IMMUNOCHEMICAL CHARACTERIZATION OF THE BETA-2 SUBUNIT OF THE GABA(A) RECEPTOR

To date three beta subunits of the GABA(A) receptor have been identified in rat brain as a result of cDNA library screening. The beta2 subunit has been reported to have a wide distribution in rat brain based on in situ hybridization studies quantifying beta2 mRNA. To study the beta2 subunit more directly, we have raised a polyclonal antibody to a synthetic peptide representing residues 315-334 of the intracellular loop of the beta2 subunit. The antibody, which had been affinity-purified, recognized the beta2 peptide but did not immunolabel homologous beta1 and beta3 subunit peptides, indicating that this antibody is specific for the beta2 subunit of the receptor. In western blots of the purified receptor, the antibody recognized a major diffuse band of 54-58 kDa and exhibited minor labeling of lower-molecular-mass polypeptides. In western blots of cortex homogenate, the antibody exhibited nervous system-specific labeling of a 55-kDa band that comigrated with the 55-kDa band of the purified receptor. Quantitative immunolabeling of this 55-kDa polypeptide permitted direct determination of the relative amounts of the beta2 subunit in different brain regions. The brainstem contained the highest relative specific activity of the beta2 subunit, followed by the inferior colliculus, olfactory lobe, and cerebellum. Lower levels of immunolabeling were seen in hypothalamus, hippocampus, thalamus, and cortex.