Photoaffinity labeling of the benzodiazepine binding site of alpha1beta3gamma2 gamma-aminobutyric acidA receptors with flunitrazepam identifies a subset of ligands that interact directly with His102 of the alpha subunit and predicts orientation of these within the benzodiazepine pharmacophore.

Photoincorporation of ligands into the benzodiazepine site of native gamma-aminobutyric acidA (GABAA) receptors provides useful information about the nature of the benzodiazepine (BZ) binding site. Photoincorporation of flunitrazepam into a single population of GABAA receptors, recombinant human alpha1beta3gamma2, was investigated to probe further the mechanism and orientation of flunitrazepam and other ligands in the BZ binding site. It was concluded that the receptor is primarily derivatized with the entire, unfragmented, flunitrazepam molecule, which undergoes a conformational change during photolysis and largely vacates the benzodiazepine binding site. Investigation of the BZ site after photoincorporation of [3H]flunitrazepam confirmed that binding of other radioligands was unaffected by incorporation of flunitrazepam. This did not correlate with their efficacy but depended on the presence of particular structural features in the molecule. It was observed that affected compounds have a pendant phenyl moiety, analogous to the 5-phenyl group of flunitrazepam, which are proposed to overlap and interact with the same residue or residues in the BZ binding site. Because the major site of flunitrazepam photoincorporation has been shown to be His102, we propose that this group of compounds interacts directly with His 102, whereas compounds of other structural types have no direct interaction with this amino acid. The orientation of ligands within the BZ binding site and their specific interaction with identified amino acids are not well understood. The data in the current study indicate that His102 interacts directly with the pendant phenyl group of diazepam, and further implications for the pharmacophore of the BZ binding site are discussed.