THE ALPHA(4) SUBUNIT OF THE GABA(A) RECEPTORS FROM RAT-BRAIN AND RETINA

A novel anti-alpha(4) antibody has been used for the purification and characterization of the alpha(4)-containing GABA(A) receptors in the r at brain and for studying the immunocytochemical distribution of the a lpha(4) subunit peptide in rat brain and retina. The anti-alpha(4) ant ibody recognized a 66 kDa peptide in brain membranes and immunoprecipi tated 10-28% of the brain GABA(A) receptors in various brain regions a s determined by [H-3]muscimol binding. The highest immunoprecipitation values were obtained in the thalamus and the lowest in the cerebellum . Surprisingly, the receptors immunoprecipitated by anti-alpha(4) show ed little or no diazepam-insensitive or diazepam-sensitive [H-3]Ro15-4 513 binding sites in any brain region. In the cerebellum, where 25% of the [H-3]Ro15-4513 binding is diazepam-insensitive, much of the latte r was immunoprecipitated by an anti-alpha(6) antibody but not by the a nti-alpha(4) antibody. Immunoblots of immunoaffinity-purified GABA(A) receptors from the cerebral cortex on immobilized anti-alpha(4) reveal ed molecular colocalization of alpha(4) and gamma(2). However, the abs ence of significant benzodiazepine binding in these GABA(A) receptors suggests that the assembly of the alpha(4) and gamma(2) subunits in th e cerebral cortex and in other brain regions is such that they do not normally form diazepam-insensitive [H-3]Ro15-4513 binding sites. This result contrasts with the presence of diazepam-insensitive [H-3]Ro15-4 513 binding sites in the GABA(A) receptors expressed in heterologous s ystems resulting from the combination of alpha(4), gamma(2) and beta(2 ) subunits. Immunocytochemistry has revealed the abundance of alpha(4) peptide immunoreactivity in the thalamus and dentate gyrus (mainly in the hilar neurons and the inner third of the granule cell layer). The alpha(4) immunoreactivity is also present in the external plexiform l ayer of the olfactory bulb and in all layers of the neocortex and pyri form cortex. In the retina, alpha(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 (C) 1996 El sevier Science Ltd.