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

Institute of Biochemistry and Molecular Medicine, University of Bern, Bühlstrasse 28, CH-3012 Bern, Switzerland.
FEBS Letters (Impact Factor: 3.34). 11/2007; 581(24):4718-22. DOI: 10.1016/j.febslet.2007.08.068
Source: PubMed

ABSTRACT 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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Available from: Erwin Sigel, Aug 27, 2015
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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 · 4.24 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 · 4.12 Impact Factor
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    • "In the NCS compound, the -Cl group is replaced by –NCS (Fig. 1). The synthesis is described elsewhere (Berezhnoy et al. 2004; Tan et al. 2007b). The compound was dissolved in DMSO (Dimethyl sulfoxide) at a concentration of 10 mM and kept at )20°C. "
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    ABSTRACT: Classical benzodiazepines, for example diazepam, interact with alpha(x)beta(2)gamma(2) GABA(A) receptors, x = 1, 2, 3, 5. Little is known about effects of alpha subunits on the structure of the binding pocket. We studied here the interaction of the covalently reacting diazepam analog 7-Isothiocyanato-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one (NCS compound) with alpha(1)H101Cbeta(2)gamma(2) and with receptors containing the homologous mutation, alpha(2)H101Cbeta(2)gamma(2), alpha(3)H126Cbeta(2)gamma(2) and alpha(5)H105Cbeta(2)gamma(2). This comparison was extended to alpha(6)R100Cbeta(2)gamma(2) receptors as this mutation conveys to these receptors high affinity towards classical benzodiazepines. The interaction was studied at the ligand binding level and at the functional level using electrophysiological techniques. Results indicate that the geometry of alpha(6)R100Cbeta(2)gamma(2) enables best interaction with NCS compound, followed by alpha(3)H126Cbeta(2)gamma(2), alpha(1)H101Cbeta(2)gamma(2) and alpha(2)H101Cbeta(2)gamma(2), while alpha(5)H105Cbeta(2)gamma(2) receptors show little interaction. Our results allow conclusions about the relative apposition of alpha(1)H101 and homologous positions in alpha(2), alpha(3), alpha(5) and alpha(6) with the position occupied by -Cl in diazepam. During this study we found evidence for the presence of a novel site for benzodiazepines that prevents modulation of GABA(A) receptors via the classical benzodiazepine site. The novel site potentially contributes to the high degree of safety to some of these drugs. Our results indicate that this site may be located at the alpha/beta subunit interface pseudo-symmetrically to the site for classical benzodiazepines located at the alpha/gamma interface.
    Journal of Neurochemistry 09/2008; 106(6):2353-63. DOI:10.1111/j.1471-4159.2008.05574.x · 4.24 Impact Factor
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