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

已知电鳐nACh受体（AChR）被蛋白激酶C（PKC）分别在<333><377>α和δ亚基上的Ser^和Ser^磷酸化，并且被cAMP依赖性蛋白激酶（PKA）分别在γ和δ亚基上的Ser^&lt;353，354&gt;和Ser^&lt;361，362&gt;磷酸化。在表达天然和突变AChRs的非洲爪蟾卵母细胞中，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AChR通道特性的影响，应用10 μ M ACh引起的单通道电流的斜率电导为31 pS，内源性PKC激活使电导增加（41 pS）<-6>，用带负电荷的氨基酸（malpha+PKC/NA 333 mdelta +PKC/NA 377）取代PKC磷酸化位点模拟了这种效应（41 pS）。值得注意的是，用更高浓度的ACh（10 μ M）预处理<-4>也将电导提高到相同水平（43 pS），并且这被PKC抑制剂阻断。这些结果表明 ...更多信息 AChR通过ACh本身激活的一种新的PKC途径磷酸化。随后，PKC磷酸化对AChR脱敏的影响进行了研究，通过分析从切除的补丁获得的合奏单通道电流。缺失δ亚基上PKC磷酸化位点的突变AChR（mdeltaDELTAPKC/Ser 377）显著延迟了脱敏速率，而缺失α亚基上PKC磷酸化位点的突变AChR（malphaDELTAPKC/Ser 333）或malpha+PKC/NA 333 mdelta +PKC/NA 377则没有影响。这为AChR脱敏受PKC自身磷酸化调节提供了额外的证据。此外，AChR通道的Ca^2+内流也受PKC自身磷酸化的调节。共表达的5-羟色胺受体介导的PKC激活降低了Ca^2+通透性。相比之下，用PKC抑制剂预处理使Ca^&lt;2+&gt;渗透性增加了2倍。mdelta+PKC/NA 377模拟PKC磷酸化的作用，而mdeltaDELTAPKC/Ser 377增强Ca^2+通透性，就像在去磷酸化状态下一样。malpha+PKC/NA 333或malphaDELTAPKC/Ser 333对Ca^2+通透性无影响，表明<377>δ亚基上的Ser^参与了PKC自磷酸化对Ca^2+通透性的调节，而PKA磷酸化则加速了失敏和PKC磷酸化的速率。除PKC磷酸化外，PKA磷酸化还可增强AChR通道Ca^2+内流，其作用位点是<353>突变型AChR γ亚基上两个PKA磷酸化位点的Ser^，表明PKC和PKA磷酸化在AChR信号转导中起重要作用。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