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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    • "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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    ABSTRACT: We present a full-length α(1)β(2)γ(2) GABA receptor model optimized for agonists and benzodiazepine (BZD) allosteric modulators. We propose binding hypotheses for the agonists GABA, muscimol and THIP and for the allosteric modulator diazepam (DZP). The receptor model is primarily based on the glutamate-gated chloride channel (GluCl) from C. elegans and includes additional structural information from the prokaryotic ligand-gated ion channel ELIC in a few regions. Available mutational data of the binding sites are well explained by the model and the proposed ligand binding poses. We suggest a GABA binding mode similar to the binding mode of glutamate in the GluCl X-ray structure. Key interactions are predicted with residues α(1)R66, β(2)T202, α(1)T129, β(2)E155, β(2)Y205 and the backbone of β(2)S156. Muscimol is predicted to bind similarly, however, with minor differences rationalized with quantum mechanical energy calculations. Muscimol key interactions are predicted to be α(1)R66, β(2)T202, α(1)T129, β(2)E155, β(2)Y205 and β(2)F200. Furthermore, we argue that a water molecule could mediate further interactions between muscimol and the backbone of β(2)S156 and β(2)Y157. DZP is predicted to bind with interactions comparable to those of the agonists in the orthosteric site. The carbonyl group of DZP is predicted to interact with two threonines α(1)T206 and γ(2)T142, similar to the acidic moiety of GABA. The chlorine atom of DZP is placed near the important α(1)H101 and the N-methyl group near α(1)Y159, α(1)T206, and α(1)Y209. We present a binding mode of DZP in which the pending phenyl moiety of DZP is buried in the binding pocket and thus shielded from solvent exposure. Our full length GABA(A) receptor is made available as Model S1.
    PLoS ONE 01/2013; 8(1):e52323. DOI:10.1371/journal.pone.0052323 · 3.23 Impact Factor
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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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    ABSTRACT: The GABA type A receptor (GABA(A)R) is the major inhibitory receptor in the mammalian central nervous system and the target of numerous pharmaceuticals. The alpha-subunit of these pentameric Cys-loop neurotransmitter-gated ion channels contributes to the binding of both GABA and allosteric modulators such as the benzodiazepines, suggesting a role for this subunit in the conformational changes associated with activation of the receptor. Herein we use the nonsense suppression methodology to incorporate a photoactivatable unnatural amino acid and photochemically cleave the backbone of the alpha subunit of the alpha(1)beta(2) GABA(A)R in a linker region that is believed to span the subunit. Proteolytic cleavage impairs GABA but not pentobarbital activation, strongly suggesting that conformational changes involving this linker region are critical to the GABA activation pathway.
    Molecular pharmacology 04/2010; 78(1):29-35. DOI:10.1124/mol.109.059832 · 4.13 Impact Factor
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    • "ttached to – C - 3 of the positive allosteric modulator diazepam . In addition , we extend the findings by Berezhnoy et al . ( 2004 ) and Tan et al . ( 2007a ) by showing that none of 15 mutated receptors newly tested here show strong covalent reaction . This confirms that only a 1 H101 and a 1 N102 are located close to the – Cl atom of diazepam ( Berezhnoy et al . 2004 ; Tan et al . 2007a ) ."
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    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 benzodiazepine-binding 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 alpha(1)S205C and alpha(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 alpha(1)H101C and alpha(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 benzodiazepine-binding site of alpha(1)beta(2)gamma(2) receptors.
    Journal of Neurochemistry 10/2009; 111(5):1264-73. DOI:10.1111/j.1471-4159.2009.06419.x · 4.28 Impact Factor
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