Characterization of the interaction of zopiclone with gamma-aminobutyric acid type A receptors

Zopiclone is a cyclopyrrolone that is used clinically as a hypnotic. Althou gh this drug is known to interact with neuronal gamma-aminobutyric acid typ e A receptors, its binding site(s) within the receptor oligomer has been re ported to be distinct from that of the classical benzodiazepines. After pho toaffinity labeling with flunitrazepam, receptors in rat cerebellar membran es showed differentially reduced affinity for flunitrazepam and zopiclone b y 50- and 3-fold, respectively. Because histidine 101 of the alpha-subunit is a major site of photolabeling, we have made specific substitutions of th is residue and studied the consequences on the binding properties of zopicl one and diazepam using recombinant alpha 1 beta 2 gamma 2-receptors transie ntly expressed in tsA201 cells. Both compounds showed similar binding profi les with receptors containing mutated alpha-subunits, suggesting a similar interaction with the residue at position 101. At alpha 1 beta 2 gamma 3-rec eptors, flunitrazepam affinity was dramatically decreased by approximately 36-fold, whereas the affinity for zopiclone was decreased only 3-fold, sugg esting a differential contribution of the gamma-subunit to the binding pock et. Additionally, we used electrophysiological techniques to examine the co ntribution of the gamma-subunit isoform in the receptor oligomer to ligand recognition using recombinant receptors expressed in Xenopus oocytes. Both compounds are agonists at alpha 1 beta 2 gamma 2- and alpha 1 beta 2 gamma 3-receptors, with flunitrazepam being more potent but less efficacious. In summary, these data suggest that histidine 101 of the alpha 1-subunit plays a similar role in ligand recognition for zopiclone, diazepam, and flunitra zepam.