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Abstract: In order to explore the molecular interaction between cyclothiazide (CTZ) and γ-aminobutyric acidA (GABAA) receptors, possibly underlying inhibition of GABAA receptor currents, [3H]-CTZ was synthesized. Binding of [3H]-CTZ to rat brain synaptic membranes could be observed only in the presence of the GABAA receptor antagonist (−)[1S,9R]-bicuculline methiodide (BMI) (EC50,BMI =500±80μM). GABA decreased [3H]-CTZ binding induced by the presence 300μM and 3mM BMI with IC50,GABA values of 300±50μM and 5.0±0.7mM, respectively. Binding of CTZ to [3H]-CTZ labeled sites was characterized by IC50,CTZ values of 0.16±0.03μM ([BMI]=300μM) and 7.0±0.5μM ([BMI]=3mM). Binding of the diastereomeric fraction [3H]-(3R,1′S,4′S,5′R +3S,1′R,4′R,5′S)-CTZ induced by 3mM BMI was quantitatively the more significant in cerebrocortical and hippocampal membranes. It was characterized by IC50,CTZ =80±15nM and IC50,GABA =13±3mМ. In the absence of BMI, CTZ (1mM) significantly decreased GABA-induced enhancement of [3H]-flunitrazepam binding. Our findings suggest that functional inhibition may occur through binding of CTZ to an allosteric site of GABAA receptors. This allosteric site is possibly emerged in the receptor conformation, stabilized by BMI binding. [Copyright &y& Elsevier]