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Antibodies directed against a nonapeptide sequence of the gamma-aminobutyrate (GABA)/benzodiazepine receptor alpha-subunit. Detection of a distinct alpha-like subunit in pig cerebral cortex but not cerebellum.

ABSTRACT: A synthetic peptide, corresponding to amino acid residues 101-109 of the bovine gamma-aminobutyrate/benzodiazepine receptor alpha-subunit, was used to raise a polyclonal antiserum. The reactivity of this antiserum towards polypeptides of both bovine and pig receptor preparations was established by immunoprecipitation and immunoblotting. Anti-peptide antibodies recognized the alpha-subunit (51 kDa) of receptor prepared from pig cerebellum or cerebral cortex. However, a polypeptide of 57 kDa was additionally recognized in cortical, but not cerebellar, preparations. This alpha-like polypeptide appeared larger than the band of polypeptides labelled irreversibly with [3H]muscimol (beta-subunit, 55-57 kDa) and corresponds to a polypeptide detected only in cortex after silver-staining or irreversible labelling with [3H]flunitrazepam. These results support the idea that the distinct regional patterns of polypeptides labelled irreversibly with [3H]flunitrazepam reflect the existence of heterologous distributions of distinct alpha-like subunits.

SUBMITTER: Kirkness EF

PROVIDER: S-EPMC1135401 | biostudies-other | 1988 Nov

REPOSITORIES: biostudies-other

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The gamma-aminobutyrate/benzodiazepine receptor from pig brain. Purification and characterization of the receptor complex from cerebral cortex and cerebellum.

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A Review of the Updated Pharmacophore for the Alpha 5 GABA(A) Benzodiazepine Receptor Model.

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The gamma-aminobutyrate/benzodiazepine receptor from pig brain. Enhancement of gamma-aminobutyrate-receptor binding by the anaesthetic propanidid.

Project description:The binding of [3H]muscimol, a gamma-aminobutyrate (GABA) receptor agonist, to a membrane preparation from pig cerebral cortex was enhanced by the anaesthetic propanidid in a concentration-dependent manner. At 0 degrees C, binding was stimulated to 220% of control values, with 50% stimulation at 60 microM-propanidid. At 37 degrees C, propanidid caused a more powerful stimulation of [3H]muscimol binding (340% of control values). Propanidid (1 mM) exerted little effect on the affinity of muscimol binding (KD approx. 10 nM), but increased the apparent number of high-affinity binding sites in the membrane by 2-fold. Enhancement of [3H]muscimol binding was observed only in the presence of Cl- ions, half-maximal activation being achieved at approx. 40 mM-Cl-. Picrotoxinin inhibited the stimulation of [3H]muscimol binding by propanidid with an IC50 (concentration causing 50% inhibition) value of approx. 25 microM. The enhancement of [3H]muscimol binding by propanidid was not additive with the enhancement produced by secobarbital. Phenobarbital inhibited the effect of propanidid and secobarbital. The GABA receptor was solubilized with Triton X-100 or with Chaps [3-[(3-cholamidopropyl)dimethylammonio]propanesulphonate]. Propanidid and secobarbital did not stimulate the binding of [3H]muscimol after solubilization with Triton X-100. However, the receptor could be solubilized by 5 mM-Chaps with retention of the stimulatory effects of propanidid and secobarbital. Unlike barbiturates, propanidid did not stimulate the binding of [3H]flunitrazepam to membranes. It is suggested that the ability to modulate the [3H]muscimol site of the GABA-receptor complex may be a common and perhaps functional characteristic of general anaesthetics.

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Arabidopsis aldehyde dehydrogenase 10 family members confer salt tolerance through putrescine-derived 4-aminobutyrate (GABA) production.

Project description:Polyamines represent a potential source of 4-aminobutyrate (GABA) in plants exposed to abiotic stress. Terminal catabolism of putrescine in Arabidopsis thaliana involves amine oxidase and the production of 4-aminobutanal, which is a substrate for NAD+-dependent aminoaldehyde dehydrogenase (AMADH). Here, two AMADH homologs were chosen (AtALDH10A8 and AtALDH10A9) as candidates for encoding 4-aminobutanal dehydrogenase activity for GABA synthesis. The two genes were cloned and soluble recombinant proteins were produced in Escherichia coli. The pH optima for activity and catalytic efficiency of recombinant AtALDH10A8 with 3-aminopropanal as substrate was 10.5 and 8.5, respectively, whereas the optima for AtALDH10A9 were approximately 9.5. Maximal activity and catalytic efficiency were obtained with NAD+ and 3-aminopropanal, followed by 4-aminobutanal; negligible activity was obtained with betaine aldehyde. NAD+ reduction was accompanied by the production of GABA and ?-alanine, respectively, with 4-aminobutanal and 3-aminopropanal as substrates. Transient co-expression systems using Arabidopsis cell suspension protoplasts or onion epidermal cells and several organelle markers revealed that AtALDH10A9 was peroxisomal, but AtALDH10A8 was cytosolic, although the N-terminal 140 amino acid sequence of AtALDH10A8 localized to the plastid. Root growth of single loss-of-function mutants was more sensitive to salinity than wild-type plants, and this was accompanied by reduced GABA accumulation.

