The benzodiazepine binding pocket of recombinant alpha1beta2gamma2 gamma-aminobutyric acidA receptors: relative orientation of ligands and amino acid side chains.

Wild-type alpha1beta2gamma2 gamma-aminobutyric acid (GABA)A receptors and receptors containing a point-mutated subunit gamma2F77Y were expressed by transient transfection in human embryonic kidney 293 cells. Mutant receptors bound the benzodiazepine binding site ligand [3H]flumazenil with similar, subnanomolar affinity as wild-type receptor. Displacement studies with diazepam showed that the affinity for this compound was reduced 250-fold on mutation, indicating that the tyrosine hydroxyl group interferes with diazepam binding. This differential behavior then was used to find the chemical entity presumably interacting with the phenyalanine residue in position 77 of the gamma2 subunit of wild-type receptors. Thirty-four substances were analyzed in this respect. Our results suggest that the phenyl substituent of diazepam is located close to gammaF77. Similarly, we investigated the possible location of alpha1T206 and gamma2M130. Electrophysiological data obtained with the wild-type receptor furthermore suggest a simple overlap between positive allosteric modulators acting at the benzodiazepine binding site with its antagonists.