The gamma 2 subunit is an integral component of the gamma-aminobutyric acidA receptor but the alpha 1 polypeptide is the principal site of the agonist benzodiazepine photoaffinity labeling reaction.
Polyclonal antibodies were raised to a synthetic peptide whose amino acid sequence was derived from the novel gamma-aminobutyric acidA (GABAA) receptor subunit, gamma 2. These anti-gamma 2 1-15 Cys antibodies reacted specifically with the GABAA receptor purified from adult bovine cerebral cortex in an enzyme-linked immunosorbent assay. Anti-gamma 2 1-15 Cys antibodies specifically immunoprecipitated [3H]flunitrazepam photoaffinity-labeled native receptor in parallel with anti-alpha 1 324-341 antibodies. Immunoprecipitation of sodium dodecyl sulphate (SDS) denatured photoaffinity-labeled receptor by anti-gamma 2 1-15 Cys antibodies, however, resulted in a significant decrease in the maximum percentage of radioactivity immunoprecipitated compared to that by anti-alpha 1 324-341 antibodies. In immunoblots, anti-gamma 2 1-15 Cys antibodies reacted with a broad band in the molecular weight range Mr 43,000-49,000 which was distinct from that recognized by anti-alpha 1 324-341 antibodies. The anti-alpha 1 324-341 immunoreactive band was the main subunit irreversibly photoaffinity labeled by [3H]flunitrazepam, i.e. Mr 53,000. These results demonstrate for the first time that the gamma 2 subunit is an integral component of the GABAA receptor but it is the alpha 1 subunit that is the principal site of the agonist benzodiazepine photoaffinity labeling reaction. It supports a role of both the alpha 1 and gamma 2 polypeptides in the formation of the central benzodiazepine binding site within a GABAA receptor oligomer.
Stephenson
F A
FA
Department of Pharmaceutical Chemistry, School of Pharmacy, London, Great Britain.
Duggan
M J
MJ
Pollard
S
S
D000345:Affinity Labels; D000818:Animals; D001921:Brain / Q000378:metabolism; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D005445:Flunitrazepam / Q000378:metabolism*; D015151:Immunoblotting; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D011963:Receptors, GABA-A / Q000302:isolation & purification / Q000378:metabolism*
D000345:Affinity Labels; D046911:Macromolecular Substances; D011963:Receptors, GABA-A; D005445:Flunitrazepam