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.
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.
Department of Physiology, College of Veterinary Medicine, Konkuk University, Seoul, Korea.
Byung-Hwan
BH
Lee
Sun-Hye
SH
Choi
Mi Kyung
MK
Pyo
Tae-Joon
TJ
Shin
Sung-Hee
SH
Hwang
Bo-Ra
BR
Kim
Jun-Ho
JH
Lee
Hyewhon
H
Rhim
Hyoung-Choon
HC
Kim
Seung-Yeol
SY
Nah
D000595:Amino Acid Sequence; D000818:Animals; D004110:Diltiazem / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D005260:Female; D005707:Gallic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D015640:Ion Channel Gating / Q000187:drug effects; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D017434:Protein Structure, Tertiary; D044406:Receptors, Serotonin, 5-HT3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014633:Valine / Q000235:genetics*; D014982:Xenopus laevis
D044406:Receptors, Serotonin, 5-HT3; C006014:8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate; D005707:Gallic Acid; D004110:Diltiazem; D014633:Valine