Mutagenesis and derivatization of human vesicle monoamine transporter 2 (VMAT2) cysteines identifies transporter domains involved in tetrabenazine binding and substrate transport

The vesicle monoamine transporter (VMAT2) concentrates monoamine neurotrans mitter into synaptic vesicles. Photoaffinity labeling, chimera analysis, an d mutagenesis have identified functionally important amino acids and provid ed some information regarding structure and ligand binding sites. To extend these studies, we engineered functional human VMAT2 constructs with reduce d numbers of cysteines. Subsets of cysteines were discovered, which restore function to an inactive cysteine-less human VMAT2. Replacement of three tr ansmembrane (TM) cysteines together (net removal/replacement of three atoms ) significantly enhanced monoamine transport. Cysteine modification studies involving single and combination cysteine mutants with methanethiosulfonat e ethylamine revealed that [H-3]dihydrotetrabenazine binding is >90% inhibi ted by modification of two sets of cysteines, The primary target (responsib le for similar to 80% of inhibition) is Cys(439) in TM 11. The secondary ta rget (responsible for similar to 20% of inhibition) is one or more of the f our non-TM cysteines, [H-3]Dihydrotetrabenazine protects against modificati on of Cys(439) by a 10,000-fold molar excess of methanethiosulfonate ethyla mine, demonstrating that Cys(439) is either at the tetrabenazine binding si te, or conformationally linked to tetrabenazine binding. Supporting a direc t effect, the position of tetrabenazine-protectable Cys439 is consistent wi th previous mutagenesis, chimera, and photoaffinity labeling data, demonstr ating involvement of TM 10-12 in a tetrabenazine binding domain.