Multiple distinct subunits of the gamma-aminobutyric acid-A receptor protein show different ligand-binding affinities

M Bureau and RW Olsen

Department of Pharmacology, School of Medicine, University of California, Los Angeles 90024.

The purified gamma-aminobutyric acid/benzodiazepine receptor protein from mammalian brain contains at least four discrete polypeptides (Mr 51,000, 53,000, 55,000 and 58,000) by a variety of visualization techniques and in three species (rat, cow, and human). These polypeptide bands vary in their affinity for gamma-aminobutyric acid analogs as shown by inhibition of [3H]muscimol binding, demonstrated by photoaffinity labeling and gel electrophoresis in sodium dodecyl sulfate. One-dimensional peptide maps of proteolytic digests revealed that distinct fragments were produced, indicating that the four polypeptides represent discrete sequences. The four bands were identified by Western blotting with subunit-specific monoclonal antibodies as two species each of previously identified alpha and beta subunits. [3H]Muscimol photolabeled all four bands (beta and alpha) to varying degrees not proportional to the extent of protein staining. The Mr 58,000 beta subunit subtype showed a higher affinity for 4,5,6,7- tetrahydro-isoxazolo-[5,4-c]pyridin-3-ol than the Mr 56,000 beta subtype, whereas the Mr 56,000 beta and Mr 51,000 alpha bands were more enhanced by pentobarbital than the Mr 58,000 band. Furthermore, the alpha subunit pattern revealed by photoaffinity labeling with [3H]flunitrazepam was significantly different for three regions of bovine brain, showing only one major band in cerebellum at Mr 51,000, two major bands in cortex at Mr 53,000 and 51,000, and three bands in hippocampus at Mr 55,000 as well as Mr 53,000 and 51,000. Because the ratio of the amounts of the various polypeptides varies with brain region and the pharmacological properties of the peptides vary, it is likely that a family of oligomeric gamma-aminobutyric acid/benzodiazepine receptors exists in the brain. This is consistent with the reported variable expression of different subunit subtype mRNAs and with brain region-dependent variation in pharmacology and binding behavior.

Volume 37, Issue 4, pp. 497-502, 04/01/1990
Copyright © 1990 by American Society for Pharmacology and Experimental Therapeutics

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