Previous reports have shown that diltiazem and TMB, calcium channel antagonists, inhibit 5-hydroxytryptamine type 3A (5-HT(3A)) receptor-mediated currents (I(5-HT)) in cell lines and in heterologously expressed Xenopus oocytes. In the present study, we sought to elucidate the molecular mechanisms underlying diltiazem- and TMB-induced 5-HT(3A) receptor regulations. We used the two-microelectrode voltage clamp technique to investigate the effect of diltiazem and TMB on 5-HT-mediated ion currents 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, diltiazem and TMB dose-dependently inhibited peak I(5-HT) with an IC(50) of 71.4+/-4.9 and 4.5+/-0.3 microM, respectively. Among various mutants of TM2, mutation V291A greatly attenuated and abolished the TMB- and diltiazem-induced inhibition of peak I(5-HT), respectively. Mutation V291A also induced constitutively active ion currents in the absence of 5-HT. Diltiazem and TMB inhibited constitutively active ion currents in a dose-dependent manner. The IC(50) values of constitutively active ion currents in V291A receptors were 165.3+/-11.1 and 6.6+/-0.5 microM for diltiazem and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), respectively. Results of site-directed mutagenesis experiments suggest that the Val291 residue could be a candidate for common interaction site for diltiazem- and TMB-8-mediated 5-HT(3A) receptor regulations.