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Papers In Press, published online ahead of print May 24, 2001
J. Biol. Chem, 10.1074/jbc.M103947200
Submitted on May 2, 2001
Revised on May 24, 2001
Accepted on May 24, 2001

Mutagenesis and derivatization of human VMAT2 cysteines identifies transporter domains involved in tetrabenazine binding and substrate transport

David S. Thiriot and Arnold E. Ruoho

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

Corresponding Author: aeruoho{at}facstaff.wisc.edu

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 (MTSEA) revealed that 3H-dihydrotetrabenazine (3H-TBZOH) binding is >90% inhibited by modification of two sets of cysteines. The primary target (responsible for approximately 80% of inhibition) is Cys 439 in TM 11. The secondary target (responsible for approximately 20% of inhibition) is one or more of the four non-TM cysteines. 3H-TBZOH protects against modification of Cys 439 by a 10,000-fold molar excess of MTSEA, demonstrating that Cys 439 is either at the tetrabenazine binding site, or conformationally linked to tetrabenazine binding. Supporting a direct effect, the position of TBZ-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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