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J. Biol. Chem., Vol. 276, Issue 29, 27304-27315, July 20, 2001

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Mutagenesis and Derivatization of Human Vesicle Monoamine Transporter 2 (VMAT2) Cysteines Identifies Transporter Domains Involved in Tetrabenazine Binding and Substrate Transport^*

David S. Thiriot and Arnold E. Ruoho^§

From the Department of Pharmacology, University of Wisconsin-Madison Medical School, Madison, Wisconsin 53706-1532

The vesicle monoamine transporter (VMAT2) concentrates monoamine neurotransmitter into synaptic vesicles. Photoaffinity labeling, chimera analysis, and mutagenesis have identified functionally important amino acids and provided some information regarding structure and ligand binding sites. To extend these studies, we engineered functional human VMAT2 constructs with reduced numbers of cysteines. Subsets of cysteines were discovered, which restore function to an inactive cysteine-less human VMAT2. Replacement of three transmembrane (TM) cysteines together (net removal/replacement of three atoms) significantly enhanced monoamine transport. Cysteine modification studies involving single and combination cysteine mutants with methanethiosulfonate ethylamine revealed that [³H]dihydrotetrabenazine binding is >90% inhibited by modification of two sets of cysteines. The primary target (responsible for ~80% of inhibition) is Cys⁴³⁹ in TM 11. The secondary target (responsible for ~20% of inhibition) is one or more of the four non-TM cysteines. [³H]Dihydrotetrabenazine protects against modification of Cys⁴³⁹ by a 10,000-fold molar excess of methanethiosulfonate ethylamine, demonstrating that Cys⁴³⁹ is either at the tetrabenazine binding site, or conformationally linked to tetrabenazine binding. Supporting a direct effect, the position of tetrabenazine-protectable Cys 439 is consistent with previous mutagenesis, chimera, and photoaffinity labeling data, demonstrating involvement of TM 10-12 in a tetrabenazine binding domain.

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