Abstract
We explored the interaction of a nitrogen mustard derivative of acetylcholine with the human M2 muscarinic receptor expressed in Chinese hamster ovary cells using the muscarinic radioligand, [3H]N-methylscopolamine (NMS). Acetylcholine mustard caused a concentration-dependent, first-order loss of [3H]NMS binding at 37°C, with the half-maximal rate constant occurring at 24 μM and a maximal rate constant of 0.16 min-1. We examined the effects of various ligands on the rate of alkylation of M2 receptors by acetylcholine mustard. N-methylscopolamine and 4-(trimethylamino)-2-butynyl-(3-chlorophenyl)carbamate (McN-A-343) competitively slowed the rate of alkylation, whereas the inhibition by gallamine reached a plateau at high concentrations, indicating allosteric inhibition. In contrast, 17-β-hydroxy-17-α-ethynyl-5-α-androstano[3,2-β]-pyrimido[1,2-α]benzimidazole (WIN 51708) had no effect. We also measured the inhibition of [3H]NMS binding by acetylcholine mustard at 0°C, conditions under which there is little or no detectable covalent binding. In these experiments, the dissociation constant of the aziridinium ion of acetylcholine mustard was estimated to be 12.3 μM. In contrast, the parent mustard and alcoholic hydrolysis product of acetylcholine mustard were without effect. Our results show that measurement of the effects of ligands on the rate of inactivation of the orthosteric site by a small site-directed electrophile is a powerful method for discriminating competitive inhibition from allosterism.
Footnotes
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This work was supported by a National Institutes of Health Grant GM 69829 and a Predoctoral Fellowship from the PhRMA Foundation (to K.W.F.).
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.108.141234.
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ABBREVIATIONS: NMS, N-methylscopolamine; AChM, acetylcholine mustard; McN-A-343, 4-(trimethylamino)-2-butynyl-(3-chlorophenyl)carbamate; WIN 51708, 17-β-hydroxy-17-α-ethynyl-5-α-androstano[3,2-β]pyrimido[1,2-α]benzimidazole; KRB, Krebs-Ringer bicarbonate; CHO, Chinese hamster ovary; hM2, human M2;EC50, concentration of agonist eliciting half-maximal response.
- Received May 16, 2008.
- Accepted August 4, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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