Binding of .alpha.-bungarotoxin to synthetic peptides corresponding to residues 173-204 of the .alpha. subunit of Torpedo, calf, and human acetylcholine receptor and restoration of high-affinity binding by sodium dodecyl sulfate

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Abstract
In order to investigate structure-function relationships of a segment of the acetylcholine receptor .alpha. subunit, binding of .alpha.-bungarotoxin to synthetic peptides corresonding to residues 173-204 of Torpedo, calf, and human .alpha. subunits was compared using a solid-phase radioassay. The affinities of 125I-.alpha.-bungarotoxin for the calf and human peptides were 15- and 150-fold less, respectively, than for the Torpedo peptide. On the basis of nonconservative substitutions in the calf and human sequences, aromatic residues (Tyr-181, Trp-187, and Tyr-189) are important for the higher affinity binding of the Torpedo peptide. Substitution of negatively charged Glu-180 with uncharged Gln in the calf peptide did not significantly affect toxin binding, indicating Glu-180 alone does not comprise the anionic subsite on the receptor to which the cationic quaternary ammonium groups of cholinergic agents bind. d-Tubocurarine competed toxin binding to the modified calf 32-mer which lacks Glu-180 and Asp-195 present in Torpedo. Thus, the negative subsite could be formed by another negatively charged residue or by more than one amino acid side chain. It is possible that the positive charges on cholinergic ligands are countered by a negative electrostatic potential provided by polar groups, such as the hydroxyl group of tyrosine, present on several residues in this region, and the negative charges present on any of residues 175, 180, 195, or 200. Equilibrium saturation binding of .alpha.-bungarotoxin to Torpedo peptide 173-204 revealed a minor binding component with an apparent KD of 4.2 nM and a major component with a KD of 63 nM. In the presence of 0.01% sodium dodecyl sulfate, one binding component with a KD of 7.8 nM was detected. This compares with an affinity of KD = 0.41 nM for toxin binding to native acetylcholine receptor in the solid-phase assay. Sodium dodecyl sulfate may stabilize a conformation of the peptide that is conducive to high-affinity binding.

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