Received for publication October 12, 2007.
Revised December 22, 2007.
Accepted for publication December 27, 2007.

Alpha 2 Subunit Specificity of Cyclothiazide Inhibition on Glycine Receptors

Abstract

In the mammalian cortex 2 subunit-containing glycine receptors (GlyRs) mediate tonic inhibition, but the precise functional role of this form of GlyRs is difficult to establish because of the lack of subtype-selective antagonist. In this study, we found that cyclothiazide (CTZ), an epilepitogenic agent, potently inhibited GlyR-mediated current (I_Gly) in cultured rat hippocampal neurons. The inhibition was glycine concentration-dependent, suggesting a competitive mechanism. Notably, when heterologously expressed in HEK293T cells, GlyRs containing the 2 but not 1 or 3 subunit 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-terminal of the 2 subunit confers the specificity of CTZ action. Thus CTZ is a potent and selective inhibitor of 2-GlyRs, and threonine 59 site plays a critical role in the susceptibility of GlyR to CTZ inhibition.

Key words: Glycine, Ion channel regulation, Mutagenesis/Chimeric approaches