标题：Mutations of Arginine 222 in Pre-transmembrane Domain I of Mouse 5-HT3A Receptor Abolish 20(R)- But Not 20(S)-Ginsenoside Rg3 Inhibition of 5-HT-Mediated Ion Currents
摘要：Ginsenosides, active ingredients of Panax ginseng, exist as stereoisomers depending on the position of the hydroxyl group on carbon-20; i.e. 20( R )-ginsenoside and 20( S )-ginsenoside are epimers. We previously investigated the structure–activity relationship of the ginsenoside Rg3 stereoisomers, 20- R -protopanaxatriol-3-[ O -β- D -glucopyranosyl (1→2)-β-glucopyranoside], (20( R )-Rg3) and 20- S -protopanaxatriol-3-[ O -β- D -glucopyranosyl (1→2)-β-glucopyranoside], (20( S )-Rg3) in regulating 5-HT3A receptor-mediated ion currents ( I 5-HT) expressed in Xenopus oocytes and found that 20( S )-Rg3 rather than 20( R )-Rg3 was more stronger inhibitor of I 5-HT. In the present study, we further investigated the effects of 20( R )-Rg3 and 20( S )-Rg3 on mouse 5-HT3A receptor channel activity after site-directed mutations of 5-HT3A receptor facilitation site, which is located at pre-transmembrane domain I (pre-TM1). 5-HT3A receptor was expressed in Xenopus oocytes, and I 5-HT was measured using two-electrode voltage clamp technique. In wild-type, both 20( R )-Rg3 and 20( S )-Rg3 inhibited I 5-HT with concentration-dependent and reversible manner. Induction of 5-HT3A receptor facilitation by point mutations of pre-TM1 amino acid residue R222 to R222A, R222D, R222E or R222T not only decreased EC50 values for I 5-HT compared to wild-type but also abolished 20( R )-Rg3-induced inhibition of I 5-HT. Those mutations also shifted the IC50 values by 20( S )-Rg3 into right direction by 2- to 4-folds compared with wild-type. These results indicate that 5-HT3A receptor facilitation differentially affects 20( R )-Rg3- and 20( S )-Rg3-mediated 5-HT3A receptor channel regulation.
关键词：ginseng;ginsenoside stereoisomer;5-HT3A receptor;ligand-gated ion channel;Xenopus oocyte
