Relative positioning of diazepam in the benzodiazepine‐binding‐pocket of GABA_A receptors

Abstract

GABA A receptors are the major inhibitory neurotransmitter receptors in the brain. Some of them are targets of benzodiazepines that are widely used in clinical practice for their sedative/hypnotic, anxiolytic, muscle relaxant and anticonvulsant effects. In order to rationally separate these different drug actions, we need to understand the interaction of such compounds with the benzodiazepine-binding pocket. With this aim, we mutated residues located in the benzodiazepinebinding site individually to cysteine. These mutated receptors were combined with benzodiazepine site ligands carrying a cysteine reactive group in a defined position. Proximal apposition of reaction partners will lead to a covalent reaction. We describe here such proximity-accelerated chemical coupling reactions of a 1 S205C and a 1 T206C with a diazepam derivative modified at the C-3 position with a reactive isothiocyanate group (-NCS). We also provide new data that identify a 1 H101C and a 1 N102C as exclusive sites of the reaction of a diazepam derivative where the -Cl atom is replaced by a -NCS group. Based on these observations we propose a relative positioning of diazepam within the benzodiazepinebinding site of a 1 b 2 c 2 receptors.

Pentameric ligand-gated ion channel ELIC is activated by GABA and modulated by benzodiazepines

“…Although Brflurazepam occupies a pocket that resembles the location of the α/γ interface in GABA A receptors, the bromine substituent on position 7 of the phenyl moiety in flurazepam points out of the benzodiazepine pocket in ELIC. In contrast, previous photolabeling studies with flunitrazepam (44) or experiments with isothiocyanate substitutions at position 7 of diazepam (45,46) position this moiety near α1H101 of loop A in GABA A receptors, a location deep inside the pocket (I79 in ELIC). Presumably, the divergent amino acid composition between ELIC and the high-affinity α/γ benzodiazepine site in GABA A receptors causes differences in the pocket architecture and results in different benzodiazepine-binding poses in ELIC.…”

Structure, Function, and Modulation of GABAA Receptors

“…Direct apposition of the reactive group and cysteine leads to a covalent reaction. From this work, we know that the C1 atom of diazepam is located close to ␣ 1 His-101 (75) and that the 3Ј-atom is located close to ␣ 1 Ser-205 and ␣ 1 Thr-206 (76). Very recently, diazepam has been docked into a multitude of conformational variants of a large number of homology models of the GABA A receptor, and the docking poses were rated according to all available experimental observations (77).…”

Influence of GABAA Receptor α Subunit Isoforms on the Benzodiazepine Binding Site

“…3-NCS was able to displace [ 3 H]Ro15–1788 or [ 3 H]flunitrazepam from wild-type α 1 β 2 γ 2 receptors. The K i values were 340±16 nM and 240±55 nM [32], respectively. The K i values for displacement of [ 3 H]Ro 15–1788 by 3-NCS in α 2 β 2 γ 2 were 1550±250 nM (n = 3) and in α 5 β 1 γ 2 receptors 9840±1480 nM (n = 3).…”

“…Diazepam is arranged in the binding pocket of α 1 β 2 γ 2 receptors such as to allow interaction of a reactive group replacing the –Cl atom with a reactive residue in place of α 1 H101 [28]–[31] and interaction of a reactive group attached to the 3′-atom with a reactive residues in place of α 1 S205 or α 1 S206 [32]. It has also been shown that a reactive residue in place of the –Cl atom interacts with a reactive residue in place the histidine homologous to α 1 H101 in α 2 , α 3 and the corresponding arginine residue in α 6 but not to the histidine residue in α 5 [33].…”