More AChR is phosphorylated via a novel PKC pathway activated by ACh itself. Subsequently the effect of PKC phosphorylation on AChR desensitization was examined by analyzing the ensemble single channel currents obtained from excised patches. The mutant AChR lacking PKC phosphorylation site on the delta subunit (mdeltaDELTAPKC/Ser377) significantly delayd the rate of desensitization, whereas deletion of that on the alpha subunit (malphaDELTAPKC/Ser333) or malpha+PKC/NA333mdelta+PKC/NA377 had no effect. This provides an additional evidence that AChR desensitization is regulated by PKC autophosphorylation. Furthermore, Ca^<2+> influx through AChR channel is also regulated by PKC autophosphorylation. The activation of PKC mediated by coexpressed serotonin receptor reduced Ca^<2+> permeability. By contrast, pretreatment with a PKC inhibitor increased Ca^<2+> permeability 2-fold. mdelta+PKC/NA377 mimicked the effect of PKC phosphorylation, while mdeltaDELTAPKC/Ser377 enhanced Ca^<2+> permeability just as in a dephosphorylated state. malpha+PKC/NA333 or malphaDELTAPKC/Ser333 showed no effect on it, indicating that Ser^<377> on the delta subunit is responsible for regulation of Ca^<2+> permeability due to PKC autophosphorylation.Otherwise, PKA phosphorylation accelerated the rate of desensitization as well as PKC phosphorylation. Apart from PKC phosphorylation, Ca^<2+> influx through AChR channel was enhanced by PKA phosphorylation, and its responsible site was detected to be Ser^<353> of two PKA phosphorylation sites on the gamma subunit by mutant AChRs.The results presented here demonstrate that PKC and PKA phosphorylation are crucial for signal transduction in AChR. Less