The benzodiazepine binding pocket of recombinant alpha 1 beta 2 gamma 2 gamma-aminobutyric acid(A) receptors: Relative orientation of ligands and amino acid side chains

Wild-type alpha 1 beta 2 gamma 2 gamma-aminobutyric acid (GABA)(A) receptor s and receptors containing a point-mutated subunit gamma 2F77Y were express ed by transient transfection in human embryonic kidney 293 cells. Mutant re ceptors bound the benzodiazepine binding site ligand [H-3]flumazenil with s imilar, subnanomolar affinity as wild-type receptor. Displacement studies w ith diazepam showed that the affinity for this compound was reduced 250-fol d on mutation, indicating that the tyrosine hydroxyl group interferes with diazepam binding. This differential behavior then was used to find the chem ical entity presumably interacting with the phenyalanine residue in positio n 77 of the gamma 2 subunit of wild-type receptors. Thirty-four substances were analyzed in this respect. Our results suggest that the phenyl substitu ent of diazepam is located close to gamma F77. Similarly, we investigated t he possible location of alpha 1T206 and gamma 2M130. Electrophysiological d ata obtained with the wild-type receptor furthermore suggest a simple overl ap between positive allosteric modulators acting at the benzodiazepine bind ing site with its antagonists.