Analysis of ligand binding to the synthetic dodecapeptide 185-196 of the acetylcholine receptor alpha subunit.

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Abstract
A synthetic dodecapeptide corresponding to residues 185-196 of the Torpedo acetylcholine receptor .alpha. subunit, which contains the adjacent cysteine residues at positions 192 and 193, was recently shown by us to contain the essential elements for .alpha.-bungarotoxin binding. In the present study, we have used Sepharose-linked peptides for quantitative analysis of the cholinergic binding properties of this and other synthetic peptides. Sepharose-linked peptides corresponding to residues 1-20, 126-143, 143-158, 169-181, 185-196, 193-210, and 394-409 of the .alpha. subunit of Torpedo acetylcholine receptor, as well as a peptide corresponding to residues 185-196 of the .alpha. subunit of human acetylcholine receptor, were tested for their toxin-binding capacity. Of these immobilized peptides, only peptide 185-196 of the Torpedo acetylcholine receptor bound toxin significantly, thus verifying that this synthetic peptide contains essential components of the receptor toxin-binding site. Analysis of toxin binding to the peptide yielded a dissociation constant of 3.5 .times. 10-5 M. This binding was inhibited by various cholinergic ligands. The inhibition potency obtained was .alpha.-bungarotoxin > Naja naja siamensis toxin > d-tubocurarine > decamethonium > acetylcholine > carbamoylcholine. This pharmaceutical profile resembles that of the nicotinic acetylcholine receptor and therefore suggests that the synthetic dodecapeptide also includes the neurotransmitter binding site. Reduction and carboxymethylation of the cysteine residues on peptide 185-196 inhibit its capacity to bind toxin, demonstrating that an intact disulfide is required for toxin binding. A decrease in toxin binding was also obtained following chemical modification of the tryptophan residue at position 187, thus implying its possible involvement in toxin binding. The failure to detect binding of toxin to the corresponding human sequence 185-196, in which the tryptophan residue is replaced by serine, supports this hypothesis.