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

Byung-Hwan Lee; Sun-Hye Choi; Mi Kyung Pyo; Tae-Joon Shin; Sung-Hee Hwang; Bo-Ra Kim; Jun-Ho Lee; Hyewhon Rhim; Hyoung-Choon Kim; Seung-Yeol Nah

doi:10.1248/bpb.32.861

Regular Articles

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

Byung-Hwan Lee, Sun-Hye Choi, Mi Kyung Pyo, Tae-Joon Shin, Sung-Hee Hwang, Bo-Ra Kim, Jun-Ho Lee, Hyewhon Rhim, Hyoung-Choon Kim, Seung-Yeol Nah

Author information

Byung-Hwan Lee
Department of Physiology, College of Veterinary Medicine, Konkuk University
Sun-Hye Choi
Department of Physiology, College of Veterinary Medicine, Konkuk University
Mi Kyung Pyo
Department of Physiology, College of Veterinary Medicine, Konkuk University
Tae-Joon Shin
Department of Physiology, College of Veterinary Medicine, Konkuk University
Sung-Hee Hwang
Department of Physiology, College of Veterinary Medicine, Konkuk University
Bo-Ra Kim
Department of Physiology, College of Veterinary Medicine, Konkuk University
Jun-Ho Lee
Department of Physiology, College of Oriental Medicine Kyung-Hee University
Hyewhon Rhim
Biomedical Research Center, KIST
Hyoung-Choon Kim
Neurotoxicology Program, College of Pharmacy, Korea Institute of Drug Abuse, Kangwon National University
Seung-Yeol Nah
Department of Physiology, College of Veterinary Medicine, Konkuk University

Keywords: diltiazem, 5-hydroxytryptamine type 3A receptor, common interaction site, 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester

JOURNAL FREE ACCESS

2009 Volume 32 Issue 5 Pages 861-867

DOI https://doi.org/10.1248/bpb.32.861

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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.

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