title: Modification of benzodiazepine receptors supports the distinctive role of histidine residues.
abstract: 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.
authors.affiliation: Department of Molecular Pharmacology, Hungarian Academy of Sciences, Budapest.
authors.forename: G
authors.initials: G
authors.lastname: Maksay
mesh_terms: D000818:Animals; D001386:Azides / Q000494:pharmacology; D001569:Benzodiazepines / Q000494:pharmacology; D001921:Brain / Q000502:physiology; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004852:Epoxy Compounds / Q000494:pharmacology; D006639:Histidine / Q000032:analysis / Q000502:physiology*; D066298:In Vitro Techniques; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D011963:Receptors, GABA-A / Q000737:chemistry / Q000187:drug effects*; D013329:Structure-Activity Relationship; D013774:Tetranitromethane / Q000494:pharmacology; D014316:Tritium
chemical_list: D001386:Azides; D004852:Epoxy Compounds; D011963:Receptors, GABA-A; D014316:Tritium; D001569:Benzodiazepines; D006639:Histidine; C007366:diepoxybutane; C042957:Ro 15-4513; D013774:Tetranitromethane; D004047:Diethyl Pyrocarbonate