Characterization of γ‐Aminobutyric Acid‐Benzodiazepine Receptor Complexes by Protection Against Inactivation by Group‐Specific Reagents

The chemical topography of the γ‐aminobutyric acid (GABA) and benzodiazepine (BZ) receptors was investigated in a thoroughly washed cortical membrane preparation of the rat. Chemical modification by several amino‐ and tyrosyl‐selective reagents and the protection from it by direct and allosteric ligands of the GABA‐BZ receptor complex were used to identify the residues at the binding sites. Inhibition of specific GABA binding by p ‐diazobenzenesulfonic acid (DSA), tetrani‐tromethane (TNM), and N ‐acetylimidazole and the selective and complete protection from it by GABA and muscimol suggest the presence of a tyrosine residue at the GABA A site. TNM, like DSA, selectively decreased the number of the low‐affinity GABA receptors, and this could be completely protected only by GABA concentrations that can saturate the low‐affinity sites. TNM pre‐treatment also abolished the muscimol enhancement of [ 3 H]diazepam binding, which suggests that the low‐affinity GABA receptor sites are responsible for this enhancement. Inhibition of GABA binding by pyridoxal‐5‐phosphate (PLP) and the selective protection by GABA and muscimol support the presence of a lysine residue at the GABA A receptor site. Complete and selective protection from diethylpyrocarbonate (DEP) inhibition of [ 3 H]diazepam binding by flurazepam suggests the presence of a histidine residue at the BZ site. Flurazepam selectively protected from inhibition of [ 3 H]diazepam binding by N ‐bromosuccinimide and N ‐acetylimidazole, but not that by DSA and TNM, which does not allow a unanimous conclusion regarding the presence of tyrosine or tryptophan residues at the BZ site.