Distribution of alpha-bungarotoxin binding sites over residues 173-204 of the alpha subunit of the acetylcholine receptor.

The binding of alpha-bungarotoxin to several synthetic peptides comprising different segments of the region 173-204 of the alpha subunit of the Torpedo acetylcholine receptor was investigated to further localize the neurotoxin-binding site on the primary sequence. When tested in a solid phase microwell assay system, a 32-amino acid peptide corresponding to residues 173-204 (32-mer) bound 125I-alpha-bungarotoxin with the same affinity (4.2 x 10(-8) M as determined from IC50 values) as the isolated alpha subunit (4.6 x 10(-8) M). The relative affinities of other antagonists (alpha-cobratoxin, d-tubocurarine) maintained the same rank order in this assay system as has been demonstrated with the intact receptor. Agonists competed with binding of toxin at millimolar concentrations but lost all rank order of potency. These findings demonstrate that peptide 173-204 contains many of the antagonist-binding determinants present on denatured alpha subunit but has lost specificity of agonist binding. To further localize the toxin-binding site, alpha-bungarotoxin binding to seven shorter peptides corresponding to portions of the 32-mer was investigated. 125I-alpha-Bungarotoxin bound to alpha subunit peptides 179-192, 181-198, 185-196, 186-196, and 193-204, but not to alpha subunit peptides 173-180 and 194-204. In a second assay, all of the peptides competed with binding of 125I-acetylcholine receptor to immobilized alpha-bungarotoxin. The apparent affinity was highest for the 173-204 32-mer (1.4 x 10(-7) M) and lowest for peptides 173-180 and 194-204 (greater than 10(-4) M). The affinity of the other peptides was intermediate (approximately 10(-5) M) and about 100-fold less than that of the 32-mer. The affinity of alpha-bungarotoxin was 3.5 x 10(-10) M, of isolated, native acetylcholine receptor, 3.2 x 10(-9) M, and of isolated denatured subunit, 1.2 x 10(-8) M, with this assay. The retention of some toxin-binding capacity by the shorter peptides indicates toxin-binding determinants are distributed over the entire length of the 32-mer. The determinants with higher affinity are located in the central region of the 32-mer between residues 179 and 196.