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Therapeutic potential of alpha 5 subunit containing GABA<sub>A</sub> receptors in Alzheimer's disease.

Project description: Not available

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Enhanced production of gamma-aminobutyrate (GABA) in recombinant Corynebacterium glutamicum strains from empty fruit bunch biosugar solution.

Project description:BACKGROUND:Recent interest has been focused on the production of platform chemicals from renewable biomass due to increasing concerns on global warming and depletion of fossil fuel reserves. Microbial production of platform chemicals in biorefineries has been suggested to be a promising solution for these problems. Gamma-aminobutyrate (GABA), a versatile bulk chemical used in food and pharmaceutical industry, is also used as a key monomer for nylon 4. GABA can be biologically produced by decarboxylation of glutamate. RESULTS:In this study, we examined high glutamate-producing Corynebacterium glutamicum strains as hosts for enhanced production of GABA from glucose and xylose as carbon sources. An Escherichia coli gadB mutant with a broad pH range of activity and E. coli xylAB genes were expressed under the control of a synthetic H36 promoter. When empty fruit bunch (EFB) solution was used as carbon source (45 g/L glucose and 5 g/L xylose), 12.54 ± 0.07 g/L GABA was produced by recombinant C. glutamicum H36GD1852 expressing E. coli gadB mutant gene and xylAB genes. Batch fermentation of the same strain resulted in the production of 35.47 g/L of GABA when EFB solution was added to support 90 g/L glucose and 10 g/L xylose. CONCLUSIONS:This is the first report of GABA production by recombinant C. glutamicum strains from co-utilization of glucose and xylose from EFB solution. Recombinant C. glutamicum strains developed in this study should be useful for an efficient and sustainable production of GABA from lignocellulosic biomasses.

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Project description:The GABA(A) receptor mutation gamma(2)R43Q causes absence epilepsy in humans. Homology modeling suggests that gamma(2)Arg43, gamma(2)Glu178, and beta(2)Arg117 participate in a salt-bridge network linking the gamma(2) and beta(2) subunits. Here we show that several mutations at these locations exert similar long-distance effects on other intersubunit interfaces involved in GABA and benzodiazepine binding. These mutations alter GABA-evoked receptor kinetics by slowing deactivation, enhancing desensitization, or both. Kinetic modeling and nonstationary noise analysis for gamma(2)R43Q reveal that these effects are due to slowed GABA unbinding and slowed recovery from desensitization. Both gamma(2)R43Q and beta(2)R117K also speed diazepam dissociation from the receptor's benzodiazepine binding interface, as assayed by the rate of decay of diazepam-induced potentiation of GABA-evoked currents. These data demonstrate that gamma(2)Arg43 and beta(2)Arg117 similarly regulate the stability of both the GABA and benzodiazepine binding sites at the distant beta/alpha and alpha/gamma intersubunit interfaces, respectively. A simple explanation for these results is that gamma(2)Arg43 and beta(2)Arg117 participate in interactions between the gamma(2) and beta(2) subunits, disruptions of which alter the neighboring intersubunit binding sites in a similar fashion. In addition, gamma(2)Arg43 and gamma(2)Glu178 regulate desensitization, probably mediated within the transmembrane domains near the pore. Therefore, mutations at the gamma/beta intersubunit interface have specific long-distance effects that are propagated widely throughout the GABA(A) receptor protein.

| S-EPMC2802431 | biostudies-literature

Beyond risk: Prospective effects of GABA Receptor Subunit Alpha-2 (GABRA2) × Positive Peer Involvement on adolescent behavior.

Project description:Research on Gene × Environment interactions typically focuses on maladaptive contexts and outcomes. However, the same genetic factors may also impact susceptibility to positive social contexts, leading to adaptive behavior. This paper examines whether the GABA receptor subunit alpha-2 (GABRA2) single nucleotide polymorphism rs279858 moderates the influence of positive peer affiliation on externalizing behavior and various forms of competence. Regions of significance were calculated to determine whether the form of the interaction supported differential susceptibility (increased sensitivity to both low and high positive peer affiliation) or vantage sensitivity (increased sensitivity to high positive peer affiliation). It was hypothesized that those carrying the homozygous minor allele (GG) would be more susceptible to peer effects. A sample (n = 300) of primarily male (69.7%) and White (93.0%) adolescents from the Michigan Longitudinal Study was assessed from ages 12 to 17. There was evidence for prospective Gene × Environment interactions in three of the four models. At low levels of positive peer involvement, those with the GG genotype were rated as having fewer adaptive outcomes, while at high levels they were rated as having greater adaptive outcomes. This supports differential susceptibility. Conceptualizing GABRA2 variants as purely risk factors may be inaccurate. Genetic differences in susceptibility to adaptive environmental exposures warrants further investigation.

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