Identification of the alpha subunit half-cystine specifically labeled by an affinity reagent for the acetylcholine...

Nicotinic acetylcholine receptors contain a readily reducible disulfide bond at the periphery of the acetylcholine binding site. Following reduction of this disulfide, the binding site is susceptible to affinity labeling by electrophilic reagents with quaternary ammonium moieties. We reduced purified receptor from Torpedo californica electric tissue and affinity alkylated it with 4-(N-maleimido)benzyltri[3H]methylammonium iodide. The label was incorporated solely into the alpha subunit of the receptor. Isolated, labeled alpha subunit was cleaved with CNBr, and the fragments were separated by reverse-phase high-performance liquid chromatography. A uniquely labeled CNBr fragment was isolated, and its partial sequence was determined by automated Edman degradation. This CNBr fragment was cleaved at tryptophan residues, the subfragments were separated, and the labeled subfragments were partially sequenced. From our protein sequence information, we identify the labeled CNBr fragment as residues 179 to 207 of the sequence of alpha predicted from the cDNA sequence (Noda, M., Takahashi, H., Tanabe, T., Toyosato, M., Furutani, Y., Hirose, T., Asai, M., Inayama, S., Miyata, T., and Numa, S. (1982) Nature (Lond.) 299, 793-797). From the cycle of the Edman degradation in which radioactive residues are released, we conclude that Cys 192 and, possibly in addition, Cys 193 are the residues specifically labeled by 4-(N-maleimido)benzyltri[3H]methylammonium iodide. They are, therefore, close to the acetylcholine binding site.