Abstract

Zopiclone is a cyclopyrrolone that is used clinically as a hypnotic. Although this drug is known to interact with neuronal γ-aminobutyric acid type A receptors, its binding site(s) within the receptor oligomer has been reported to be distinct from that of the classical benzodiazepines. After photoaffinity labeling with flunitrazepam, receptors in rat cerebellar membranes showed differentially reduced affinity for flunitrazepam and zopiclone by 50- and 3-fold, respectively. Because histidine 101 of the α-subunit is a major site of photolabeling, we have made specific substitutions of this residue and studied the consequences on the binding properties of zopiclone and diazepam using recombinant α1β2γ2-receptors transiently expressed in tsA201 cells. Both compounds showed similar binding profiles with receptors containing mutated α-subunits, suggesting a similar interaction with the residue at position 101. At α1β2γ3-receptors, flunitrazepam affinity was dramatically decreased by approximately 36-fold, whereas the affinity for zopiclone was decreased only 3-fold, suggesting a differential contribution of the γ-subunit to the binding pocket. Additionally, we used electrophysiological techniques to examine the contribution of the γ-subunit isoform in the receptor oligomer to ligand recognition using recombinant receptors expressed in Xenopusoocytes. Both compounds are agonists at α1β2γ2- and α1β2γ3-receptors, with flunitrazepam being more potent but less efficacious. In summary, these data suggest that histidine 101 of the α1-subunit plays a similar role in ligand recognition for zopiclone, diazepam, and flunitrazepam.