Identification of the Bovine γ-Aminobutyric Acid Type A Receptor α Subunit Residues Photolabeled by the Imidazobenzodiazepine [3H]Ro15-4513

Sawyer, Gregory W.; Chiara, David C.; Olsen, Richard W.; Cohen, Jonathan B.

doi:10.1074/jbc.m209281200

Cited by 53 publications

(50 citation statements)

References 48 publications

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“…Photoaffinity labeling has successfully been used to locate the benzodiazepine (Davies et al, 1996;Duncalfe et al, 1996;Sawyer et al, 2002;Wieland et al, 1992) and muscimol (Smith & Olsen, 1994) binding sites on the GABA A receptor, and sites for etomidate (Husain et al, 2006;Ziebell et al, 2004) and octanol (Pratt et al, 2000) on the Torpedo nicotinic acetylcholine receptor. In principle photoaffinity labeling techniques should provide a comprehensive search of all possible protein targets and should not be confounded by either multiple regions of protein contributing to a binding site or by the existence of multiple binding sites.…”

Section: E Insights From Photoaffinity Labelsmentioning

confidence: 99%

Mechanisms of neurosteroid interactions with GABAA receptors

Akk¹,

Covey²,

Evers³

et al. 2007

Pharmacology & Therapeutics

148

166

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Neuroactive steroids have some of their most potent actions by augmenting the function of GABA A receptors. Endogenous steroid actions on GABA A receptors may underlie important effects on mood and behavior. Exogenous neuroactive steroids have potential as anesthetics, anticonvulsants, and neuroprotectants. We have taken multiple approaches to understand more completely the interaction of neuroactive steroids with GABA A receptors. We have developed many novel steroid analogues in this effort. Recent work has resulted in synthesis of new enantiomer analogue pairs, novel ligands that probe various properties of the steroid pharmacophore, fluorescent neuroactive steroid analogues, and photoaffinity labels. Using these tools, combined with receptor binding and electrophysiological assays, we have begun to untangle the complexity of steroid actions at this important class of ligand-gated ion channel.

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Section: E Insights From Photoaffinity Labelsmentioning

confidence: 99%

Mechanisms of neurosteroid interactions with GABAA receptors

Akk¹,

Covey²,

Evers³

et al. 2007

Pharmacology & Therapeutics

148

166

View full text Add to dashboard Cite

show abstract

“…Photoaffinity labeling, which provides an experimental approach to directly identify amino acids contributing to a drug binding site without previous assumptions about the protein points of contact (Kotzyba-Hibert et al, 1995), has been used extensively to identify amino acids contributing to ligand binding sites in the Torpedo nAChR (Mourot et al, 2006) and for GABA A R agonists and benzodiazepines (Smith and Olsen, 1994;Duncalfe et al, 1996;Sawyer et al, 2002). We now use 2-(3-methyl-3H-diaziren-3-yl) ethyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate ([ 3 H]azietomidate), a photoreactive analog of etomidate that retains anesthetic and GABA A R modulatory properties (Husain et al, 2003), to provide the first identification of amino acids contributing to an anesthetic binding site in a GABA A R. The two labeled residues, ␣M1-14 (␣Met-236) and , are shown by homology modeling to border a pocket in the TMD located at the same subunit interfaces that contain the GABA binding site in the extracellular domain.…”

Section: Introductionmentioning

confidence: 99%

Identification of a GABA_AReceptor Anesthetic Binding Site at Subunit Interfaces by Photolabeling with an Etomidate Analog

Li¹,

Chiara²,

Sawyer³

et al. 2006

J. Neurosci.

275

308

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General anesthetics, including etomidate, act by binding to and enhancing the function of GABA type A receptors (GABA A Rs), which mediate inhibitory neurotransmission in the brain. Here, we used a radiolabeled, photoreactive etomidate analog ([ 3 H]azietomidate), which retains anesthetic potency in vivo and enhances GABA A R function in vitro, to identify directly, for the first time, amino acids that contribute to a GABA A R anesthetic binding site. For GABA A Rs purified by affinity chromatography from detergent extracts of bovine cortex, [ 3 H]azietomidate photoincorporation was increased by GABA and inhibited by etomidate in a concentration-dependent manner (IC 50 ϭ 30 M). Protein microsequencing of fragments isolated from proteolytic digests established photolabeling of two residues: one within the ␣M1 transmembrane helix at ␣1Met-236 (and/or the homologous methionines in ␣2,3,5), not previously implicated in etomidate function, and one within the ␤M3 transmembrane helix at ␤3Met-286 (and/or the homologous methionines in ␤1,2), an etomidate sensitivity determinant. The pharmacological specificity of labeling indicates that these methionines contribute to a single binding pocket for etomidate located in the transmembrane domain at the interface between ␤ and ␣ subunits, in what is predicted by structural models based on homology with the nicotinic acetylcholine receptor to be a water-filled pocket ϳ50 Å below the GABA binding site. The localization of the etomidate binding site to an intersubunit, not an intrasubunit, binding pocket is a novel conclusion that suggests more generally that the localization of drug binding sites to subunit interfaces may be a feature not only for GABA and benzodiazepines but also for etomidate and other intravenous and volatile anesthetics.

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“…Mutagenesis studies identified the cleft between a and c subunits as the binding pocket for benzodiazepines [12][13][14]. a 1 H101 was identified as the target of photoaffinity labeling by [ 3 H] flunitrazepam [15] and a 1 Y209 as the target of [ 3 H] Ro15-4513 [16]. Pharmacophore modeling attempted to describe the shape of the binding pocket [17,18].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2007

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Benzodiazepines are widely used drugs exerting sedative, anxiolytic, muscle relaxant, and anticonvulsant effects by acting through specific high affinity binding sites on some GA-BA 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 a 1 b 2 c 2 GABA A receptor containing a key residue for the interaction of benzodiazepines: a 1 H101. We describe a direct interaction of a 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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Identification of the Bovine γ-Aminobutyric Acid Type A Receptor α Subunit Residues Photolabeled by the Imidazobenzodiazepine [3H]Ro15-4513

Cited by 53 publications

References 48 publications

Mechanisms of neurosteroid interactions with GABAA receptors

Mechanisms of neurosteroid interactions with GABAA receptors

Identification of a GABA_AReceptor Anesthetic Binding Site at Subunit Interfaces by Photolabeling with an Etomidate Analog

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

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Identification of the Bovine γ-Aminobutyric Acid Type A Receptor α Subunit Residues Photolabeled by the Imidazobenzodiazepine [3H]Ro15-4513

Cited by 53 publications

References 48 publications

Mechanisms of neurosteroid interactions with GABAA receptors

Mechanisms of neurosteroid interactions with GABAA receptors

Identification of a GABAAReceptor Anesthetic Binding Site at Subunit Interfaces by Photolabeling with an Etomidate Analog

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

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Identification of a GABA_AReceptor Anesthetic Binding Site at Subunit Interfaces by Photolabeling with an Etomidate Analog

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