Mustard analogs of acetylcholine: irreversible partial agonists at Gq‐linked muscarinic receptors

Abstract

Mustard analogs of many receptor ligands have been used to irreversibly label or inactivate receptors. In aqueous solution, mustard groups cyclize to form aziridinium ions that closely mimic quaternary amines. Such a reactive intermediate may therefore bind in the same orientation within the same binding pocket of the receptor that accommodates the related reversible ligand. Once oriented, it is capable of alkylating any of a number of nearby amino acid side chains (including those of cysteine, glutamate, aspartate, and others) and might, in principle, induce irreversible activation of the receptor. However, acetylcholine mustard has been reported by several laboratories to possess agonist activity at muscarinic receptors only until alkylation is achieved, at which point the receptor becomes inactivated. Therefore, we were surprised to find that agonist activity persisted after irreversible binding of acetylcholine mustard and its analog, acetylethylcholine mustard, at Gq‐linked muscarinic receptors (M1, M3, and M5) expressed in CHO cells. The cells were preincubated with cyclized agonist mustards and thoroughly washed prior to assay; the observed responses could be prevented by including atropine in the preincubation phase, but were not inhibited by the presence of atropine in the assay phase. The metabolism of inositol phosphates (IP) was enhanced robustly, though to a lesser degree than that induced by acetylcholine. Acetylethylcholine mustard was less efficacious than acetylcholine mustard, especially at the M5 subtype. For both mustards, the order of efficacy was M1>M3>M5. The most interesting results were obtained measuring the receptor‐stimulated release of labeled arachidonic acid (AA) from intact cells. After preincubation as above, the basal AA release was found to be elevated to a small but significant degree; we have previously shown amiodarone to be a positive allosteric modulator (PAM) at these receptors and inclusion of amiodarone in the assay phase dramatically increased the response of cells that had been preincubated with the agonist mustards. Once again, atropine in the assay itself did not prevent this effect of amiodarone, but atropine in the preincubation phase prevented it completely. At the M1 subtype, the well‐known M1‐specific PAM BQCA enhanced the mustard‐induced activity in a manner similar to that observed for amiodarone. These studies confirm that amiodarone, BQCA, and other ligands investigated act at an allosteric site and suggest that receptors that have been alkylated by acetylcholine mustard and acetylethylcholine mustard do adopt active conformations that can be further enhanced by appropriate allosteric ligands.Support or Funding InformationSupported AG005214 to JEThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.