Identification of ginsenoside interaction sites in 5-HT_3A receptors

We previously demonstrated that 20(S)-ginsenoside Rg₃ (Rg₃), 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 Rg₃-induced 5-HT_3A receptor regulation. We used the two-microelectrode voltage-clamp technique to investigate the effect of Rg₃ 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, Rg₃ dose-dependently inhibited peak I_5-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 I_5-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-HT_3A 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-HT_3A receptor channel activity in the open state at different site(s) from those of TMB-8 and diltiazem.