THE ALPHA 4 SUBUNIT OF THE GABAA RECEPTORS FROM RAT BRAIN AND RETINA
1996
Abstract A novel anti-α 4 antibody has been used for the purification and characterization of the α 4 -containing GABA A receptors in the rat brain and for studying the immunocytochemical distribution of the α 4 subunit peptide in rat brain and retina. The anti-α 4 antibody recognized a 66 kDa peptide in brain membranes and immunoprecipitated 10–28% of the brain GABA A receptors in various brain regions as determined by [ 3 H]muscimol binding. The highest immunoprecipitation values were obtained in the thalamus and the lowest in the cerebellum. Surprisingly, the receptors immunoprecipitated by anti-α 4 showed little or no diazepam-insensitive or diazepam-sensitive [ 3 H]Ro15-4513 binding sites in any brain region. In the cerebellum, where 25% of the [ 3 H]Ro15-4513 binding is diazepam-insensitive, much of the latter was immunoprecipitated by an anti-α 6 antibody but not by the anti-α 4 antibody. Immunoblots of immunoaffinity-purified GABA A receptors from the cerebral cortex on immobilized anti-α 4 revealed molecular colocalization of α 4 and γ 2 . However, the absence of significant benzodiazepine binding in these GABA A receptors suggests that the assembly of the α 4 and γ 2 subunits in the cerebral cortex and in other brain regions is such that they do not normally form diazepam-insensitive [ 3 H]Ro15-4513 binding sites. This result contrasts with the presence of diazepam-insensitive [ 3 H]Ro15-4513 binding sites in the GABA A receptors expressed in heterologous systems resulting from the combination of α 4 , γ 2 and β 2 subunits. Immunocytochemistry has revealed the abundance of α 4 peptide immunoreactivity in the thalamus and dentate gyrus (mainly in the hilar neurons and the inner third of the granule cell layer). The α 4 immunoreactivity is also present in the external plexiform layer of the olfactory bulb and in all layers of the neocortex and pyriform cortex. In the retina, α 4 is concentrated on ganglion cells (including some giant ganglion cells), the inner plexiform layer and to a lesser extent in the outer plexiform layer. Copyright © 1996 Elsevier Science Ltd.
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