Transmembrane Reorientation of the Substrate-Binding Site in Vesicular Acetylcholine Transporter

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Abstract
Active transport of acetylcholine (ACh) by vesicular ACh transporter (VAChT) is driven by a proton-motive force established by V-ATPase. A published microscopic kinetics model predicts the ACh-binding site is primarily oriented toward the outside for nontransporting VAChT and toward the inside for transporting VAChT. The allosteric ligand [3H]vesamicol cannot bind when the ACh-binding site is outwardly oriented and occupied by ACh, but it can bind when the ACh site is inwardly oriented. The kinetics model was tested in the paper reported here using rat VAChT expressed in PC12A1237 cells. Equilibrium titrations of [3H]vesamicol binding and ACh competition show that ATP blocks competition between vesamicol and ACh in over one-half of the VAChT. NaCl did not mimic ACh chloride, and bafilomycin A1 and FCCP completely blocked the ATP effect, which shows that it is mediated by a proton-motive force. The data are consistent with reorientation of over one-half of the ACh-binding sites from the outside to the inside of vesicles upon activation of transport. The observations support the proposed microscopic kinetics model, and they should be useful in characterizing effects of mutations on the VAChT transport cycle.

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