Abstract

Investigating how a test drug alters the reaction of a site-directed electrophile with a receptor is a powerful method for determining whether the drug acts competitively or allosterically, provided that the binding site of the electrophile is known. In this study, therefore, we mutated nucleophilic residues near and within the orthosteric pockets of M₁ and M₂ muscarinic receptors to identify where acetylcholine mustard and 4-[(2-bromoethyl)methyl-amino]-2-butynyl-N-(3-chlorophenyl)carbamate (BR384) bind covalently. BR384 is the nitrogen mustard analog of [4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyl]trimethylammonium chloride (McN-A-343). Mutation of the highly conserved aspartic acid in M₁ (Asp105) and M₂ (Asp103) receptors to asparagine largely prevented receptor alkylation by acetylcholine mustard, although modest alkylation still occurred at M₂ D103N at high concentrations of the mustard. Receptor alkylation by BR384 was also greatly inhibited in the M₁ D105N mutant, but some alkylation still occurred at high concentrations of the compound. In contrast, BR384 rapidly alkylated the M₂ D103N mutant. Its affinity was reduced to one tenth, however. The alkylation of M₂ D103N by BR384 was competitively inhibited by N-methylscopolamine and allosterically inhibited by gallamine. Mutation of a variety of other nucleophilic residues, some in combination with D103N, had little effect on M₂ receptor alkylation by BR384. Our results suggest that BR384 alkylates at least one residue other than the conserved aspartic acid at the ligand-binding site of M₁ and M₂ receptors. This additional residue seems to be located within or near the orthosteric-binding pocket and is not part of the allosteric site for gallamine.