On the Benzodiazepine Binding Pocket in GABAA Receptors

Abstract

Benzodiazepines are used for their sedative/hypnotic, anxiolytic, muscle relaxant, and anticonvulsive effects. They exert their actions through a specific high affinity binding site on the major inhibitory neurotransmitter receptor, the ␥-aminobutyric acid, type A (GABA A ) receptor channel, where they act as positive allosteric modulators. To start to elucidate the relative positioning of benzodiazepine binding site ligands in their binding pocket, GABA A receptor residues thought to reside in the site were individually mutated to cysteine and combined with benzodiazepine analogs carrying substituents reactive to cysteine. Direct apposition of such reactive partners is expected to lead to an irreversible site-directed reaction. We describe here the covalent interaction of ␣ 1 H101C with a reactive group attached to the C-7 position of diazepam. This interaction was studied at the level of radioactive ligand binding and at the functional level using electrophysiological methods. Covalent reaction occurs concomitantly with occupancy of the binding pocket. It stabilizes the receptor in its allosterically stimulated conformation. Covalent modification is not observed in wild type receptors or when using mutated ␣ 1 H101C-containing receptors in combination with the reactive ligand pre-reacted with a sulfhydryl group, and the modification rate is reduced by the binding site ligand Ro15-1788. We present in addition evidence that ␥ 2 Ala-79 is probably located in the access pathway of the ligand to its binding pocket.

GABAA receptor subtypes: the “one glass of wine” receptors

“…Because the only difference between the alcohol antagonist Ro15-4513 and the non-alcohol antagonistic flumazenil is fluorine, instead of the larger azido moiety, at the C7 position of the benzodiazepine ring structure, the Ro15-4513 azido group is likely to occupy the ETOH site on these receptors. This view is fully consistent with reports that the C7 position is situated close to a residue critical for benzodiazepine binding (α1-H101 (rat numbering: (Dunn et al, 1999;Smith and Olsen, 2000;Berezhnoy et al, 2004). Most amazingly the homologous residue in α6 is α6R100 that we find to dramatically enhance alcohol sensitivity in recombinant α6β3δ receptors in vitro and in vivo when mutated to glutamine (Q) (Hanchar et al, 2005).…”

Two neighboring residues of loop A of the α1 subunit point towards the benzodiazepine binding site of GABAA receptors

“…1). Synthesis and sample preparation for both compounds are detailed elsewhere [19,20]. A receptors in HEK 293 cells, radioactive ligand binding assay and detection of a covalent reaction cDNAs, transfection into HEK 293 cells, radioactive ligand binding assay and detection of a covalent reaction are detailed elsewhere [19,20].…”

“…Synthesis and sample preparation for both compounds are detailed elsewhere [19,20]. A receptors in HEK 293 cells, radioactive ligand binding assay and detection of a covalent reaction cDNAs, transfection into HEK 293 cells, radioactive ligand binding assay and detection of a covalent reaction are detailed elsewhere [19,20]. Basically the latter included (a) incubation of membranes expressing wild type or mutant receptors with the reactive compound, (b) extensive washing of the membranes in order to remove non-reacted agent, and (c) a radioactive ligand binding assay to determine residual binding.…”

“…modification of receptor function by the Imid-NCS compound cRNA synthesis, Xenopus laevis oocyte injection and electrophysiological experiments have been previously described [19][20][21][22][23]. Covalent modification of a 1 N102Cb 2 c 2 receptors by Imid-NCS compound was tested as follows: after obtaining a reproducible response to the application of GABA, the Imid-NCS compound freshly diluted to 1 lM in perfusion medium was applied for 3 min.…”

“…The benzodiazepine binding site is constituted of six loops from A to F. The important residue a 1 H101 forming part of loop A has previously been shown to molecularly interact with diazepam [19] and imidazobenzodiazepine derivatives [20]. We investigated here the direct surrounding of this residue.…”