A fluorescent-based assay for live cell, spatially resolved assessment of vesicular monoamine transporter 2-mediated neurotransmitter transport.
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.
Center for Neurodegenerative Disease, Emory University, Atlanta, GA 30322, United States; Department of Environmental Health, Rollins School of Public Health, Emory University, Atlanta, GA 30322, United States.
Alison I
AI
Bernstein
Kristen A
KA
Stout
Gary W
GW
Miller
D018759:Adrenergic Uptake Inhibitors / Q000494:pharmacology; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D010879:Piperazines / Q000494:pharmacology; D021381:Protein Transport / Q000502:physiology; D013747:Tetrabenazine / Q000494:pharmacology; D014162:Transfection; D014316:Tritium / Q000378:metabolism; D050493:Vesicular Monoamine Transport Proteins / Q000235:genetics / Q000378:metabolism*; D000097573:Red Fluorescent Protein
D018759:Adrenergic Uptake Inhibitors; D004298:Dopamine; D050483:Dopamine Plasma Membrane Transport Proteins; D008164:Luminescent Proteins; D010879:Piperazines; D013747:Tetrabenazine; D014316:Tritium; D050493:Vesicular Monoamine Transport Proteins; D000097573:Red Fluorescent Protein; C461261:(1-(2-(bis(4-fluorophenyl)methoxy)ethyl)-4-(3-hydroxy-3-phenylpropyl) piperazinyl decanoate)