Characterisation of δ-subunit containing GABAA receptors from rat brain

Abstract

Polyclonal antibodies have been raised in rabbits against the predicted cytoplasmic loop region of the δ-subunit of the GABA_A receptor. These specifically identify the expressed fragment by Western blot but do not cross react with analogous polypeptides from the γ₁, γ₂ or γ₃-subunits. Polyclonal antisera immunoprecipitated [³H]muscimol binding sites from several brain regions consistent with the reported distribution of δ-subunit mRNA and also detected the δ-subunit by Western blot, identifying a polypeptide of 55KDa. Receptors immunoprecipitated from rat brain with the δ-antisera exhibited an atypical profile with respect to their radioligand binding properties. Receptors immunoprecipitated from all regions tested bound [³H]muscimol, but did not bind benzodiazepine site ligands [³H]Ro 15,1788 or [³H]flunitrazepam with high affinity. Receptors containing a δ-subunit accounted for 10.7 ± 2% of all GABA_A receptors ([³H]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 δ-subunit. The pharmacology of the GABA (γ-aminonutyric acid) binding site on receptors immunoprecipitated from cerebellum with γ₂ and δ-antisera was compared. The rank order of potency of a series of 6 compounds to compete for [³H]muscimol binding sites was similar in these two populations, but muscimol had a significantly higher affinity for receptors containing the δ-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 δ-subunit with γ-subunits was investigated by purifying receptor on a γ₁, γ₂ or γ₃-immunoaffinity resins, separating the subunit components by polyacrylamide gel electrophoresis, transferring to nitrocellulose and probing with δ-subunit specific antibodies. The δ-subunit could not be detected in combination with any γ-subunit.