Cysteine mutagenesis of the human vesicle monoamine transporter identifies ligand binding sites and a disulfide bond

Thiriot, D.S.; Sievert, M.K.; Ruoho, A.E.

FASEB Journal 17(4-5): Abstract 645

2003


ISSN/ISBN: 0892-6638
Accession: 034673371

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Abstract
Cysteine mutants of VMAT2 were constructed to obtain information on the structure, function, and ligand binding sites of human VMAT2. A subset of three cysteines were identified which restore wild-type ligand binding affinity and serotonin transport to inactive, cysteine-less VMAT2. Cysteine-modification with methanethiosulfonate ethylamine demonstrated that [3H]dihydrotetrabenazine ([3H]TBZOH) binding is >90% inhibited by reaction at two "classes" of cysteines. The primary target (responsible for approximately 80% of inhibition) is Cys 439 in transmembrane (TM) 11, demonstrating that Cys 439 is either at or conformationally linked to the tetrabenazine binding site. The involvement of this region of VMAT2 (TM 10-12) in tetrabenazine binding is consistent with previous data. Thrombin cleavage sites allowed identification of an intramolecular disulfide bond between the N-and C-terminal halves of VMAT2. Cleavage of VMAT2 between TM6 and TM7, in the absence of reducing agent, resulted in VMAT2 migrating as "full-length" transporter on SDS-PAGE. Addition of dithiothreitol resulted in complete conversion from full-length to thrombin-cleaved size, demonstrating a tethering of the two halves by a DTT-reversible covalent bond. Replacement of either Cys 126 or Cys 333 with serine eliminated the DTT-reversible intramolecular covalent bond.