Biol. Pharm. Bull., 30(9),1721-1726, September 2007

Regular Articles

Mutations of Arginine 222 in Pre-transmembrane Domain I of Mouse 5-HT_3A Receptor Abolish 20(R)- But Not 20(S)-Ginsenoside Rg₃ Inhibition of 5-HT-Mediated Ion Currents

Byung-Hwan LEE,^a,# Jun-Ho LEE,^a,# In-Soo YOON,^a Joon-Hee LEE,^a Sun-Hye CHOI,^a Tae-Joon SHIN,^a Mi Kyung PYO,^a Woo-Sung CHOI,^a Sang-Mok LEE,^a Yoongho LIM,^b Hyewhon RHIM,^c and Seung-Yeol NAH^*,a

^a Ginsentology Research Laboratory and Department of Physiology, College of Veterinary Medicine, Konkuk University; ^b Bio/Molecular Informatics Center, Konkuk University; Seoul 143-701, Korea: and ^c Life Sciences Division, KIST; Seoul 136-701, Korea. * To whom correspondence should be addressed. e-mail: synah@konkuk.ac.kr ^# These authors contributed equally to this work.

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-HT_3A 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-HT_3A receptor channel activity after site-directed mutations of 5-HT_3A receptor facilitation site, which is located at pre-transmembrane domain I (pre-TM1). 5-HT_3A 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-HT_3A 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-HT_3A receptor facilitation differentially affects 20(R)-Rg₃- and 20(S)-Rg₃-mediated 5-HT_3A receptor channel regulation.

Key words ginseng; ginsenoside stereoisomer; 5-HT_3A receptor; ligand-gated ion channel; Xenopus oocyte