Engineering the human vesicle monoamine transporter to study structure, function, and ligand binding sites

The Vesicle Monoamine Transporter (VMAT2) concentrates monoamine neurotransmitter into synaptic vesicles. Cysteine-replacement and engineered protease site mutants were constructed to obtain information on the structure, function, and ligand binding sites of human VMAT2. The human VMAT2 sequence contains ten naturally-occurring cysteines. Deriving information from the sequence of C. elegans VMAT, a subset of three cysteines were identified which restore wild-type ligand binding affinity and serotonin transport to inactive, cysteine-less VMAT2. However, replacement of only these three cysteines together results in significant enhancement of transport activity. Cysteine-modification studies involving treatment of single and combination cysteine mutants 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. 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 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. To obtain structural information, thrombin cleavage sites were strategically engineered into deglycosylated, HA-tagged human VMAT2. Insertion of these protease sites did not disrupt ligand binding or serotonin transport. These protease sites allowed identification of an intramolecular disulfide bond between the N- and C-terminal halves of the transporter. After thrombin cleavage between TM6 and TM7, in the absence of reducing agent, VMAT2 migrated on SDS-polyacrylamide gels as full-length transporter. Addition of dithiothreitol resulted in complete conversion from full-length to thrombin-cleaved size, demonstrating tethering by a DTT-reversible covalent bond. The identity of the disulfide-bound cysteine pair was indicated by the observation that replacement of Cys 126 or Cys 333 with serine both significantly reduced 3H-serotonin transport. Replacement of either Cys 126 or Cys 333 was found to eliminate the DTT-reversible intramolecular covalent bond. Therefore, human VMAT2 Cys 126 in loop 1/2 and Cys 333 in loop 7/8 form a disulfide bond which contributes to efficient monoamine transport.