Localization and synthesis of the acetylcholine-binding site in the α-chain of the Torpedo californica acetylcholine receptor

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Abstract
The sequence of the .alpha.-chain of the acetylcholine receptor of T. californica was determined by recent cloning studies. The integrity of the disulfide bond between Cys-128 and Cys-142 is important for the maintenance of the binding activity of the receptor, thus implicating the regions around the disulfide bridge in binding with acetylcholine. A synthetic peptide containing this loop region (residues 125-147) was synthesized. Solid-phase radiometric binding assays demonstrated a high binding of 125I-labeled .alpha.-bungarotoxin to the synthetic peptide. The free peptide bound well to [3H]acetylcholine. Additional experiments demonstrated that pretreatment of peptide 125-147 with 2-mercaptoethanol destroyed its binding activity, clearly showing that the integrity of the disulfide structure was essential for binding. Unlabeled acetylcholine also inhibited the binding of labeled acetylcholine to the synthetic peptide. The region 125-147, therefore, contains essential elements of the acetylcholine binding site of the Tropedo receptor.