Covalent modification of a critical sulfhydryl group in the acetylcholine receptor: cysteine-222 of the .alpha.-subunit

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Abstract
Chemical modification of the Torpedo californica acetylcholine receptor (AcChR) by the fluorescent agent N-(1-pyrenyl)maleimide (PM) under nonreducing conditions resulted in the labeling of cysteine residues in all subunits and marked inhibition of the AcChR ion channel opening [Clarke, J. H., and Martinez-Carrion, M. (1986) J. Biol. Chem. 261, 10063-10072]. The PM alkylation kinetics are not affected by the presence of agonists or a competitive antagonist. The PM-labeled .alpha.-subunit has been purified and digested with both CNBr and trypsin. The resulting fragments from both cleavages were fractionated by high-performance liquid chromatography. The amino acid analysis and sequencing data of the PM-labeled peptides identified cysteine-222 as the only residue labeled by PM on the .alpha.-subunit primary structure. Cysteine-222 is located in the middle of a hydrophobic domain designated M1, which contains a homologous class of cysteines (Cys-241 in the aligned sequences) conserved in the four subunits of the AcChR. Because of its reactivity and fluorescent properties of the bound probe, .alpha.Cys-222 seems to be a free sulfhydryl group accessible through a hydrophobic pocket, and these properties should be incorporated into proposed folding models for the .alpha.-subunit.