IMMUNOCYTOCHEMICAL LOCALIZATION OF THE BETA(2) SUBUNIT OF THE GAMMA-AMINOBUTYRIC ACID(A) RECEPTOR IN THE RAT-BRAIN

An antiserum to the beta(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 beta(2) subunit fused to staphylococcal protein A (SPA). The antiserum immunoprecipitated both the solubilized and the affinity-purified GABA(A) 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 GABA(A) 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 GABA(A) receptor subunits. (C) 1994 Wiley-Liss, Inc.