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

Institute of Biochemistry and Molecular Medicine, University of Bern, Bühlstrasse 28, CH-3012 Bern, Switzerland.
FEBS Letters (Impact Factor: 3.17). 11/2007; 581(24):4718-22. DOI: 10.1016/j.febslet.2007.08.068
Source: PubMed


Benzodiazepines are widely used drugs exerting sedative, anxiolytic, muscle relaxant, and anticonvulsant effects by acting through specific high affinity binding sites on some GABA(A) receptors. It is important to understand how these ligands are positioned in this binding site. We are especially interested here in the conformation of loop A of the alpha(1)beta(2)gamma(2) GABA(A) receptor containing a key residue for the interaction of benzodiazepines: alpha(1)H101. We describe a direct interaction of alpha(1)N102 with a diazepam- and an imidazobenzodiazepine-derivative. Our observations help to better understand the conformation of this region of the benzodiazepine pocket in GABA(A) receptor.

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    • "QM partial charges were calculated using Jaguar 7.6 default settings. Subsequently, DZP was manually docked to the BZD binding site according to experimental evidence: Docked DZP should 1) have the Cl-substituent positioned in the vicinity of or pointing towards α1H101, α1N102 [67], α1G157, α1V202, and α1V211 [68], 2) have the C-3 atom positioned in the vicinity of or directed towards α1S205 and α1T206 [69], 3) have the N-methyl substituent directed towards an exit from the binding cavity [70], and 4) have the pending phenyl ring positioned in a lipophilic cavity [71], [72]. Following manual positioning of DZP, the α1-γ2 interface was allowed to adapt to DZP using the side chain prediction tool in Prime in which backbone and residue sampling within 4 Å of DZP was performed. "
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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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    • " model - ing . It should however be noted that sequence identity of these two proteins only amounts to about 18% and that the binding pocket is to a large extent made up by loop structures Fig . 7 Positioning of diazepam in the benzodiazepine - binding pocket of a 1 b 2 c 2 receptors . The – Cl atom ( dark green ) points to a 1 H101 and a 1 N102 ( Tan et al . 2007a ) and to a 1 G157 , a 1 V202 and a 1 V211 ( Tan et al . 2007b ) , whereas the opposite hydrogen ( yellow ) attached to the - C - 3 atom points to a 1 S205 and a 1 T206 . An immobilized diazepam affinity column has been used to isolate the GABA A receptors form solubilized bovine brain ( Sigel et al . 1982 , 1983 ) . Diazepam was at - ta"
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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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    • "A caveat is that affinity-labeling could " capture " a minor orientation that does not contribute appreciably to the action of reversibly bound BZDs. DZ-NCS also modifies ␣ 1 N102C and ␥ 2 A79C, albeit with lower efficiency (Tan et al., 2007a), and an NCS analog of Ro 15-4513, which lacks the pendant phenyl and may have greater freedom to orient in the binding pocket, reacts with ␣ 1 residues 101, 157, 202, and 211. Confidence that DZ-NCS covalently linked to ␣1 residue 101 occupies the binding pocket similarly to reversibly bound DZ is increased because ␣ 1 His101 is a known contact residue that is critical for pharmacological activity of BZDs (McKernan et al., 1995; Davies et al., 1996; Duncalfe et al., 1996; Smith and Olsen, 2000) and because covalent linkage of DZ-NCS results in irreversible potentiation comparable with that produced by DZ (Berezhnoy et al., 2004). "
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    ABSTRACT: Positive allosteric modulation of the GABA(A) receptor (GABA(A)R) via the benzodiazepine recognition site is the mechanism whereby diverse chemical classes of therapeutic agents act to reduce anxiety, induce and maintain sleep, reduce seizures, and induce conscious sedation. The binding of such therapeutic agents to this allosteric modulatory site increases the affinity of GABA for the agonist recognition site. A major unanswered question, however, relates to how positive allosteric modulators dock in the 1,4-benzodiazepine (BZD) recognition site. In the present study, the X-ray structure of an acetylcholine binding protein from the snail Lymnea stagnalis and the results from site-directed affinity-labeling studies were used as the basis for modeling of the BZD binding pocket at the alpha(1)/gamma(2) subunit interface. A tethered BZD was introduced into the binding pocket, and molecular simulations were carried out to yield a set of candidate orientations of the BZD ligand in the binding pocket. Candidate orientations were refined based on known structure-activity and stereospecificity characteristics of BZDs and the impact of the alpha(1)H101R mutation. Results favor a model in which the BZD molecule is oriented such that the C5-phenyl substituent extends approximately parallel to the plane of the membrane rather than parallel to the ion channel. Application of this computational modeling strategy, which integrates site-directed affinity labeling with structure-activity knowledge to create a molecular model of the docking of active ligands in the binding pocket, may provide a basis for the design of more selective GABA(A)R modulators with enhanced therapeutic potential.
    Molecular pharmacology 06/2009; 76(2):440-50. DOI:10.1124/mol.109.054650 · 4.13 Impact Factor
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