The alpha 4 subunit of the GABAA 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 GABAA receptors in the rat brain and for studying the immunocytochemical distribution of the alpha 4 subunit peptide in rat brain and retina. The anti-alpha 4 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-alpha 4 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-alpha 6 antibody but not by the anti-alpha 4 antibody. Immunoblots of immunoaffinity-purified GABAA receptors from the cerebral cortex on immobilized anti-alpha 4 revealed molecular colocalization of alpha 4 and gamma 2. However, the absence of significant benzodiazepine binding in these GABAA receptors suggests that the assembly of the alpha 4 and gamma 2 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 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 layer of the olfactory bulb and in all layers of the neocortex and pyriform 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.
Khan
Z U
ZU
Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri-Kansas City 64110-2499, USA.
Gutiérrez
A
A
Mehta
A K
AK
Miralles
C P
CP
De Blas
A L
AL
D000818:Animals; D015153:Blotting, Western; D001923:Brain Chemistry / Q000187:drug effects*; D018755:GABA Agonists / Q000378:metabolism; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D009118:Muscimol / Q000378:metabolism; D011233:Precipitin Tests; D051381:Rats; D011963:Receptors, GABA-A / Q000187:drug effects / Q000378:metabolism*; D012160:Retina / Q000187:drug effects / Q000378:metabolism*
D018755:GABA Agonists; D011963:Receptors, GABA-A; D009118:Muscimol