New transport assay demonstrates vesicular acetylcholine transporter has many alternative substrates

The acetylcholine-binding site in vesicular acetylcholine transporter faces predominantly toward the outside of the vesicle when resting but predominantly toward the inside when transporting. Transport-related reorientation is detected by an ATP-induced decrease in the ability of saturating substrate to displace allosterically bound [3H]vesamicol. The assay was used here to determine whether structurally diverse compounds are transported by rat VAChT expressed in PC12A123.7 cells. Competition by ethidium, tetraphenylphosphonium and other monovalent organic cations with [3H]vesamicol is decreased when ATP is added, and the effect depends on proton-motive force. The results indicate that many organic molecules carrying +1 charge are transported, even though the compounds do not resemble acetylcholine in structural details.