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.
Footnotes
This work was supported by the National Institutes of Health National Institute of General Medical Sciences [Grant GM69829] and the National Institutes of Health National Institute of Neurological Disorders and Stroke [Grant NS057742].
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.110.065367.
-
ABBREVIATIONS:
- AChM
- acetylcholine mustard
- ACh
- acetylcholine
- BR384
- 4-[(2-bromoethyl)methyl-amino]-2-butynyl N-(3-chlorophenyl)carbamate
- McN-A-343
- [4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyl]trimethylammonium chloride
- NMS
- N-methylscopolamine
- WIN 51,708
- 17-β-hydroxy-17-α-ethynyl-5-α-androstano[3,2-β]pyrimido[1,2-α]benzimidazole.
- CHO
- Chinese hamster ovary
- HEK
- human embryonic kidney
- TM
- transmembrane domain
- E2
- extracellular loop 2
- E3
- extracellular loop 3.
- Received April 5, 2010.
- Accepted July 19, 2010.
- Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|