Prevention of ginsenoside-induced desensitization of Ca2+-activated Cl- current by microinjection of inositol hexakisphosphate in Xenopus laevis oocytes - Involvement of GRK2 AND beta-arrestin I

Abstract

We demonstrated that ginsenosides, the active ingredient of Panax ginseng, enhance endogenous Ca2+-activated Cl- currents via Galpha(q/11)-phospholipase C-beta3 pathway in Xenopus laevis oocytes. Moreover, prolonged treatment of ginsenosides induced Cl- channel desensitization. However, the molecular mechanisms involved in ginsenoside-induced Cl- channel desensitization have not yet been determined precisely. To provide answers to these questions, we investigated the changes in ginsenoside-induced Cl- channel desensitization after intraoocyte injection of inositol hexakisphosphate (InsP(6)), which is known to bind beta-arrestins and interfere with beta-arrestin- induced receptor down-regulation, and cRNAs coding beta-arrestin I/II and G-protein receptor kinase 2 (GRK2), which is known to phosphorylate G protein-coupled receptors and attenuate agonist stimulations. When control oocytes were stimulated with ginsenosides, the second, third, and fourth responses to ginsenosides were 69.6 +/- 4.1, 9.2 +/- 2.3, and 2.6 +/- 2.2% of the first responses, respectively. Preintraoocyte injection of InsP(6) before ginsenoside treatment restored ginsenoside effect to initial response levels in a concentration, time-, and structurally specific manner, in that inositol hexasulfate had no effect. The EC50 was 13.9 +/- 8.7 muM. Injection of cRNA coding beta-arrestin I but not beta-arrestin II blocked InsP(6) effect on prevention of ginsenoside-induced Cl- channel desensitization. Injection of cRNA coding GRK2 abolished ginsenoside effect enhancing Cl- current. However, the GRK2-caused loss of ginsenoside effect on Cl- current was prevented by coinjection of GRK2 with GRK2-K220R, a dominant-negative mutant of GRK. These results indicate that ginsenoside-induced Cl- channel desensitization is mediated via activation of GRK2 and beta-arrestin I.