Identification of ginsenoside interaction sites in 5-HT3A receptors

We previously demonstrated that 20(S)-ginsenoside Rg3 (Rg3), one of the active components of Panax ginseng, non-competitively inhibits 5-HT3A receptor channel activity on extracellular side of the cell. Here, we sought to elucidate the molecular mechanisms underlying Rg3-induced 5-HT3A receptor regulation. We used the two-microelectrode voltage-clamp technique to investigate the effect of Rg3 on 5-HT-mediated ion currents (I5-HT) in Xenopus oocytes expressing wild-type or 5-HT3A receptors harboring mutations in the gating pore region of transmembrane domain 2 (TM2). In oocytes expressing wild-type 5-HT3A receptors, Rg3 dose-dependently inhibited peak I5-HT with an IC50 of 27.6 ± 4.3 μM. Mutations V291A, F292A, and I295A in TM2 greatly attenuated or abolished the Rg3-induced inhibition of peak I5-HT. Mutation V291A but not F292A and I295A induced constitutively active ion currents with decrease of current decay rate. Rg3 accelerated the rate of current decay with dose-dependent manner in the presence of 5-HT. Rg3 and TMB-8, an open channel blocker, dose-dependently inhibited constitutively active ion currents. The IC50 values of constitutively active ion currents in V291A mutant receptor were 72.4 ± 23.1 and 6.5 ± 0.7 μM for Rg3 and TMB-8, respectively. Diltiazem did not prevent Rg3-induced inhibition of constitutively active ion currents in occlusion experiments. These results indicate that Rg3 inhibits 5-HT3A receptor channel activity through interactions with residues V291, F292, and I295 in the channel gating region of TM2 and further demonstrate that Rg3 regulates 5-HT3A receptor channel activity in the open state at different site(s) from those of TMB-8 and diltiazem.