V. Barbiturate and benzodiazepine modulation of GABA receptor binding and function

Olsen, Richard W.; Yang, Junyuan; King, R. G.; Dilber, A.; Stauber, G. B.; Ransom, Richard W.

doi:10.1016/0024-3205(86)90320-6

Cited by 91 publications

(34 citation statements)

References 45 publications

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“…Barbiturates modulate the GABA A receptor to enhance GABA‐mediated inhibition primarily by increasing the channel mean open duration. In vitro radioligand binding studies with [ 3 H]‐dihydropicrotoxinin, [ 35 S]‐t‐butylbicyclophosphorothionate ([ 35 S]‐TBPT, a convulsant that binds to the picrotoxinin‐binding site of the GABA‐receptor complex), and [ 3 H]‐diazepam have revealed differences in efficacy among a number of barbiturate derivatives (Leeb‐Lundberg & Olsen 1982; Johnston 1983; Lohse et al ., 1987; Olsen et al ., 1986; Richter & Holtman 1982). For example, it has been found that the relative potencies (IC 50 values) for the inhibition of [ 35 S]‐TBPT binding increase in the order rac ‐methohexitone, rac ‐thiopentone, rac ‐pentobarbitone, rac ‐secobarbitone, rac ‐hexobarbitone, amobarbitone and phenobarbitone (Lohse et al ., 1987).…”

Section: Discussionmentioning

confidence: 99%

Stereoselective interaction of thiopentone enantiomers with the GABA_A receptor

Cordato

Chebib

Mather

et al. 1999

British J Pharmacology

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1 As pharmacokinetic di erences between the thiopentone enantiomers seem insu cient to explain the *2 fold greater potency for CNS e ects of (7)-S-over (+)-R-thiopentone, this study was performed to determine any enantioselectivity of thiopentone at the GABA A receptor, the primary receptor for barbiturate hypnotic e ects. 2 Two electrode voltage clamp recording was performed on Xenopus laevis oocytes expressing human GABA A receptor subtype a 1 b 2 g 2 to determine relative di erences in potentiation of the GABA response by rac-, (+)-R-and (7)-S-thiopentone, and rac-pentobarbitone. Changes in the cellular environment pH and in GABA concentrations were also evaluated. 3 With 3 mM GABA, the EC 50 values were (7)-S-thiopentone (mean 26.0+s.e.mean 3.2 mM, n=9 cells) 4rac-thiopentone (35.9+4.2 mM, n=6, P=0.1) 4(+)-R-thiopentone (52.5+5.0 mM, n=8, P50.02) 4rac-pentobarbitone (97.0+11.2 mM, n=11, P50.01). Adjustment of environment pH to 7.0 or 8.0 did not alter the EC 50 values for (+)-R-or (7)-S-thiopentone. 4 Uninjected oocytes responded to 4100 mM (7)-S-and R-thiopentone. This direct response was abolished by intracellular oocyte injection of 1,2-bis(2-aminophenoxy)ethane-N,N,N1,N1-tetraacetic acid (BAPTA), a Ca 2+ chelating agent. With BAPTA, the EC 50 values were (7)-S-thiopentone (20.6+3.2 mM, n=8) 5(+)-R-thiopentone (36.2+3.2 mM, n=9, P50.005). 5 (7)-S-thiopentone was found to be *2 fold more potent than (+)-R-thiopentone in the potentiation of GABA at GABA A receptors expressed on Xenopus oocytes. This is consistent with the di erences in potency for CNS depressant e ects found in vivo.

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Section: Discussionmentioning

confidence: 99%

Stereoselective interaction of thiopentone enantiomers with the GABA_A receptor

Cordato

Chebib

Mather

et al. 1999

British J Pharmacology

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“…discussion in [7,21,31,401 Again, it remains possible that two species exist and the larger one binds TBPS and benzodiazepines but the smaller one carries the bulk of the benzodiazepine sites. The similar stoichiometries for the two binding activities in membranes and in solution and the co-chromatography of the two suggest that they reside on the same protein complex that shows differential target sizes for the two activities.…”

Section: Discussionmentioning

confidence: 99%

Convulsant/barbiturate activity on the soluble γ‐aminobutyric acid – benzodiazepine receptor complex

King

Nielsen

Stauber

et al. 1987

European Journal of Biochemistry

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Cage convulsant t-butyl bicyclophosphoro[3sS]thionate binding activity in rat brain membrane homogenates was solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]propane sulfonate (Chaps) and shown to co-purify with the benzodiazepine -y-aminobutyric acid (GABA) receptor complex on gel filtration and affinity chromatography. Whereas convulsant binding activity, but not GABA and benzodiazepine receptor binding, was eliminated by solubilization in other detergents like sodium deoxycholate or Triton X-1 00, or by addition of Triton X-100 to the extracts solubilized in the zwitterionic detergent, convulsant activity was not irreversibly lost or selectively unstable, but could be restored by exchanging the protein back into the detergent Chaps. The GABA -benzodiazepine receptor activity solubilized in Chaps alone, containing convulsant activity, and a sample in Chaps supplemented with Triton X-100 and lacking convulsant activity, did not differ in size as measured by gel filtration column chromatography or by radiation inactivation target size analysis. This suggests that convulsant binding activity does not require any additional protein subunits or other macromolecules nor any unique aggregation state relative to GABA and benzodiazepine receptor binding, and that all three activities reside on the same protein complex. As in intact brain, the target size for convulsant binding activity was 3 -5 times that of benzodiazepine binding activity, suggesting that an oligomeric protein structure of the receptor complex with intact strong subunit interactions present in the native membrane environment is needed for convulsant activity, and that this and other properties are more preserved in Chaps than in other detergents.Neuronal membrane chloride channels activated by the major inhibitory neurotransmitter, y-aminobutyric acid (GABA), are inhibited by convulsant drugs such as the naturally occurring picrotoxinin [l -31. This inhibitory action is mediated through the binding of convulsants to a receptor site that is part of an oligomeric protein complex consisting of GABA receptor sites, benzodiazepine receptor sites, and barbiturate receptor sites coupled to a chloride channel [3 -71. Binding sites for these convulsant drugs have been assayed in vitro with the radioactive picrotoxin analog, [ 3 H ]~-dihydropicrotoxinin [8,9] propane sulfonate (Chaps), and to co-purify with the GABA -benzodiazepine receptor complex [17,24]. Likewise, GABA enhancement of benzodiazepine binding is preserved to a greater extent in Chaps-solubilized preparations than in other detergents [25].Pretreatment of membranes with Triton X-100 causes perturbation of the GABA -benzodiazepine receptor complex which leads to an increase in the affinity of [3H]

