α2 subunit specificity of cyclothiazide inhibition on glycine receptors

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