CHARACTERIZATION OF DELTA-SUBUNIT CONTAINING GABA(A) RECEPTORS FROM RAT-BRAIN

Polyclonal antibodies have been raised in rabbits against the predicted cytoplasmic loop region of the S-subunit of the GABA, receptor. These specifically identify the expressed fragment by Western blot but do not cross react with analogous polypeptides from the gamma(1), gamma(2) or gamma(3)-subunits. Polyclonal antisera immunoprecipitated [H-3]muscimol binding sites from several brain regions consistent with the reported distribution of delta-subunit mRNA and also detected the delta-subunit by Western blot, identifying a polypeptide of 55 KDa. Receptors immunoprecipitated from rat brain with the delta-antisera exhibited an atypical profile with respect to their radioligand binding properties. Receptors immunoprecipitated from all regions tested bound [H-3]muscimol, but did not bind benzodiazepine site ligands [H-3]Ro 15,1788 or [H-3]flunitrazepam with high affinity. Receptors containing a delta-subunit accounted for 10.7 +/- 2% of all GABA(A) receptors ([H-3]muscimol binding sites) in the rat central nervous system as deduced from quantitative immunoprecipitation experiments, the largest population being in the cerebellum where approximately 27% of all receptors contained a delta-subunit. The pharmacology of the GABA (gamma-aminobutyric acid) binding site on receptors immunoprecipitated from cerebellum with gamma(2) and delta-antisera was compared. The rank order of potency of a series of 6 compounds to compete for [H-3]muscimol binding sites was similar in these two populations, but muscimol had a significantly higher affinity for receptors containing the delta-subunit. These receptors therefore comprise a novel population of GABA(A) receptors which do not bind benzodiazepines but have a 5-fold higher affinity for muscimol. Coexistence of the delta-subunit with gamma-subunits was investigated by purifying receptor on a gamma(1), gamma(2) or gamma(3)-immunoaffinity resins, separating the subunit components by polyacrylamide gel electrophoresis, transferring to nitrocellulose and probing with delta-subunit specific antibodies. The delta-subunit could not be detected in combination with any gamma-subunit.