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National Institute of Environmental Health Sciences

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Publication Detail

Title: A fluorescent-based assay for live cell, spatially resolved assessment of vesicular monoamine transporter 2-mediated neurotransmitter transport.

Authors: Bernstein, Alison I; Stout, Kristen A; Miller, Gary W

Published In J Neurosci Methods, (2012 Aug 15)

Abstract: The vesicular monoamine transporter 2 (VMAT2; Slc18a2) packages monoamines into synaptic vesicles. Monoamine homeostasis is highly regulated and dysfunction may play a role in Parkinson's disease, Huntington's disease, drug addiction, and neuropsychiatric disorders. The primary function of VMAT2 is to sequester monoamine neurotransmitters into vesicles for subsequent release; it also sequesters toxicants away from cytosolic sites of action. Identification of compounds that modify the action of VMAT2 may be useful as therapeutic agents for preventing or reversing monoamine-related toxicity. Current methods for measuring VMAT2 function are unable to assess uptake in intact cells. Here, we adapted the Neurotransmitter Uptake Assay (Molecular Devices) to develop a measure of VMAT2 function in live whole cells. This assay contains a fluorescent compound, which is transported into cells by the plasma membrane monoamine transporters and has been marketed as a rapid, high-throughput, plate reader based assay for function of these plasma membrane transporters. We demonstrate a modified version of this assay that can be used to visualize and measure transport into vesicles by VMAT2. HEK293 cell lines stably expressing the dopamine transporter and a mCherry-VMAT2 fusion protein were generated. Confocal microscopy confirmed that the fluorescent compound is transported into mCherry-positive compartments. Furthermore, the VMAT2-specific inhibitor tetrabenazine (TBZ) blocks uptake into the mCherry-positive compartment. Confocal images can be analyzed to generate a measure of VMAT2 activity. In summary, we demonstrate a method for spatially resolved analysis of VMAT2-mediated uptake in live intact cells.

PubMed ID: 22698664 Exiting the NIEHS site

MeSH Terms: Adrenergic Uptake Inhibitors/pharmacology; Dopamine Plasma Membrane Transport Proteins/metabolism; Dopamine/metabolism*; Dose-Response Relationship, Drug; Gene Expression Regulation/drug effects; HEK293 Cells; Humans; Luminescent Proteins/genetics; Luminescent Proteins/metabolism; Piperazines/pharmacology; Protein Transport/physiology; Tetrabenazine/pharmacology; Transfection; Tritium/metabolism; Vesicular Monoamine Transport Proteins/genetics; Vesicular Monoamine Transport Proteins/metabolism*

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