COVALENT LABELING OF MUSCARINIC ACETYLCHOLINE-RECEPTORS BY TRITIATED ARYLDIAZONIUM PHOTOPROBES

p-dimethylamino (A) and p-dibutylamino (B) benzenediazonium salts, pre viously characterized as efficient labels of membrane-bound and solubi lized muscarinic receptor sites, are endowed with overall interesting photochemical and alkylating properties that allow their use as struct ural probes of the muscarinic ligand binding domain to be considered. Under reversible binding conditions, these antagonists display no bind ing selectivity towards the 5 muscarinic acetylcholine receptor (mAChR ) subtypes. They were used here, in a tritiated form, as photoaffinity labels of purified muscarinic receptors from porcine striatum, and th eir irreversible binding was assessed by SDS-polyacrylamide gel electr ophoresis (SDS-PAGE) analysis. When irradiated under energy transfer c onditions, [H-3]A and [H-3]B were both found to covalent-ly label puri fied muscarinic receptor sites in a light-dependent and atropine-prote ctable manner. The electrophoretic migration properties of the alkylat ed sites were similar to those of [H-3]propylbenzilylcholine mustard ( PrBCM)-labeled mAChRs. Specific radioactive incorporation showed a cle ar dependency on probe concentration. Labeling efficiency was rather h igh, with up to 30% and even 60% of the receptor population being phot olabeled by [H-3]A and [H-3]B, respectively. These two photoactivatabl e ligands have proven to be powerful tools for the structural analysis of other cholinergic targets (acetylcholinesterase and the nicotinic acetylcholine receptor) by allowing the characterization of a number o f different residues belonging to their acetylcholine binding domain. Altogether, these results reinforce the interest of our site-directed labeling approach because [H-3]A- and [3H]B-alkylated mAChRs may now b e considered as suitable materials to investigate the muscarinic recep tor-binding pocket through peptide mapping, sequence analyses, and ide ntification of radiolabeled amino acid residues. (C) 1997 Elsevier Sci ence Inc.