Reconstitution of the purified γ-aminobutyric acid-benzodiazepine receptor complex from bovine cerebral cortex into phospholipid vesicles

Sigel, Erwin; C, Mamalaki; Barnard, Eric A.

doi:10.1016/0304-3940(85)90419-7

Cited by 35 publications

(35 citation statements)

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“…GABA A R was purified by modification of previous protocols (Sigel and Barnard, 1984;Sawyer et al, 2002) on a benzodiazepine Ro7/1986-1 affinity column. Detailed conditions for solubilization and purification as well as the immunoblot and mass spectrometry protocols for characterization of the purified GABA A R are described in the supplemental Materials and Methods (available at www.jneurosci.org as supplemental material).…”

Section: Materials [mentioning

confidence: 99%

“…Allosteric modulation by barbiturates and etomidate are preserved by use of milder detergents such as CHAPS, but isolation of GABA A R at high purity and yield was problematic (Stephenson and Olsen, 1982;Sigel and Barnard, 1984;King et al, 1987). As an alternative purification strategy, we examined whether the use of the detergent C 12 E 9 , which was particularly effective for purification of serotonin 5-HT 3 receptors (Hovius et al, 1998), in conjunction with CHAPS would facilitate efficient purification of GABA A R with retention of allosteric modulation.…”

Section: Purification Of Brain Gaba a R With Retention Of Allosteric mentioning

confidence: 99%

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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.

276

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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Section: Materials [mentioning

confidence: 99%

Section: Purification Of Brain Gaba a R With Retention Of Allosteric mentioning

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.

276

308

View full text Add to dashboard Cite

show abstract

“…This yields two candidate orientations, one with the C5-phenyl group oriented approximately parallel to the cell membrane, and the other with the C5-phenyl oriented parallel to the ion channel. We evaluated the consistency of these orientations with respect to four criteria: 1) the capacity to accommodate a tethered DZ analog ([poly(Me-BZD)] that was used in the early affinity column purification of GABA A receptors (Sigel et al, 1983;Sigel and Barnard, 1984); 2) the effect of the ␣ 1 H101R mutation, which abolishes BZD binding (Wieland et al, 1992;Wieland and Lü ddens, 1994;Benson et al, 1998;Dunn et al, 1999); 3) the two enantiomers of 3-methyl-substituted FNZ, Ro 11-6896 and Ro 11-6893 (Niehoff et al, 1982;De Blas et al, 1985); and 4) the binding affinities of a set of active and inactive BZD derivatives (Klopman and Contreras, 1985;Zhang et al, 1994) (Fig. 2).…”

mentioning

confidence: 99%

Docking of 1,4-Benzodiazepines in the α₁/γ₂GABA_AReceptor Modulator Site

Berezhnoy

Gibbs²,

Farb³

2009

Mol Pharmacol

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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 ␣ 1 /␥ 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 structureactivity and stereospecificity characteristics of BZDs and the impact of the ␣ 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 sitedirected 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.

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“…It acts at three types of receptors, the G-protein-coupled GABA B receptor, and the GABA A and GABA C receptors, which both constitute ion channels. Two subunits of the GABA A receptor have initially been purified (Sigel et al, 1983), and their coding DNA has been cloned (Schofield et al, 1987). Numerous subunits have since been cloned (for review, see Macdonald and Olsen, 1994;Rabow et al, 1995;Barnard et al, 1998).…”

mentioning

confidence: 99%

Novel Plant Substances Acting as β Subunit Isoform-Selective Positive Allosteric Modulators of GABA_AReceptors

Baur

Simmen²,

Senn³

et al. 2005

Mol Pharmacol

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GABA A receptors are modulated by a large variety of compounds. A common chemical characteristic of most of these modulators is that they contain a cyclic entity. Three linear molecules of a polyacetylene structure were isolated from the East African medicinal plant Cussonia zimmermannii Harms and shown to allosterically stimulate GABA A receptors. Stimulation was not abolished by the absence of the ␥ 2 subunit, the benzodiazepine antagonist Ro15-1788 (8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester), or the point mutation ␤ 2 N265S that abolishes effects by loreclezole. At a concentration of 30 M, the substances by themselves elicited only tiny currents. Maximal stimulation at ␣ 1 ␤ 2 ␥ 2 amounted to 110 to 450% for the three substances, and half-maximal stimulation was observed at concentrations of 1 to 2 M. Stimulation was subunit composition-dependent and was for the substance MS-1,Maximal stimulation by MS-1 was 450% at ␣ 1 ␤ 2 ␥ 2 , 80% at ␣ 1 ␤ 1 ␥ 2 , and 150% at ␣ 1 ␤ 3 ␥ 2 . MS-1 was thus specific for receptors containing the ␤ 2 subunit. The reversal potential was unaffected by 10 M MS-1, whereas apparent picrotoxin affinity for current inhibition was increased approximately 3-fold. In summary, these positive allosteric modulators of GABA A receptors of plant origin have a novel unusual chemical structure and act at a site independent of that of benzodiazepines and loreclezole.GABA is the major inhibitory neurotransmitter in the mammalian central nervous system. It acts at three types of receptors, the G-protein-coupled GABA B receptor, and the GABA A and GABA C receptors, which both constitute ion channels. Two subunits of the GABA A receptor have initially been purified (Sigel et al., 1983), and their coding DNA has been cloned (Schofield et al., 1987). Numerous subunits have since been cloned (for review, see Macdonald and Olsen, 1994;Rabow et al., 1995;Barnard et al., 1998). These subunits show homology to subunits of the nicotinic acetylcholine receptors, the glycine receptor, and the 5HT3 receptor. The GABA A receptors are heteromeric protein complexes consisting of five subunits that are arranged around a central Cl Ϫ -selective channel (Macdonald and Olsen, 1994). The major receptor isoform of the GABA A receptor in the brain presumably consists of ␣ 1 , ␤ 2 , and ␥ 2 subunits (Laurie et al., 1992;Benke et al

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Reconstitution of the purified γ-aminobutyric acid-benzodiazepine receptor complex from bovine cerebral cortex into phospholipid vesicles

Cited by 35 publications

References 9 publications

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

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

Docking of 1,4-Benzodiazepines in the α₁/γ₂GABA_AReceptor Modulator Site

Novel Plant Substances Acting as β Subunit Isoform-Selective Positive Allosteric Modulators of GABA_AReceptors

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Reconstitution of the purified γ-aminobutyric acid-benzodiazepine receptor complex from bovine cerebral cortex into phospholipid vesicles

Cited by 35 publications

References 9 publications

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

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

Docking of 1,4-Benzodiazepines in the α1/γ2GABAAReceptor Modulator Site

Novel Plant Substances Acting as β Subunit Isoform-Selective Positive Allosteric Modulators of GABAAReceptors

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

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

Docking of 1,4-Benzodiazepines in the α₁/γ₂GABA_AReceptor Modulator Site

Novel Plant Substances Acting as β Subunit Isoform-Selective Positive Allosteric Modulators of GABA_AReceptors