Mutations of arginine 222 in pre-transmembrane domain I of mouse 5-HT3A receptor abolish 20(R)- but not 20(S)-Ginsenoside Rg(3) inhibition of 5-HT-mediated ion currents

Abstract

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 Rg(3) stereoisomers, 20-R-protopanaxatriol-3-[O-beta-D-glucopyranosyl (I -> 2)-beta-gl u copy ran oside], (20(R)-Rg(3)) and 20-S-protopanaxatriol-3-[O-beta-D-glucopyranosyl (1 -> 2)-beta-glueopyranoside], (20(S)-Rg(3)) in regulating 5-HT3A receptor-mediated ion currents (I5-HT) expressed in Xenopus oocytes and found that 20(S)-Rg(3). rather than 20(R)-Rg3 was more stronger inhibitor of I5-HP 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 compared to wildtype but also abolished 20(R)-Rg(3)-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)-Rg(3)- and 20(S)-Rg(3)-mediated 5-HT3A receptor channel regulation.