Immunochemical characterization of the beta 2 subunit of the GABAA receptor.
To date three beta subunits of the GABAA receptor have been identified in rat brain as a result of cDNA library screening. The beta 2 subunit has been reported to have a wide distribution in rat brain based on in situ hybridization studies quantifying beta 2 mRNA. To study the beta 2 subunit more directly, we have raised a polyclonal antibody to a synthetic peptide representing residues 315-334 of the intracellular loop of the beta 2 subunit. The antibody, which had been affinity-purified, recognized the beta 2 peptide but did not immunolabel homologous beta 1 and beta 3 subunit peptides, indicating that this antibody is specific for the beta 2 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 beta 2 subunit in different brain regions. The brainstem contained the highest relative specific activity of the beta 2 subunit, followed by the inferior colliculus, olfactory lobe, and cerebellum. Lower levels of immunolabeling were seen in hypothalamus, hippocampus, thalamus, and cortex.
Machu
T K
TK
Department of Pharmacology, University of Colorado Health Sciences Center, Denver 80262.
Olsen
R W
RW
Browning
M D
MD
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism*; D001933:Brain Stem / Q000378:metabolism; D002531:Cerebellum / Q000378:metabolism; D002540:Cerebral Cortex / Q000378:metabolism*; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006624:Hippocampus / Q000378:metabolism; D017403:In Situ Hybridization; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009928:Organ Specificity; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D051381:Rats; D011963:Receptors, GABA-A / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification
D000906:Antibodies; D046911:Macromolecular Substances; D012333:RNA, Messenger; D011963:Receptors, GABA-A