On the Benzodiazepine Binding Pocket in GABAA Receptors

Department of Pharmacology, University of Bern, CH-3010 Bern, Switzerland.
Journal of Biological Chemistry (Impact Factor: 4.57). 02/2004; 279(5):3160-8. DOI: 10.1074/jbc.M311371200
Source: PubMed


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 gamma-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 alpha(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 alpha(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 gamma(2)Ala-79 is probably located in the access pathway of the ligand to its binding pocket.

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    • "have identified that Gln182-Arg197 region of γ2 subunit is part of the allosteric pathway that allows propagation of structural changes induced by positive benzodiazepine modulators through the protein to the channel domain. Furthermore, several amino acid residues on α and γ subunits have been identified that are crucial for benzodiazepine activity and form benzodiazepine binding pocket [86] [103] [104] [105]. It is considered that antagonists bind in a pocket partly overlapping with the agonist site, although they can extend further into the solvent accessible cavity [106]. "
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    • "As described in the methods section DZP was manually positioned in a binding mode (Figure 8) satisfying the listed criteria deduced from experimental literature data followed by optimization of side chains in the binding pocket. The resulting model agrees to a large extent with the binding mode of DZP, recently described by Richter et al. [95] despite being built on different templates and is further in agreement with available mutational data (Table 2) indicating that residues α1F99, [67], [96] α1H101, [73], [97] α1Y159, [96], [98] α1Y209, [96], [98], [99] γ2F77, [100]–[102] and γ2M130 [96], [102] line the binding pocket. As was the case for the GABA othosteric binding site, the GluCl template contributes to the homology model with a higher similarity than the previously available templates. "
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    • "These residues can be associated with the canonical " loops " first identified in studies of the nAChR. His101 on loop A is essential to BZD binding (Buhr et al., 1996; Kucken et al., 2003; Berezhnoy et al., 2004), whereas a number of residues on loop E, beginning with Asn115, contribute to the complementary GABA binding component of the ␣ subunit (Cromer et al., 2002; Kloda and Czajkowski, 2007). These residues are connected through a linker that is unstructured in both the acetylcholine binding protein and the cryo-electron microscopy nAChR structures. "
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