A Role for the Val291 Residue within the Transmembrane Domain 2 in Diltiazem- and TMB-8 [3,4,5-Trimethoxybenzoic Acid 8-(Diethylamino)octyl Ester]-Mediated 5-Hydroxytryptamine Type 3A Receptor Regulations

Abstract

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 IC₅₀ of 71.4±4.9 and 4.5±0.3 μM, 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 IC₅₀ values of constitutively active ion currents in V291A receptors were 165.3±11.1 and 6.6±0.5 μM 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.