Structural Determinants of α-Bungarotoxin Binding to the Sequence Segment 181–200 of the Muscle Nicotinic Acetylcholine Receptor α Subunit: Effects of Cysteine/Cystine Modification and Species-Specific Amino Acid Substitutions

The sequence segment 181–200 of the Torpedo nicotinic acetylcholine receptor (nAChR) α subunit forms a binding site for α-bungarotoxin (α-BTX) [e.g., see Conti-Tronconi, B. M., Tang, F., Diethelm, B. M., Spencer, S. R., Reinhardt-Maelicke, S., & Maelicke, A. (1990) Biochemistry 29, 6221–6230]. Synthetic peptides corresponding to the homologous sequences of human, calf, mouse, chicken, frog, and cobra muscle nAChR αl subunits were tested for their ability to bind ¹²⁵I-α-BTX, and differences in α-BTX affinity were determined by using solution (IC₅₀S) and solid-phase (K_ds) assays. Panels of overlapping peptides corresponding to the complete αl subunit of mouse and human were also tested for α-BTX binding, but other sequence segments forming the α-BTX site were not consistently detectable. The Torpedo αl(181–200) and the homologous frog and chicken peptides bound α-BTX with higher affinity (K_ds ~ 1–2 μM, IC₅₀s ~ 1–2 μM) than the human and calf peptides (K_ds ~ 3–5 μM, IC₅₀s ~ 15 μM). The mouse peptide bound α-BTX weakly when attached to a solid support (K_d ~ 8 μM) but was effective in competing for ¹²⁵I-α-BTX in solution (IC₅₀ ~ 1 μM). The cobra nAChR αl-subunit peptide did not detectably bind α-BTX in either assay. Amino acid substitutions were correlated with α-BTX binding activity of peptides from different species. The role of a putative vicinal disulfide bound between Cys-192 and −193, relative to the Torpedo sequence, was determined by modifying the peptides with sulfhydryl reagents. Reduction and alkylation of the peptides decreased α-BTX binding, whereas oxidation of the peptides had little effect. Modifications of the cysteine/cystine residues of the cobra peptide failed to induce α-BTX binding activity. These results indicate that while the adjacent cysteines are likely to be involved in forming the toxin/α1 -subunit interface a vicinal disulfide bound was not required for α-BTX binding.