Cyclothiazide: A subunit-specific inhibitor of GABA_C receptors

We tested the effects of cyclothiazide (CTZ), an agent used to block desensitization of AMPA-type glutamate receptors, on heterologously expressed GABA_C receptors formed by homomeric ρ subunits. CTZ inhibition of GABA_C receptors was subunit specific; it produced a dose-dependent reduction of the GABA-elicited current on homomeric ρ2 receptors with an IC 50 of about 12 μm, but had no significant effect on homomeric ρ1 receptors. This differential sensitivity was attributable to a single amino acid located on the second transmembrane domain of the ρ subunits. Mutating the residue at this position from serine to proline on the ρ2 subunit eliminated CTZ sensitivity, whereas switching proline to serine on the ρ1 subunit made the receptor CTZ sensitive. The inhibitory properties of CTZ were consistent with its action as a channel blocker on the receptors formed by ρ2 subunits. The effect showed a small degree of voltage dependence, and was due mainly to a non-competitive mechanism that reduced the maximum response elicited by GABA. In addition, the prominent membrane current rebound when co-application of GABA and CTZ was terminated suggests that the binding site for CTZ on the GABA_C receptor is distinct from that for GABA, and that CTZ acts as a non-competitive antagonist on the GABA_C receptor. CTZ inhibited the open channel of the GABA_C receptor with a time constant of about 0.4 s, but the kinetics were ∼10-fold slower when GABA is absent. The ability of CTZ to interact with various types of neurotransmitter receptors indicates that the drug has multiple actions in the CNS.