Separation of a1, a2 and a3 subunits of the GABAA-benzodiazepine receptor complex by immunoaffinity chromatography

Zezula, Jürgen; Fuchs, Karoline; Sieghart, Werner

doi:10.1016/0006-8993(91)91556-g

Cited by 55 publications

(20 citation statements)

References 13 publications

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“…The antisera were produced in rabbits to synthetic peptide sequences that are specific for the respective GABA A receptor subunits. Antisera to the following peptide sequences were used, and their production and characterization have been described previously: ␣1(1-9) (Zezula et al, 1991); ␣4(1-14) and ␣4(379 -421) (Bencsits et al, 1999), used for immunohistochemistry and immunoblots, respectively; ␦(1-44) ; and ␥2(319 -366) (Tretter et al, 1997). The specificity of the affinity-purified antisera has been demonstrated in immunochemical studies by Jechlinger et al (1998) and in immunohistochemical studies by Sperk and colleagues Pirker et al, 2000).…”

Section: Antiseramentioning

confidence: 99%

GABA_A receptor changes in δ subunit‐deficient mice: Altered expression of α4 and γ2 subunits in the forebrain

Peng¹,

Hauer

Mihalek

et al. 2002

J of Comparative Neurology

229

221

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The delta subunit is a novel subunit of the pentameric gamma-aminobutyric acid (GABA)(A) receptor that conveys special pharmacological and functional properties to recombinant receptors and may be particularly important in mediating tonic inhibition. Mice that lack the delta subunit have been produced by gene-targeting technology, and these mice were studied with immunohistochemical and immunoblot methods to determine whether changes in GABA(A) receptors were limited to deletion of the delta subunit or whether alterations in other GABA(A) receptor subunits were also present in the delta subunit knockout (delta-/-) mice. Immunohistochemical studies of wild-type mice confirmed the restricted distribution of the delta subunit in the forebrain. Regions with moderate to high levels of delta subunit expression included thalamic relay nuclei, caudate-putamen, molecular layer of the dentate gyrus, and outer layers of the cerebral cortex. Virtually no delta subunit labeling was evident in adjacent regions, such as the thalamic reticular nucleus, hypothalamus, and globus pallidus. Comparisons of the expression of other subunits in delta-/- and wild-type mice demonstrated substantial changes in the alpha4 and gamma2 subunits of the GABA(A) receptor in the delta-/- mice. gamma2 Subunit expression was increased, whereas alpha4 subunit expression was decreased in delta-/- mice. Importantly, alterations of both the alpha4 and the gamma2 subunits were confined primarily to brain regions that normally expressed the delta subunit. This suggests that the additional subunit changes are directly linked to loss of the delta subunit and could reflect local changes in subunit composition and function of GABA(A) receptors in delta-/- mice.

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Section: Antiseramentioning

confidence: 99%

GABA_A receptor changes in δ subunit‐deficient mice: Altered expression of α4 and γ2 subunits in the forebrain

Peng¹,

Hauer

Mihalek

et al. 2002

J of Comparative Neurology

229

221

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“…The ␣ 3 subunit is also contained in a significant proportion of GABA A receptors (22,24,25). To investigate whether 3␤-HSD-containing neurons also express GABA A receptors, we have used a specific polyclonal antibody against the ␣ 3 subunit (17,18,26) and a monoclonal antibody against the ␤ 2 ͞␤ 3 subunits that also cross-reacts with the ␣ 1 subunit (24,27,28). In a recent report, this latter antibody has been used successfully to localize the GABA A receptor complex in the brain of the frog Rana pipiens (29).…”

Section: Discussionmentioning

confidence: 99%

γ-Aminobutyric acid, acting through γ-aminobutyric acid type A receptors, inhibits the biosynthesis of neurosteroids in the frog hypothalamus

do-Rego

Mensah-Nyagan

Beaujean

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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Most of the actions of neurosteroids on the central nervous system are mediated through allosteric modulation of the ␥-aminobutyric acid type A (GABA A) receptor, but a direct effect of GABA on the regulation of neurosteroid biosynthesis has never been investigated. In the present report, we have attempted to determine whether 3␤-hydroxysteroid dehydrogenase (3␤-HSD)-containing neurons, which secrete neurosteroids in the frog hypothalamus, also express the GABAA receptor, and we have investigated the effect of GABA on neurosteroid biosynthesis by frog hypothalamic explants. Double immunohistochemical labeling revealed that most 3␤-HSD-positive neurons also contain GABAA receptor ␣3 and ␤2͞␤3 subunit-like immunoreactivities. Pulse-chase experiments showed that GABA inhibited in a dose-dependent manner the conversion of tritiated pregnenolone into radioactive steroids, including 17-hydroxy-pregnenolone, progesterone, 17-hydroxyprogesterone, dehydroepiandrosterone, and dihydrotestosterone. The effect of GABA on neurosteroid biosynthesis was mimicked by the GABA A receptor agonist muscimol but was not affected by the GABA B receptor agonist baclofen. The selective GABAA receptor antagonists bicuculline and SR95531 reversed the inhibitory effect of GABA on neurosteroid formation. The present results indicate that steroid-producing neurons of the frog hypothalamus express the GABAA receptor ␣3 and ␤2͞␤3 subunits. Our data also demonstrate that GABA, acting on GABA A receptors at the hypothalamic level, inhibits the activity of several key steroidogenic enzymes, including 3␤-HSD and cytochrome P450C17 (17␣-hydroxylase).neuroactive steroids ͉ 3␤-hydroxysteroid dehydrogenase ͉ GABA A-benzodiazepine receptor complex

