The α₄ subunit of the GABA(A) receptors from rat brain and retina

A novel anti-α₄ antibody has been used for the purification and characterization of the α₄-containing GABA(A) 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 GABA(A) 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 GABA(A) 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 GABA(A) 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 GABA(A) 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.