Cyclothiazide: a subunit‐specific inhibitor of GABA_C receptors

Abstract

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.

Mechanisms Underlying Lateral GABAergic Feedback onto Rod Bipolar Cells in Rat Retina

“…As shown previously (Singer and Diamond, 2003), blocking AMPAR desensitization with cyclothiazide (CTZ, 50 µM) increased reciprocal GABA release and caused a slower, GABA C R-mediated component to emerge in the reciprocal IPSC (Figure 6A). Although CTZ has been reported to antagonize both GABA A R and GABA C Rs (Deng and Chen, 2003; Xie et al, 2008), in our hands CTZ reduced only slightly GABA A R activation and exerted no effect on GABA C R activation in response to exogenous GABA puffs (Supplementary Figure 2A–D). We also observed that CTZ potentiated GABA release from A17 amacrine cells when stimulated by exogenous glutamate puffs (to 173 ± 17 % of control response; p = 0.00137; Supplementary Figure 2; Grimes et al 2009).…”

Pharmacological Analysis of the Activation and Receptor Properties of the Tonic GABACR Current in Retinal Bipolar Cell Terminals

“…At a concentration of 300 µM, cyclothiazide abolishes GABA responses mediated by ρ2 homomers but has no significant effect on the responses of ρ1 homomers [42]. We therefore examined the effect of cyclothiazide on GABA C R-mediated currents in BCTs.…”

“…Amino-acid substitutions in the pore-forming second transmembrane domain, in particular a switch at the 2′ position from proline in ρ1 to serine in ρ2, underlies subunit differences in properties such as deactivation rate, channel conductance and sensitivity to GABA [24], [63], [64]. This amino-acid substitution also underlies the difference in sensitivity to both picrotoxin and cyclothiazide of ρ1 and ρ2 receptors [41], [42].…”

Neurochemicals for the Investigation of GABAC Receptors

“…Cyclothiazide is a negative modulator of perch q2 GABA C receptors (IC 50 12 lM) without effect on human q1 receptors [79]. In addition to its widespread use as a positive modulator of AMPA receptors, cyclothiazide is a potent negative modulator of GABA A receptors [80].…”