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2 Subunit Specificity of Cyclothiazide Inhibition on Glycine Receptors

Institute of Neuroscience and State Key Laboratory of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China (X.-B.Z., G.-C.S., L.-Y.L., F.Y., T.-L.X.); and School of Life Sciences, University of Science and Technology of China, Hefei, China (X.-B.Z., T.-L.X.)

In the mammalian cortex, 2 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 2 but not 1 or 3 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 2 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 2 subunit confers the specificity of CTZ action. Thus, CTZ is a potent and selective inhibitor of 2-GlyRs, and threonine at position 59 plays a critical role in the susceptibility of GlyR to CTZ inhibition.

Address correspondence to: Tian-Le Xu, Institute of Neuroscience, Chinese Academy of Sciences, 320 Yue-yang Road, Shanghai 200031, China. E-mail: tlxu{at}ion.ac.cn