Identification of ginsenoside interaction sites in 5-HT3A receptors.
We previously demonstrated that 20(S)-ginsenoside Rg(3) (Rg(3)), one of the active components of Panax ginseng, non-competitively inhibits 5-HT(3A) receptor channel activity on extracellular side of the cell. Here, we sought to elucidate the molecular mechanisms underlying Rg(3)-induced 5-HT(3A) receptor regulation. We used the two-microelectrode voltage-clamp technique to investigate the effect of Rg(3) on 5-HT-mediated ion currents (I(5-HT)) in Xenopus oocytes expressing wild-type or 5-HT(3A) receptors harboring mutations in the gating pore region of transmembrane domain 2 (TM2). In oocytes expressing wild-type 5-HT(3A) receptors, Rg(3) dose-dependently inhibited peak I(5-HT) with an IC(50) of 27.6+/-4.3microM. Mutations V291A, F292A, and I295A in TM2 greatly attenuated or abolished the Rg(3)-induced inhibition of peak I(5-HT). Mutation V291A but not F292A and I295A induced constitutively active ion currents with decrease of current decay rate. Rg(3) accelerated the rate of current decay with dose-dependent manner in the presence of 5-HT. Rg(3) and TMB-8, an open channel blocker, dose-dependently inhibited constitutively active ion currents. The IC(50) values of constitutively active ion currents in V291A mutant receptor were 72.4+/-23.1 and 6.5+/-0.7microM for Rg(3) and TMB-8, respectively. Diltiazem did not prevent Rg(3)-induced inhibition of constitutively active ion currents in occlusion experiments. These results indicate that Rg(3) inhibits 5-HT(3A) receptor channel activity through interactions with residues V291, F292, and I295 in the channel gating region of TM2 and further demonstrate that Rg(3) regulates 5-HT(3A) receptor channel activity in the open state at different site(s) from those of TMB-8 and diltiazem.
Lee
Byung-Hwan
BH
Ginsentology Research Laboratory, Department of Physiology, College of Veterinary Medicine, Institute of Biomedical Science and Technology, Konkuk University, Seoul 143-701, South Korea.
Lee
Jun-Ho
JH
Lee
Sang-Mok
SM
Jeong
Sang Min
SM
Yoon
In-Soo
IS
Lee
Joon-Hee
JH
Choi
Sun-Hye
SH
Pyo
Mi Kyung
MK
Rhim
Hyewhon
H
Kim
Hyoung-Chun
HC
Jang
Choon-Gon
CG
Lee
Byoung-Cheol
BC
Park
Chul-Seung
CS
Nah
Seung-Yeol
SY
D000818:Animals; D002121:Calcium Channel Blockers / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D005707:Gallic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D036145:Ginsenosides / Q000737:chemistry / Q000494:pharmacology*; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D008958:Models, Molecular; D009154:Mutation / Q000502:physiology; D009865:Oocytes; D018408:Patch-Clamp Techniques / Q000379:methods; D044406:Receptors, Serotonin, 5-HT3 / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D012701:Serotonin / Q000494:pharmacology; D014982:Xenopus laevis
D002121:Calcium Channel Blockers; D036145:Ginsenosides; D044406:Receptors, Serotonin, 5-HT3; C097367:ginsenoside Rg3; D012701:Serotonin; C006014:8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate; D005707:Gallic Acid