|Budget Amount *help
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1995: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1994: ¥1,000,000 (Direct Cost: ¥1,000,000)
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
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