GABAA receptors display association of γ2-subunit with α1- and β2/3-subunits

Abstract

Recombinant GABAA (γ-aminobutyrate-Type A) receptors that are sensitive to benzodiazepine receptor ligands can be generated by coexpression of α-, β-, and γ 2-subunit cDNAs (Pritchett, D. B., Sontheimer, H., Shivers, B. D., Ymer S., Kettenmann, H., Schofield, P. R., and Seeburg, P. H. (1989) Nature 338, 582-585; Pritchett, D. B., Luddens, H., and Seeburg, P. H. (1989) Science 245, 1389-1392; Malherbe, P., Sigel, E., Baur, R., Perssohn, E., Richards, J. G., and Mohler, H. (1990) J. Neurosci. 10, 2330-2337). However, in brain tissue, only α- and β-subunit proteins have so far been detected. To identify the size and distribution of the γ 2-subunit protein in brain tissue, polyclonal antibodies were prepared against two synthetic peptides corresponding to amino acids 1-15 and 336-350 of the cDNA-derived rat γ 2-subunit sequence. On Western blots, both anti-γ 2-subunit antisera selectively labeled a 43-kDa protein. γ 2-Subunit immunoreactivity was detected immunohistochemically in various brain regions, e.g. in the olfactory bulb, cerebral cortex, islands of Calleja, hippocampus, substantia nigra, and cerebellum. Immunoprecipitation with both antisera identified the γ 2-subunit immunoreactivity in 40 and 50% of the native GABAA receptors purified from bovine and rat brains, respectively. Monoclonal antibody bd24 selectively recognizes the α 1-subunit, whereas bd17 recognizes both the β 2- and β 3-subunits (Ewert, M., Shivers, B. D., Luddens, H., Mohler, H., and Seeburg, P. H. (1990) J. Cell Biol. 110, 2043-2048). Since either of these monoclonal antibodies (bd17 and bd24) precipitated ≈ 90% of the GABAA receptors, the γ 2-subunit is frequently associated with the α 1-subunit and the β 2- and/or β 3-subunit in vivo.