title: Alpha2 subunit specificity of cyclothiazide inhibition on glycine receptors.
abstract: In the mammalian cortex, alpha2 subunit-containing glycine receptors (GlyRs) mediate tonic inhibition, but the precise functional role of this type of GlyRs is difficult to establish because of the lack of subtype-selective antagonist. In this study, we found that cyclothiazide (CTZ), an epileptogenic agent, potently inhibited GlyR-mediated current (I(Gly)) in cultured rat hippocampal neurons. The inhibition was glycine concentration-dependent, suggesting a competitive mechanism. Note that GlyRs containing the alpha2 but not alpha1 or alpha3 subunits, when being heterologously expressed in human embryonic kidney 293T cells, were inhibited by CTZ, indicating subunit specificity of CTZ action. In addition, the degree of CTZ inhibition on I(Gly) in rat spinal neurons declined with time in culture, in parallel with a decline of alpha2 subunit expression, which is known to occur during spinal cord development. Furthermore, site-directed mutagenesis indicates that a single-amino acid threonine at position 59 near the N terminus of the alpha2 subunit confers the specificity of CTZ action. Thus, CTZ is a potent and selective inhibitor of alpha2-GlyRs, and threonine at position 59 plays a critical role in the susceptibility of GlyR to CTZ inhibition.
authors.affiliation: Institute of Neuroscience, Chinese Academy of Sciences, 320 Yue-yang Road, Shanghai 200031, China.
authors.forename: Xiao-Bing
authors.initials: XB
authors.lastname: Zhang
authors.forename: Guang-Chun
authors.initials: GC
authors.lastname: Sun
authors.forename: Lu-Ying
authors.initials: LY
authors.lastname: Liu
authors.forename: Fang
authors.initials: F
authors.lastname: Yu
authors.forename: Tian-Le
authors.initials: TL
authors.lastname: Xu
mesh_terms: D000818:Animals; D000959:Antihypertensive Agents / Q000494:pharmacology*; D001581:Benzothiadiazines / Q000494:pharmacology*; D002460:Cell Line; D002478:Cells, Cultured; D006624:Hippocampus / Q000187:drug effects / Q000196:embryology / Q000378:metabolism; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D050505:Mutant Proteins / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D010852:Picrotoxin / Q000494:pharmacology; D020671:Posterior Horn Cells / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D018009:Receptors, Glycine / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013379:Substrate Specificity / Q000187:drug effects; D013654:Taurine / Q000494:pharmacology; D013912:Threonine
chemical_list: D000959:Antihypertensive Agents; D001581:Benzothiadiazines; D050505:Mutant Proteins; D010852:Picrotoxin; D018009:Receptors, Glycine; D013654:Taurine; D013912:Threonine; C439504:GLRA2 protein, human; C004639:cyclothiazide