Immunocytochemical localization of the beta 2 subunit of the gamma-aminobutyric acidA receptor in the rat brain.

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.