Expression and function of the rat vesicular monoamine transporter 2
ABSTRACT The vesicular monoamine transporters (VMATs) are essential proteins, involved in the storage of monoamines in the central nervous system and in endocrine cells, in a process that involves exchange of 2H(+) with one substrate molecule. The VMATs interact with various native substrates and clinically relevant drugs and display the pharmacological profile of multidrug transporters. Vesicular transporters suffer from a lack of biochemical and structural data due to the difficulties in their expression. In this work we present the high-level expression of rat VMAT2 (rVMAT2) in a stable a human embryonic kidney cell line (HEK293), generated using the resistance to the neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) conferred by the protein. In addition, we describe novel procedures for the solubilization and purification of active protein, and its reconstitution into proteoliposomes. The partially purified protein in detergent binds the inhibitor tetrabenazine and, after reconstitution, displays high levels of Deltamu(H+)-driven electrogenic transport of serotonin. The reconstituted purified rVMAT2 has wild-type affinity for serotonin, and its turnover rate is approximately 0.4 substrate molecule/s.
SourceAvailable from: Mariafrancesca Scalise[Show abstract] [Hide abstract]
ABSTRACT: Proteoliposomes represent a suitable and up to date tool for studying membrane transporters which physiologically mediate absorption, excretion, trafficking and reabsorption of nutrients and metabolites. Using recently developed reconstitution strategies, transporters can be inserted in artificial bilayers with the same orientation as in the cell membranes and in the absence of other interfering molecular systems. These methodologies are very suitable for studying kinetic parameters and molecular mechanisms. After the first applications on mitochondrial transporters, in the last decade, proteoliposomes obtained with optimized methodologies have been used for studying plasma membrane transporters and defining their functional and kinetic properties and structure/function relationships. A lot of information has been obtained which has clarified and completed the knowledge on several transporters among which the OCTN sub-family members, transporters for neutral amino acid, B0AT1 and ASCT2, and others. Transporters can mediate absorption of substrate-like derivatives or drugs, improving their bioavailability or can interact with these compounds or other xenobiotics, leading to side/toxic effects. Therefore, proteoliposomes have recently been used for studying the interaction of some plasma membrane and mitochondrial transporters with toxic compounds, such as mercurials, H2O2 and some drugs. Several mechanisms have been defined and in some cases the amino acid residues responsible for the interaction have been identified. The data obtained indicate proteoliposomes as a novel and potentially important tool in drug discovery.Pharmaceutics 09/2013; 5(3):472-497. DOI:10.3390/pharmaceutics5030472
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ABSTRACT: Vesicular monoamine transporter 2 (VMAT2) transports monoamines into storage vesicles in a process that involves exchange of the charged monoamine with two protons. VMAT2 is a member of the DHA12 family of multidrug transporters that belongs to the major facilitator superfamily (MFS) of secondary transporters. Tetrabenazine (TBZ) is a non-competitive inhibitor of VMAT2 that is used in the treatment of hyperkinetic disorders associated with Huntington's disease and Tourette's syndrome. Previous biochemical studies suggested that the recognition site for TBZ and monoamines is different. However, the precise mechanism of TBZ interaction with VMAT2 remains unknown. Here we used a random mutagenesis approach and selected TBZ-resistant mutants. The mutations clustered around the lumenal opening of the transporter and mapped to either conserved proline or glycine, or to residues immediately adjacent to conserved proline and glycine. Directed mutagenesis provides further support for the essential role of the latter residues. Our data strongly suggest that the conserved α-helix breaking residues identified in this work play an important role in conformational rearrangements required for TBZ binding and substrate transport. Our results provide a novel insight into the mechanism of transport and TBZ binding by VMAT2.Journal of Biological Chemistry 09/2013; DOI:10.1074/jbc.M113.502971 · 4.60 Impact Factor
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ABSTRACT: Vesicular monoamine transporter 2 (VMAT2) is an essential component of the monoaminergic neurotransmission system in the brain as it transports monoamine neurotransmitters from the neuronal cytosol into the synaptic vesicles and thus contributes to modulation of neurotransmitter release. Considering the continuing interest in VMAT2 as a drug target, as well as a target for the design of imaging probes, we have developed a fluorescent substrate well suited for the study of VMAT2 in cell culture. Herein, we report the synthesis and characterization of a new fluorescent probe, FFN206, as an excellent VMAT2 substrate capable of detecting VMAT2 activity in intact cells using fluorescence microscopy, with subcellular localization to VMAT2-expressing acidic compartments without apparent labeling of other organelles. VMAT2 activity can also be measured via microplate reader. The apparent Km of FFN206 at VMAT2 was found to be 1.16 ± 0.10 μM, similar to that of dopamine. We further report the development and validation of a cell-based fluorescence assay amenable to high-throughput screening (HTS) using VMAT2-transfected HEK cells (Z'-factor of 0.7-0.8), enabling rapid identification of VMAT2 inhibitors and measurement of their inhibition constants over a broad range of affinities. FFN206 thus represents a new tool for optical examination of VMAT2 function in cell culture.ACS Chemical Biology 07/2013; 8(9). DOI:10.1021/cb400259n · 5.36 Impact Factor