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“…The specificity of each GABA A receptor subunit antibody was reported previously. 16,41,61,70,79 The monoclonal anti-actin antibody used for western blot analysis, as a control for total protein, binds to a highly conserved region of actin, an abundant cytoskeletal protein.…”

Section: Polyclonal Gaba a Receptor Subunit Antibodiesmentioning

confidence: 99%

Benzodiazepine-mediated regulation of α1, α2, β1–3 and γ2 GABAA receptor subunit proteins in the rat brain hippocampus and cortex

et al. 1999

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Abstract-Prolonged flurazepam exposure regulates the expression of selected (a1, b2, b3) GABA A receptor subunit messenger RNAs in specific regions of the hippocampus and cortex with a time-course consistent with benzodiazepine tolerance both in vivo and in vitro. In this report, the immunostaining density of six specific GABA A receptor subunit (a1, a2, b1-3 and g2) antibodies was measured in the hippocampus and cortex, among other brain areas, in slide-mounted brain sections from flurazepam-treated and control rats using quantitative computer-assisted image analysis techniques. In parallel with the localized reduction in a1 and b3 subunit messenger RNA expression detected in a previous study, relative a1 and b3 subunit antibody immunostaining density was significantly decreased in flurazepam-treated rat hippocampal CA1, CA3 and dentate dendritic regions, and in specific cortical layers. Quantitative western blot analysis showed that b3 subunit protein levels in crude homogenates of the hippocampal dentate region from flurazepam-treated rats, an area which showed fairly uniform decreases in b3 subunit immunostaining (16-21%), were reduced to a similar degree (18%). The latter findings provide independent support that relative immunostaining density may provide an accurate estimate of protein levels. Consistent with the absence of the regulation of their respective messenger RNAs immediately after ending flurazepam administration, no changes in the density of a2, b1 or b2 subunit antibody immunostaining were found in any brain region. g2 subunit antibody staining was changed only in the dentate molecular layer.The selective changes in GABA A receptor subunit antibody immunostaining density in the hippocampus suggested that a change in the composition of GABA A receptors involving specific subunits (a1 and b3) may be one mechanism underlying benzodiazepine anticonvulsant tolerance. ᭧ 1999 IBRO. Published by Elsevier Science Ltd.Key words: tolerance, flurazepam, quantitative immunohistochemistry, dentate gyrus, limbic system, CA1 pyramidal cells.Although effective anxiolytics and hypnotics, the clinical use of benzodiazepines as anticonvulsants is limited by tolerance development during prolonged administration. A large body of evidence has indicated that benzodiazepine tolerance is related to a reduction in GABA A receptor-mediated fast inhibitory synaptic transmission (for reviews see Refs 2 and 27). The native GABA A receptor is a pentamer composed of combinations of homologous proteins derived from five subunit families with multiple variants (a1-6, b1-4, g1-3, d1 and e1). 8,11,34 The GABA A receptor macromolecule contains binding domains for GABA and several allosteric modulators, including a well-characterized benzodiazepine binding site. 59Benzodiazepines potentiate GABA A receptor-mediated inhibitory function by increasing the opening probability of the integral Cl Ϫ channel, 52,62 and may increase Cl Ϫ channel conductance. 13Although the synaptic mechanisms mediating benzodiazepine tolerance have not been...

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Separation of a1, a2 and a3 subunits of the GABAA-benzodiazepine receptor complex by immunoaffinity chromatography

Cited by 55 publications

References 13 publications

GABA_A receptor changes in δ subunit‐deficient mice: Altered expression of α4 and γ2 subunits in the forebrain

GABA_A receptor changes in δ subunit‐deficient mice: Altered expression of α4 and γ2 subunits in the forebrain

γ-Aminobutyric acid, acting through γ-aminobutyric acid type A receptors, inhibits the biosynthesis of neurosteroids in the frog hypothalamus

Benzodiazepine-mediated regulation of α1, α2, β1–3 and γ2 GABAA receptor subunit proteins in the rat brain hippocampus and cortex

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Separation of a1, a2 and a3 subunits of the GABAA-benzodiazepine receptor complex by immunoaffinity chromatography

Cited by 55 publications

References 13 publications

GABAA receptor changes in δ subunit‐deficient mice: Altered expression of α4 and γ2 subunits in the forebrain

GABAA receptor changes in δ subunit‐deficient mice: Altered expression of α4 and γ2 subunits in the forebrain

γ-Aminobutyric acid, acting through γ-aminobutyric acid type A receptors, inhibits the biosynthesis of neurosteroids in the frog hypothalamus

Benzodiazepine-mediated regulation of α1, α2, β1–3 and γ2 GABAA receptor subunit proteins in the rat brain hippocampus and cortex

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GABA_A receptor changes in δ subunit‐deficient mice: Altered expression of α4 and γ2 subunits in the forebrain

GABA_A receptor changes in δ subunit‐deficient mice: Altered expression of α4 and γ2 subunits in the forebrain