This is the first report of a mutation that selectively reduces propofol sensitivity without altering the action of etomidate. The reduction in propofol sensitivity is consistent with the loss of a hydrogen bond within the propofol binding site. These results also suggest a possible orientation of the propofol molecule within its binding site.

Zopiclone is a cyclopyrrolone that is used clinically as a hypnotic. Although this drug is known to interact with neuronal gamma-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 alpha-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 alpha1beta2gamma2-receptors transiently expressed in tsA201 cells. Both compounds showed similar binding profiles with receptors containing mutated alpha-subunits, suggesting a similar interaction with the residue at position 101. At alpha1beta2gamma3-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 gamma-subunit to the binding pocket. Additionally, we used electrophysiological techniques to examine the contribution of the gamma-subunit isoform in the receptor oligomer to ligand recognition using recombinant receptors expressed in Xenopus oocytes. Both compounds are agonists at alpha1beta2gamma2- and alpha1beta2gamma3-receptors, with flunitrazepam being more potent but less efficacious. In summary, these data suggest that histidine 101 of the alpha1-subunit plays a similar role in ligand recognition for zopiclone, diazepam, and flunitrazepam.

GABA A receptors that contain either the a4-or a6-subunit isoform do not recognize classical 1,4-benzodiazepines (BZDs). However, other classes of BZD site ligands, including b-carbolines, bind to these diazepam-insensitive receptor subtypes. Some b-carbolines [e.g. ethyl b-carboline-3-carboxylate (b-CCE) and methyl 6,7-dimethoxy-4-ethyl-b-carboline-3-carboxylate (DMCM)] display a higher affinity for a4-compared to a6-containing receptors. In order to identify the structural determinants that underlie these affinity differences, we constructed chimeric a6/a4 subunits and co-expressed these with wild-type rat b2 and c2 L subunits in tsA201 cells for radioligand binding analysis. After identification of candidate regions, site-directed mutagenesis was used to narrow the ligand selectivity to a single amino acid residue (a6N204/a4I203). Substitutions at a6N204 did not alter the affinity of the imidazobenzodiazepine Ro15-4513. A homologous mutation in the diazepam-sensitive a1 subunit (S205N) resulted in a 7-8-fold reduction in affinity for the b-carbolines examined. Although the binding of the classical agonist flunitrazepam was relatively unaffected by this mutation in the a1 subunit, the affinity for Ro15-1788 and Ro15-4513 was decreased by 19-fold and 38-fold respectively. The importance of this residue, located in the Loop C region of the extracellular N-terminus of the subunit protein, emphasizes the differential interaction of ligands with the a subunit in diazepam-sensitive and -insensitive receptors. c-Aminobutyric acid type A (GABA A ) receptors are the major inhibitory receptors in the mammalian CNS. These receptors are members of the ligand-gated ion channel (LGIC) family that includes the nicotinic acetylcholine, glycine and serotonin type 3 (5-HT 3 ) receptors (Ortells and Lunt 1995). Each GABA A receptor is a heteropentamer of homologous subunits that assemble to form a GABA-gated, chloride-selective ion pore. Several different classes of receptor subunits have been cloned (a1-6, b1-3, c1-3, d, e, p and h) (for reviews, see Barnard et al. 1998;Korpi et al. 2002) and different combinations of these subunits in the oligomer determine the pharmacology of the receptor subtype. Although the number of subtypes remains unknown, most receptors appear to be formed by a, b and c subunits in a 2 : 2 : 1 stoichiometry (Tretter et al. 1997;Farrar et al. 1999). Allosteric modulation is a hallmark feature of the GABA A receptor and this is best exemplified by compounds that act at the benzodiazepine (BZD) binding site. Ligands of many chemical classes recognize this site and these compounds display a spectrum of efficacy from positive to negative allosteric modulation of GABA-gated chloride currents (Barnard et al. 1998).All receptors in the LGIC family share common structural features, including a similar subunit topology and overall receptor conformation. In all members of the family, agonist binding sites are thought to lie at subunit-subunit interfaces and to be formed by six distinct stretches or 'loop...

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