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

已知鱼雷NACH受体(AChR)分别被α和Delta亚基上的蛋白激酶C(PKC)和353,354；353,354和361,362；用双电极电压钳和单通道膜片钳技术研究了这些蛋白激酶的磷酸化对非洲爪哇卵母细胞AChR通道特性的影响。应用10^lt；-6&gt；MACh诱导的单通道电流的斜率电导为31pS。内源性PKC激活增加了电导(41pS)，用带负电荷的氨基酸(Malpha PKC/NA333mDelta PKC/NA377)取代PKC磷酸化位点模拟了这种影响(41pS)。值得注意的是，用较高浓度的ACh(10^&lt；-4&gt；M)预处理后，电导也增加到相同水平(43ps)，这一作用可被PKC抑制剂阻断。这些结果表明，…更多的AChR是通过ACh自身激活的新的PKC途径被磷酸化的。随后，通过分析切除斑块获得的整体单通道电流，研究了PKC磷酸化对AChR脱敏的影响。缺失增量亚基PKC磷酸化位点的突变AChR(mdeltaDELTAPKC/Ser377)显著延缓脱敏速度，而α亚基(MalphaDELTAPKC/Ser333)或Malpha PKC/NA333mDelta PKC/NA377缺失则无明显影响。这为AChR脱敏受PKC自身磷酸化调控提供了进一步的证据。此外，通过AChR通道的钙内流也受PKC自身磷酸化的调节。共表达的5-羟色胺受体介导的PKC活化降低了钙通透性。相反，使用蛋白激酶C抑制剂可使钙离子通透性增加2倍。MdeltaPKC/NA377类似于PKC的磷酸化作用，而mdeltaDELTAPKC/Ser377与去磷酸化状态下一样，增强了细胞内钙离子的通透性。Malpha PKC/NA333或MalphaDELTAPKC/Ser333对其无影响，表明Delta亚基上的Ser^lt；377；gt；负责调节PKC自磷酸化引起的Ca^2&gt；通透性的调节。反之，PKA的磷酸化加速了脱敏和PKC的磷酸化。除了PKC的磷酸化外，通过AChR通道的钙内流也被PKA的磷酸化所促进，突变的AChR检测到其负责的部位是两个PKA亚基的磷酸化位点中的353位和353位。较少

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