Acetylcholine mustard labels the binding site aspartate in muscarinic acetylcholine receptors

• Published in: The Journal of biological chemistry 1994-02, Vol.269 (6), p.4092-4097
• Main Authors: SPALDING, T. A, BIRDSALL, N. J. M, CURTIS, C. A. M, HULME, E. C
• Format: Article
• Language: English
• Subjects: Acetylcholine - analogs & derivatives; Acetylcholine - chemistry; Acetylcholine - pharmacology; Alkylation; Animals; Aspartic Acid - chemistry; Binding Sites; Biological and medical sciences; Cell receptors; Cell structures and functions; Cyanogen Bromide - chemistry; Disulfides - chemistry; Enzyme Activation; Fundamental and applied biological sciences. Psychology; GTP Phosphohydrolases - metabolism; Humans; Molecular and cellular biology; Monoamines receptors (catecholamine, serotonine, histamine, acetylcholine); Prosencephalon - chemistry; Rats; Receptors, Muscarinic - chemistry; Recombinant Proteins
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)41747-9

Acetylcholine mustard (AChM) is an analogue of acetylcholine (ACh) in which the onium headgroup is replaced by a chemically reactive aziridinium moiety. AChM aziridinium has agonist activity, but, having bound, reacts with and blocks the muscarinic receptor (mAChR) binding site. Purified mAChRs from rat forebrain have been specifically labeled with [3H]AChM. The linkage formed is cleaved by hydroxylamine, is found within cyanogen bromide (CNBr) peptides with molecular masses of approximately 2.4 and 3.9 kDa, and is close to a disulfide-bonded cysteine. Edman degradation reveals a site of label attachment 26 residues C-terminal to a CNBr cleavage site. As in the case of the alkylating antagonist analogue [3H]propylbenzilylcholine mustard, these findings indicate that a conserved aspartic acid residue in transmembrane helix 3 of the mAChRs, corresponding to Asp-105 (m1 sequence), is the site of label attachment.