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Recombinant ␣1␤2␥2 ␥-aminobutyric acid type A (GABA A ) receptors were functionally expressed in Xenopus oocytes. Upon the mutation F77L, diazepam and Ro 15-1788 retained the ability to interact with the benzodiazepine binding site, but zolpidem lost this ability. To quantify these data, radioligand binding experiments were performed using membrane preparations of transiently transfected human embryonic kidney 293 cells. The amino acid ␥77, phenylalanine, was also mutated to tyrosine, tryptophan, and isoleucine. Although there was little effect on Ro 15-1788 binding upon mutation to tyrosine, the loss in affinity for diazepam was from 12 to 2,720 nM. The change to leucine, in contrast, resulted in little change in the diazepam affinity, whereas there was a strongly reduced affinity for zolpidem from 17 to 4,870 nM and for methyl 6,7-dimethoxy-4-ethyl-␤-carboline-3-carboxylate (DMCM) from 1.9 to 1,780 nM, respectively. The change to tryptophan resulted in two-phasic displacement curves, and only about 50% of the [ 3 H]flunitrazepam binding could be displaced by zolpidem, DMCM, and Ro 15-1788, respectively, whereas midazolam and diazepam still resulted in 100% displacement, indicating the presence of two sites upon expression of this mutant receptor. Functional expression in Xenopus oocytes showed that all mutant channels displayed a comparatively small change (<4.3-fold) in their apparent agonist affinity and that these channels could still be functionally modulated by ligands of the benzodiazepine binding site. We conclude that subtle changes in ␥F77 drastically affect benzodiazepine pharmacology and that this residue probably interacts directly with most ligands of the benzodiazepine binding site and therefore defines part of the benzodiazepine binding pocket.GABA A 1 receptors are the major ion channels in mammalian brain conferring neuronal inhibition. Two subunits have initially been purified (1), and their coding DNA has been cloned (2). Later, a total of 15 mammalian subunits have been cloned (for reviews, see Refs. 3-7). They are homologous to subunits of the nicotinic acetylcholine receptor, of the glycine receptor, and the serotonin type 3 receptor, and it is assumed that the natural receptor is a pentameric protein (8).The GABA A receptor is the site of action of benzodiazepines and related compounds ( Fig. 1; for review, see Ref. 6). There is a widespread use of some benzodiazepines for their anxiolytic, sedative, muscle relaxant, and anticonvulsive properties, and the structural determinants underlying benzodiazepine action are of interest. Clinically used, sedative benzodiazepines act as positive allosteric modulators of the receptor. The ␣1 subunit has been described as the major subunit that is photoaffinity labeled by [ 3 H]flunitrazepam (9), and one major labeled amino acid has been identified (10). In agreement with these observations, in vitro binding studies identified several amino acid residues in ␣1, ␣3, ␣4, and ␣6 to be involved in benzodiazepine binding (11)(12)(13)(14). In addition, a ␥...

Ligands of the benzodiazepine binding site allosterically modulate gamma-aminobutyric acidA receptors. Their binding pocket is made up of amino acid residues located on both alpha and gamma subunits. We transiently expressed wild-type alpha1beta2gamma2 and mutant GABAA receptors in human embryonic kidney 293 cells and determined their binding properties. Receptors containing the mutant alphaY209A showed approximately 40-fold decrease in affinity for [3H]Ro 15-1788 and diazepam, whereas zolpidem displayed no measurable affinity. Receptors containing the mutant alphaY209F showed a small-to-moderate decrease in affinity for [3H]Ro 15-1788, diazepam, zolpidem, methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, and Cl 218872, amounting to 2-8-fold. Receptors containing the mutant alphaY209Q appeared in the surface membrane of transfected cells, bound [3H]muscimol with wild-type affinity, but failed to bind [3H]Ro 15-1788 or [3H]flunitrazepam with detectable affinity. If these mutant receptors were expressed in Xenopus laevis oocytes, the apparent affinity for GABA was only slightly decreased, whereas the ability of the currents to be stimulated by low concentrations of flunitrazepam was abolished. Receptors containing a point mutant of another amino acid residue, alphaT206A, surprisingly showed an increase in affinity of 5- and 16-fold, for the negative allosteric modulator methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate and the partial positive allosteric modulator Cl 218872, respectively, whereas there was only a small decrease in affinity for Ro 15-1788, diazepam, and zolpidem, amounting to 2-, 4-, and 5-fold. Both alpha206 and alpha209 are thus both important in determining the binding affinities for ligands of the benzodiazepine binding site. The residues are spaced at an interval of three amino acids and may be part of an alpha helix.

Wild-type alpha1beta2gamma2 gamma-aminobutyric acid (GABA)A receptors and receptors containing a point-mutated subunit gamma2F77Y were expressed by transient transfection in human embryonic kidney 293 cells. Mutant receptors bound the benzodiazepine binding site ligand [3H]flumazenil with similar, subnanomolar affinity as wild-type receptor. Displacement studies with diazepam showed that the affinity for this compound was reduced 250-fold on mutation, indicating that the tyrosine hydroxyl group interferes with diazepam binding. This differential behavior then was used to find the chemical entity presumably interacting with the phenyalanine residue in position 77 of the gamma2 subunit of wild-type receptors. Thirty-four substances were analyzed in this respect. Our results suggest that the phenyl substituent of diazepam is located close to gammaF77. Similarly, we investigated the possible location of alpha1T206 and gamma2M130. Electrophysiological data obtained with the wild-type receptor furthermore suggest a simple overlap between positive allosteric modulators acting at the benzodiazepine binding site with its antagonists.