Abstract
In the mammalian cortex, α2 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 (IGly) in cultured rat hippocampal neurons. The inhibition was glycine concentration-dependent, suggesting a competitive mechanism. Note that GlyRs containing the α2 but not α1 or α3 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 IGly in rat spinal neurons declined with time in culture, in parallel with a decline of α2 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 α2 subunit confers the specificity of CTZ action. Thus, CTZ is a potent and selective inhibitor of α2-GlyRs, and threonine at position 59 plays a critical role in the susceptibility of GlyR to CTZ inhibition.
Footnotes
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This study was supported by the National Natural Science Foundation of China (grant 30621062), by the National Basic Research Program of China (grant 2006CB500803), and by the Knowledge Innovation Project from the Chinese Academy of Sciences (grant KSCX2-YW-R-35).
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ABBREVIATIONS: GlyR, glycine receptor; CNS, central nervous system; CTZ, cyclothiazide; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; SDH, spinal dorsal horn; HEK, human embryonic kidney; PTX, picrotoxin; WT, wild type; DIV, day(s) in vitro.
- Received October 12, 2007.
- Accepted December 27, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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