The α4 Subunit of the GABAA Receptors from Rat Brain and Retina

Abstract

A novel anti-α4 antibody has been used for the purification and characterization of the α4-containing GABAA receptors in the rat brain and for studying the immunocytochemical distribution of the α4 subunit peptide in rat brain and retina. The anti-α4 antibody recognized a 66 kDa peptide in brain membranes and immunoprecipitated 10–28% of the brain GABAA receptors in various brain regions as determined by [3H]muscimol binding. The highest immunoprecipitation values were obtained in the thalamus and the lowest in the cerebellum. Surprisingly, the receptors immunoprecipitated by anti-α4 showed little or no diazepam-insensitive or diazepam-sensitive [3H]Ro15-4513 binding sites in any brain region. In the cerebellum, where 25% of the [3H]Ro15-4513 binding is diazepam-insensitive, much of the latter was immunoprecipitated by an anti-α6 antibody but not by the anti-α4 antibody. Immunoblots of immunoaffinity-purified GABAA receptors from the cerebral cortex on immobilized anti-α4 revealed molecular colocalization of α4 and γ2. However, the absence of significant benzodiazepine binding in these GABAA receptors suggests that the assembly of the α4 and γ2 subunits in the cerebral cortex and in other brain regions is such that they do not normally form diazepam-insensitive [3H]Ro15-4513 binding sites. This result contrasts with the presence of diazepam-insensitive [3H]Ro15-4513 binding sites in the GABAA receptors expressed in heterologous systems resulting from the combination of α4, γ2 and β2 subunits. Immunocytochemistry has revealed the abundance of α4 peptide immunoreactivity in the thalamus and dentate gyrus (mainly in the hilar neurons and the inner third of the granule cell layer). The α4 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, α4 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.