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

Abstract

Abstract An antiserum to the β 2 subunit of the rat gamma‐aminobutyric acid (GABA A ) 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 β 2 subunit fused to staphylococcal protein A (SPA). The antiserum immunoprecipitated both the solubilized and the affinity‐purified GABA A receptors. The anti‐β 2 antibodies were affinity purified on immobilized β 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 GABA A receptors from bovine cerebral cortex. Immunocytochemistry with the affinity‐purified antibody has revealed for the first time the localization of the β 2 subunit in the rat brain. A comparative study of the regional and cellular immunoreactivities of the affinity‐purified anti‐β 2 antibody and the monoclonal antibody 62‐3G1 (which recognizes both β 2 and β 3 subunits) is presented. The procedure described for generating and preparing specific anti‐β 2 subunit antibodies that are valuable for immunocytochemistry could be extended to other GABA A receptor subunits. © 1994 Wiley‐Liss, Inc.