Modification of cysteines reveals linkage to acetylcholine and vesamicol binding sites in the vesicular acetylcholine transporter of Torpedo californica

Properties of cysteinyl residues in the vesicular acetylcholine transporter (VAChT) of synaptic vesicles isolated from Torpedo californica were probed . Cysteine-specific reagents of different size and polarity were used and t he effects on [H-3]vesamicol binding determined. The vesamicol dissociation constant increased 1,000-fold after reaction with p-chloromercuriphenylsul fonate or phenylmercury acetate, but only severalfold after reaction with r elatively small methylmercury chloride or methyl-methanethiosulfonate (MMTS ). Methylmercury chloride, but not MMTS, protected binding from phenylmercu ry acetate. Thus, two classes of cysteines react to affect vesamicol bindin g. Class 1 reacts with only organomercurials, and class 2 reacts with both organomercurials and MMTS. Quantitative analysis of the competition between p-chloromercuriphenylsulfonate and VAChT ligands was possible after defini ng second-order reaction conditions. The results indicate that each cystein yl class probably contains a single residue. Acetylcholine protects cystein e 1, but apparently does not protect cysteine 2. Vesamicol, which binds to a different site than acetyl-choline does, apparently protects both cystein es, suggesting that it induces a conformational change. The relatively larg e reagent glutathione removes a substituent from cysteine 1, but not cystei ne 2, suggesting that cysteine 2 is deeper in the transporter than cysteine 1 is. The complete sequence of T. californica VAChT is given, and possible identities of cysteines 1 and 2 are discussed.