Characterisation of delta-subunit containing GABAA receptors from rat brain.
Polyclonal antibodies have been raised in rabbits against the predicted cytoplasmic loop region of the delta-subunit of the GABAA 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 [3H]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 55KDa. 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 [3H]muscimol, but did not bind benzodiazepine site ligands [3H]Ro 15,1788 or [3H]flunitrazepam with high affinity. Receptors containing a delta-subunit accounted for 10.7 +/- 2% of all GABAA 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 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 [3H]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 GABAA receptors which do not bind benzodiazepines but have a 5-fold higher affinity for muscimol.(ABSTRACT TRUNCATED AT 250 WORDS)
Quirk
K
K
Department of Biochemistry, Merck, Sharp and Dohme Research Laboratories, Harlow, Essex, UK.
Whiting
P J
PJ
Ragan
C I
CI
McKernan
R M
RM
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D001923:Brain Chemistry / Q000502:physiology*; D002531:Cerebellum / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D009118:Muscimol / Q000378:metabolism; D011233:Precipitin Tests; D011869:Radioligand Assay; D051381:Rats; D011963:Receptors, GABA-A / Q000187:drug effects / Q000378:metabolism*
D011963:Receptors, GABA-A; D009118:Muscimol