Mutations of arginine 222 in pre-transmembrane domain I of mouse 5-HT(3A) receptor abolish 20(R)- but not 20(S)-ginsenoside Rg(3) 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 Rg(3) stereoisomers, 20-R-protopanaxatriol-3-[O-beta-D-glucopyranosyl (1-->2)-beta-glucopyranoside], (20(R)-Rg(3)) and 20-S-protopanaxatriol-3-[O-beta-D-glucopyranosyl (1-->2)-beta-glucopyranoside], (20(S)-Rg(3)) in regulating 5-HT(3A) receptor-mediated ion currents (I(5-HT)) expressed in Xenopus oocytes and found that 20(S)-Rg(3) rather than 20(R)-Rg(3) was more stronger inhibitor of I(5-HT). In the present study, we further investigated the effects of 20(R)-Rg(3) and 20(S)-Rg(3) 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)-Rg(3) and 20(S)-Rg(3) 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 EC(50) values for I(5-HT) compared to wild-type but also abolished 20(R)-Rg(3)-induced inhibition of I(5-HT). Those mutations also shifted the IC(50) values by 20(S)-Rg(3) 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)-Rg(3)- and 20(S)-Rg(3)-mediated 5-HT(3A) receptor channel regulation.
Lee
Byung-Hwan
BH
Ginsentology Research Laboratory and Department of Physiology, College of Veterinary Medicine, Konkuk University, Seoul 143-701, Korea.
Lee
Jun-Ho
JH
Yoon
In-Soo
IS
Lee
Joon-Hee
JH
Choi
Sun-Hye
SH
Shin
Tae-Joon
TJ
Pyo
Mi Kyung
MK
Choi
Woo-Sung
WS
Lee
Sang-Mok
SM
Lim
Yoongho
Y
Rhim
Hyewhon
H
Nah
Seung-Yeol
SY
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D001120:Arginine / Q000235:genetics*; D036145:Ginsenosides / Q000494:pharmacology*; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D018408:Patch-Clamp Techniques; D018075:RNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D044406:Receptors, Serotonin, 5-HT3 / Q000235:genetics*; D011994:Recombinant Proteins / Q000494:pharmacology; D012701:Serotonin / Q000378:metabolism; D012702:Serotonin Antagonists / Q000494:pharmacology; D014981:Xenopus