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

Abstract

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 the effects on [3H]vesamicol binding determined. The vesamicol dissociation constant increased 1,000-fold after reaction with p-chloromercuriphenylsulfonate or phenylmercury acetate, but only severalfold after reaction with relatively small methylmercury chloride or methylmethanethiosulfonate (MMTS). Methylmercury chloride, but not MMTS, protected binding from phenylmercury acetate. Thus, two classes of cysteines react to affect vesamicol binding. 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 defining second-order reaction conditions. The results indicate that each cysteinyl class probably contains a single residue. Acetylcholine protects cysteine 1, but apparently does not protect cysteine 2. Vesamicol, which binds to a different site than acetylcholine does, apparently protects both cysteines, suggesting that it induces a conformational change. The relatively large reagent glutathione removes a substituent from cysteine 1, but not cysteine 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.