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“…The GABA A R carries binding sites for a number of therapeutic agents including the benzodiazepines, barbiturates, neurosteroids, some general anesthetics, and possibly also alcohol (1). In brain membranes, there are at least two classes of binding sites for the endogenous neurotransmitter, which differ by more than an order of magnitude in their affinity for GABA or its structural analogues (2)(3)(4). This heterogeneity in binding was originally thought to reflect the diversity of GABA A R subtypes in brain tissue.…”

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confidence: 99%

Tyrosine 62 of the γ-Aminobutyric Acid Type A Receptor β2 Subunit Is an Important Determinant of High Affinity Agonist Binding

Newell¹,

Davies²,

Bateson³

et al. 2000

Journal of Biological Chemistry

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The ␥-aminobutyric acid type A receptor (GABA A R) carries both high (K D ‫؍‬ 10 -30 nM) and low (K D ‫؍‬ 0.1-1.0 M) affinity binding sites for agonists. We have used site-directed mutagenesis to identify a specific residue in the rat ␤2 subunit that is involved in high affinity agonist binding. Tyrosine residues at positions 62 and 74 were mutated to either phenylalanine or serine and the effects on ligand binding and ion channel activation were investigated after the expression of mutant subunits with wild-type ␣1 and ␥2 subunits in tsA201 cells or in The GABA A R 1 is a member of a superfamily of ligand-gated ion channels that includes the nicotinic acetylcholine receptor (nAChR), the glycine receptor, and the serotonin type 3 receptor (1). The GABA A R carries binding sites for a number of therapeutic agents including the benzodiazepines, barbiturates, neurosteroids, some general anesthetics, and possibly also alcohol (1). In brain membranes, there are at least two classes of binding sites for the endogenous neurotransmitter, which differ by more than an order of magnitude in their affinity for GABA or its structural analogues (2-4). This heterogeneity in binding was originally thought to reflect the diversity of GABA A R subtypes in brain tissue. However, the presence of both classes of sites in a stable cell line expressing a specific subtype (5) suggests that both exist in a single receptor molecule. On the basis of biochemical studies, the reasonable correlation between the concentration of agonist required to elicit ion flux and to potentiate the binding of benzodiazepine ligands suggested that the low affinity sites are important for channel gating (see Ref. 6). However, the role(s) of the high affinity binding sites in receptor function remains unclear.All members of this receptor family are believed to be pentameric complexes formed by homologous subunits assembled to form a central ion channel (7). Recent models (see Ref. 8) predict that ligand binding sites occur at subunit-subunit interfaces. This was first demonstrated in the nAChR in which the ␣-␥ and ␣-␦ interfaces were implicated in forming nonequivalent binding sites for d-tubocurarine (9). In the GABA A R, low affinity GABA sites (i.e. those that have been implicated in channel activation) are thought to be located at the interfaces between the ␤ and ␣ subunits (10 -13), whereas the benzodiazepine binding site is predicted to occur at the homologous ␣-␥ interface (14 -19). More detailed analyses of the properties of these sites have led to a "loop model" of ligand binding sites (see Ref. 20) in which amino acid residues from at least three discontinuous regions (denoted "loops" A-C) of one subunit together with residues from at least one region of the adjacent subunit ("loop" D) form the binding pocket (see Fig. 1A).In the GABA A R, evidence for the location of the high affinity agonist site(s) is derived from a number of experimental approaches. Photoaffinity labeling studies first suggested that the ␤ subunit is a major determinant of h...

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V. Barbiturate and benzodiazepine modulation of GABA receptor binding and function

Cited by 91 publications

References 45 publications

Stereoselective interaction of thiopentone enantiomers with the GABA_A receptor

Stereoselective interaction of thiopentone enantiomers with the GABA_A receptor

Convulsant/barbiturate activity on the soluble γ‐aminobutyric acid – benzodiazepine receptor complex

Tyrosine 62 of the γ-Aminobutyric Acid Type A Receptor β2 Subunit Is an Important Determinant of High Affinity Agonist Binding

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V. Barbiturate and benzodiazepine modulation of GABA receptor binding and function

Cited by 91 publications

References 45 publications

Stereoselective interaction of thiopentone enantiomers with the GABAA receptor

Stereoselective interaction of thiopentone enantiomers with the GABAA receptor

Convulsant/barbiturate activity on the soluble γ‐aminobutyric acid – benzodiazepine receptor complex

Tyrosine 62 of the γ-Aminobutyric Acid Type A Receptor β2 Subunit Is an Important Determinant of High Affinity Agonist Binding

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Stereoselective interaction of thiopentone enantiomers with the GABA_A receptor

Stereoselective interaction of thiopentone enantiomers with the GABA_A receptor