Monoclonal antibodies and conventional antisera to the GABAA receptor/benzodiazepine receptor/Cl- channel complex.
Monoclonal antibodies (mAbs) and conventional antisera were raised to the affinity-purified GABAA receptor/benzodiazepine receptor/Cl- channel complex. The antibodies immunoprecipitated the affinity-purified complex in Triton X-100 and also reacted with the complex in a solid-phase radioimmunoassay. Immunoblots indicated that the mAb 62-3G1 reacted with the 57,000 Mr peptide subunit of the affinity-purified complex, while the antisera mainly reacted with the 51,000 Mr peptide subunit. The mAbs and the antisera also immunoprecipitated the GABAA receptor/benzodiazepine receptor/Cl- channel complex after being solubilized from cerebral cortex membranes by the zwitterionic detergent CHAPS. The immunoprecipitated complex bound 3H-muscimol, 3H-flunitrazepam (FNZ) and 35S-t-butylbicyclophosphorothionate (TBPS). The 3H-FNZ binding was stimulated by GABA, indicating that the functional interactions among the immunoprecipitated components of the complex were preserved. The mAb 62-3G1 also recognized the 57,000 Mr peptide in immunoblots with crude brain membranes. Immunocytochemistry experiments showed that the binding of both the mAb 62-3G1 and 3H-muscimol colocalized throughout the brain. The results suggest that (1) the 57,000 Mr peptide is the muscimol (GABAA receptor agonist) binding peptide of the complex, and (2) in the cerebral cortex, most of the GABAA receptors (GABARs), benzodiazepine receptors (BDZRs), and Cl- channels are physically coupled to one another.
Vitorica
J
J
Department of Neurobiology and Behavior, State University of New York, Stony Brook 11794.
Park
D
D
Chin
G
G
de Blas
A L
AL
D000818:Animals; D000911:Antibodies, Monoclonal; D001923:Brain Chemistry; D002417:Cattle; D002712:Chlorides / Q000032:analysis; D008566:Membranes / Q000032:analysis; D011963:Receptors, GABA-A / Q000032:analysis*
D000911:Antibodies, Monoclonal; D002712:Chlorides; D011963:Receptors, GABA-A