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

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 (I5-HT) expressed in Xenopus oocytes and found that 20(S)-Rg3 rather than 20(R)-Rg3 was more stronger inhibitor of I5-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 I5-HT was measured using two-electrode voltage clamp technique. In wild-type, both 20(R)-Rg3 and 20(S)-Rg3 inhibited I5-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 I5-HT compared to wild-type but also abolished 20(R)-Rg3-induced inhibition of I5-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.