Modification of benzodiazepine receptors supports the distinctive role of histidine residues.

The effect of selective protein modifying reagents was examined on benzodiazepine (BZ) receptors in synaptosomal membrane preparations of rat whole brain and cerebellum. The potency of diethyl pyrocarbonate, a histidine modifying reagent, to inactivate BZ receptor binding, correlated with the rank order of agonist-inverse agonist efficacies of BZ ligands, the binding of the partial inverse agonist [3H]Ro 15-4513 was inactivated least. Diethyl pyrocarbonate slightly enhanced the displacing potency of Ro 15-4513 and enhanced its binding in low concentrations (1-2 mM). Diazepam-sensitive and -insensitive components of [3H]Ro 15-4513 binding were separated in cerebellum. Diethyl pyrocarbonate inactivated the diazepam-sensitive component with a potency (IC50 = 1.8 mM) similar to that on the binding of other benzodiazepines, while the diazepam-insensitive component was resistant to diethyl pyrocarbonate. Tetranitromethane and 2,3-butanedione (diacetyl), reagents specific for tyrosine and arginine residues respectively, exerted concentration-dependent partial inactivation of [3H]Ro 15-4513 binding. The diazepam-insensitive component of cerebellar Ro 15-4513 binding was more sensitive to inactivation by diacetyl but less sensitive to inactivation by tetranitromethane. These findings are consistent with a distinctive role of histidine-101 in alpha 1, alpha 2, alpha 3 and alpha 5 subunits of the gamma-aminobutyric acidA receptor complex and the His is replaced by an arginine residue in the alpha 6 subunit of the diazepam-insensitive cerebellar benzodiazepine receptors. The only other point of the protein sequence where histidine residues conserved in alpha 1, alpha 2, alpha 3 and alpha 5 subunits are replaced in alpha 6 is tyrosine-214 but this residue does not appear to contribute to benzodiazepine binding.