Gα(q/11) coupled to mammalian phospholipase C beta 3-like enzyme mediates the ginsenoside effect on Ca2+-activated Cl- current in the Xenopus oocyte

초록

Recently we demonstrated that ginsenosides, the active ingredients of Panax ginseng, enhanced Ca2+-activated Cl- current in the Xenopus oocyte through a signal transduction mechanism involving the activation of pertussis toxin-insensitive G protein and phospholipase C (PLC). However, it has not yet been determined precisely which G protein subunit(s) and which PLC isoform(s) participate in the ginsenoside signaling. To provide answers to these questions, we investigated the changes in ginsenoside effect on the Cl- current after intraoocyte injections of the cRNAs coding various G protein subunits, a regulator of G protein signaling (RGS2), and Gbetagamma-binding proteins. In addition, we examined which of mammalian PLCbeta1-3 antibodies injected into the oocyte inhibited the action of ginsenosides on the Cl- current. Injection of Galpha(q) or Galpha(11) cRNA increased the basal Cl- current recorded 48 h after, and it further prevented ginsenosides from enhancing the Cl- current, whereas Galpha(12) and Galpha(oA) cRNA injection had no significant effect. The changes following Galpha(q) cRNA injection were prevented when Gbeta(1)gamma(2) and Galpha(q) subunits were co-expressed by simultaneous injection of the cRNAs coding these subunits. Injection of cRNA coding Galpha(q)Q209L, a constitutively active mutant that does not bind to Gbetagamma, produced effects similar to those of Galpha(q) cRNA injection. The effects of Galpha(q)Q209L cRNA injection, however, were not prevented by co-injection of Gbeta(1)gamma(2) cRNA. Injection of the cRNA coding RGS2, which interacts most selectively with Galpha(q/11) among various identified RGS isoforms and stimulates the hydrolysis of GTP to GDP in active GTP-bound Galpha subunit, resulted in a severe attenuation of ginsenoside effect on the Cl- current. Finally, antibodies against PLCbeta3, but not -beta1 and -beta2, markedly attenuated the ginsenoside effect examined at 3-h postinjection. These results suggest that Galpha(q/11), coupled to mammalian PLC beta3-like enzyme mediates ginsenoside effect on Ca2+-activated Cl- current in the Xenopus oocyte.

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