Lee Jun-Ho, Jeong Sang Min, Lee Byung-Hwan, Noh Hye-Sung, Kim Bo-Kyung, Kim Jai-Il, Rhim Hyewhon, Kim Hyoung-Chun, Kim Kyeong-Man, Nah Seung-Yeol
Research Laboratory for the Study of Ginseng Signal Transduction and Department of Physiology, College of Veterinary Medicine, College of Medicine, Konkuk University, Seoul 143-701 Korea.
J Biol Chem. 2004 Mar 12;279(11):9912-21. doi: 10.1074/jbc.M310824200. Epub 2003 Dec 29.
We demonstrated that ginsenosides, the active ingredient of Panax ginseng, enhance endogenous Ca(2+)-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 EC(50) was 13.9 +/- 8.7 microM. 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.
我们证明，人参的活性成分人参皂苷，通过非洲爪蟾卵母细胞中的Gα(q/11)-磷脂酶C-β3途径增强内源性Ca(2+)激活的Cl(-)电流。此外，人参皂苷的长时间处理会诱导Cl(-)通道脱敏。然而，人参皂苷诱导Cl(-)通道脱敏所涉及的分子机制尚未得到精确确定。为了回答这些问题，我们研究了在卵母细胞内注射肌醇六磷酸（InsP(6)）后人参皂苷诱导的Cl(-)通道脱敏的变化，已知InsP(6)可结合β-抑制蛋白并干扰β-抑制蛋白诱导的受体下调，以及注射编码β-抑制蛋白I/II和G蛋白受体激酶2（GRK2）的cRNA后的变化，已知GRK2可磷酸化G蛋白偶联受体并减弱激动剂刺激。当用人参皂苷刺激对照卵母细胞时，对人参皂苷的第二、第三和第四次反应分别为第一次反应的69.6±4.1%、9.2±2.3%和2.6±2.2%。在人参皂苷处理前进行卵母细胞内注射InsP(6)，以浓度、时间和结构特异性的方式将人参皂苷的作用恢复到初始反应水平，因为肌醇六硫酸没有作用。半数有效浓度（EC(50)）为13.9±8.7微摩尔。注射编码β-抑制蛋白I而非β-抑制蛋白II的cRNA可阻断InsP(6)对预防人参皂苷诱导的Cl(-)通道脱敏的作用。注射编码GRK2的cRNA可消除人参皂苷增强Cl(-)电流的作用。然而，通过将GRK2与GRK2的显性负性突变体GRK2-K220R共注射，可防止GRK2导致的人参皂苷对Cl(-)电流作用的丧失。这些结果表明，人参皂苷诱导的Cl(-)通道脱敏是通过GRK2和β-抑制蛋白I的激活介导的。
