Immunocytochemical localization of the β₂subunit of the gamma‐aminobutyric acid_Areceptor in the rat brain

Abstract

An antiserum to the beta 2 subunit of the rat gamma-aminobutyric acid (GABAA) receptor was prepared by immunizing a rabbit with a fusion protein expressed in bacteria. The fusion protein had the large, intracellular loop expanding between the putative M3 and M4 transmembrane domains of the beta 2 subunit fused to staphylococcal protein A (SPA). The antiserum immunoprecipitated both the solubilized and the affinity-purified GABAA receptors. The anti-beta 2 antibodies were affinity purified on immobilized beta 2 intracellular loop peptide. The antibodies recognized a 55-57 kDa peptide in immunoblots of either crude membranes from rat cerebral cortex or affinity-purified GABAA receptors from bovine cerebral cortex. Immunocytochemistry with the affinity-purified antibody has revealed for the first time the localization of the beta 2 subunit in the rat brain. A comparative study of the regional and cellular immunoreactivities of the affinity-purified anti-beta 2 antibody and the monoclonal antibody 62-3G1 (which recognizes both beta 2 and beta 3 subunits) is presented. The procedure described for generating and preparing specific anti-beta 2 subunit antibodies that are valuable for immunocytochemistry could be extended to other GABAA receptor subunits.

GABAergic Innervation Organizes Synaptic and Extrasynaptic GABAAReceptor Clustering in Cultured Hippocampal Neurons

“…This antibody recognizes an extracellular N-terminus epitope that is common to ␤ 2 and ␤ 3 subunits but is not present in ␤ 1 (Ewert et al, 1992). Antibodies to f usion proteins of the intracellular loops of ␤ 1 , ␤ 2 , and ␤ 3 were also raised in rabbits in our laboratory and affinity purified with purified intracellular loop of the respective isoform (Moreno et al, 1994;Li and De Blas, 1997). Subunit and isoform-specific antibodies made in several species in our laboratory have allowed us to study colocalization by triple-label immunofluorescence (see below).…”

“…Subunit and isoform-specific antibodies made in several species in our laboratory have allowed us to study colocalization by triple-label immunofluorescence (see below). All antibodies to GABA A R subunits used in this study have been thoroughly characterized, and their specificities have been determined elsewhere Vitorica et al, 1988;Moreno et al, 1994;Miralles et al, 1999). Specificity tests of GABA A R antibodies included ELISA, immunoblotting, light microscopy immunocytochemistry, displacement of immunoreactivity in these assays by specific peptides, and subunit-specific staining in host-transfected cell lines.…”

Regulation of GABAAReceptor Subunit Expression by Pharmacological Agents

“…In many brain regions, their distribution is more or less complementary (Pirker et al, 2000). In the pallidum and thalamus, ␤2 is the main ␤ subunit, whereas in the striatum, ␤3 is the most abundant ␤ subunit (Moreno et al, 1994;Miralles et al, 1999;Pirker et al, 2000). The most abundant ␥ subunit, ␥2, is expressed throughout the brain.…”

Benzodiazepine-mediated regulation of α1, α2, β1–3 and γ2 GABAA receptor subunit proteins in the rat brain hippocampus and cortex

“…1), 15,18,19,42,60,61,82 and therefore will not be described in further detail. The distribution of the b2 subunit has been described previously by Moreno et al, 40 using an antibody to an alternative epitope. The comparative distributions of the b1-3 antibodies are provided in detail in Sperk et al 61 …”