In Vitro Assays for the Functional Characterization of the Dopamine Transporter (DAT)

Shaili Aggarwal1, Ole V. Mortensen1

1 Department of Pharmacology and Physiology, Drexel University College of Medicine, Philadelphia
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 12.17
DOI:  10.1002/cpph.33
Online Posting Date:  December, 2017
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Abstract

Detailed in this unit are protocols for studying the in vitro uptake of dopamine (DA) as a means for defining the functional characteristics of dopamine transporters. All assays are performed using commercially available cell lines that transiently express the transporter under investigation. The three main assays provided are: a kinetic assay to calculate the affinity (KM) and maximal velocity (Vmax) of radiolabeled DA uptake into cells; concentration‚Äźresponse assays to measure the potencies (IC50/Ki values) of test compounds as transport inhibitors; and an efflux assay to assess the ability and potency (EC50) of a ligand to elicit reverse transport of DA accumulated in the cell. Although the methods are described using DAT and its ligands, the same procedure can be employed for studying serotonin and norepinephrine transporters as well. ¬© 2017 by John Wiley & Sons, Inc.

Keywords: dopamine transporter; monoamine; neurotransmitter; uptake

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Kinetic Uptake Assay (96‐Well Format) to Determine KM and Vmax of DAT‐Mediated DA Transport
  • Basic Protocol 2: Dose‐Response Assay (96‐Well Format) to Determine IC50 Values and Apparent Ki of Ligands at DAT
  • Basic Protocol 3: Release/Efflux Assays (24‐Well Format) to Determine the Ability of Ligands to Modulate DAT‐Mediated Efflux of Dopamine
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Kinetic Uptake Assay (96‐Well Format) to Determine KM and Vmax of DAT‐Mediated DA Transport

  Materials
  • COS‐7 cell lines (ATCC) growing in culture
  • 10× Dulbecco's phosphate‐buffered saline (DPBS; Corning; cat. no. 20031CV)
  • Trypsin 0.05%/0.53 mM EDTA for COS‐7 cells (Corning, cat. no. 25052CI)
  • Complete DMEM culture medium (see recipe)
  • pcDNA3.1‐hDAT plasmid (Addgene, cat. no. 32810; http://www.addgene.org)
  • OptiMEM serum‐free medium (Thermo Fisher, cat. no. 31985062; store up to 18 months at 4°C)
  • TransIT®‐LT1 transfection reagent (Mirus Bio LLC; store up to 12 months at 4°C)
  • PBS‐CM (see recipe)
  • Assay buffer (see recipe)
  • Test compounds
  • Dopamine (DA) dilutions (see recipe)
  • Scintillation fluid (e.g., Ecolite (+); MP Biomedicals, Inc.)
  • 96‐well plate incubation system (e.g., Falcon™ Tissue Culture‐Treated 96‐well Microplates; Thermo Fisher Scientific)
  • Plate washer (e.g., Biotek ELx50) for 96‐well plates
  • Inverted microscope
  • Sealing tape for 96‐well plates (VWR, cat. no. 60941‐070)
  • Microplate Scintillation and Luminescence Counter (Perkin Elmer)
  • GraphPad Prism for Windows
  • Microsoft Excel
  • Additional reagents and equipment for basic cell culture techniques including counting cells (unit 12.1; Phelan & May, )

Basic Protocol 2: Dose‐Response Assay (96‐Well Format) to Determine IC50 Values and Apparent Ki of Ligands at DAT

  Additional Materials (also see protocol 1)
  • Dilutions of a test compound (see recipe) to assess whether it inhibits DA transport and, if so, to determine its IC 50
  • Radioactive DA: Prepare a radioactive tritiated DA or [3H]‐DA concentration of 50 nM in the assay buffer at a concentration lower than its anticipated K M value. Prepare fresh and use within 2 hr
  • Cocaine (Sigma‐Aldrich), a known, competitive DAT inhibitor used as a standard DAT inhibitor

Basic Protocol 3: Release/Efflux Assays (24‐Well Format) to Determine the Ability of Ligands to Modulate DAT‐Mediated Efflux of Dopamine

  Additional Materials (also see protocol 1)
  • DA releasers/substrates such as DA, amphetamine, methamphetamine, etc., and/or blockers
  • 1% SDS/0.1N NaOH lysis buffer in deionized water, pH = 7.4
  • 24‐well plates, (e.g., Thermo Scientific Biolite #930186)
  • Scintillation vials (e.g., Simport™ Scientific HDPE Snaptwist™ Scintillation Vial from FisherBrand #03‐342‐3)
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Figures

Videos

Literature Cited

  Brouwer, K. L., Keppler, D., Hoffmaster, K. A., Bow, D. A., Cheng, Y., Lai, Y., … International Transporter Consortium. (2013). In vitro methods to support transporter evaluation in drug discovery and development. Clinical Pharmacology and Therapeutics, 94(1), 95–112. doi: 10.1038/clpt.2013.81.
  Cheng, Y.‐C., & Prusoff, W. H. (1973). Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochemical Pharmacology, 22(23), 3099–3108. doi: 10.1016/0006‐2952(73)90196‐2.
  Coleman, J. A., Green, E. M., & Gouaux, E. (2016). X‐ray structures and mechanism of the human serotonin transporter. Nature, 532(7599), 334–339. doi: 10.1038/nature17629 doi: 10.1038/nature17629.
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  Howell, L. L., & Negus, S. S. (2014). Monoamine transporter inhibitors and substrates as treatments for stimulant abuse. Advances in Pharmacology, 69, 129–176. doi: 10.1016/B978‐0‐12‐420118‐7.00004‐4.
  Johnson, K. A., & Goody, R. S. (2011). The original Michaelis constant: Translation of the 1913 Michaelis‐Menten paper. Biochemistry, 50(39), 8264–8269. doi: 10.1021/bi201284u.
  Koldo, H., Grouleff, J., & Schiott, B. (2015). Insights to ligand binding to the monoamine transporters—from homology modeling to LeuBAT and dDAT. Frontiers in Pharmacology, 6(208), 1–8. doi: 10.3389/fphar.2015.00208.
  Kristensen, A. S., Andersen, J., Jorgensen, T. N., Sorensen, L., Eriksen, J., Loland, C. J., … Gether, U. (2011). SLC6 neurotransmitter transporters: Structure, function, and regulation. Pharmacological Reviews, 63(3), 585–640. doi: 10.1124/pr.108.000869.
  Lin, Z., Canales, J. J., Bjorgvinsson, T., Thomsen, M., Qu, H., Liu, Q. R., … Caine, S. B. (2011). Monoamine transporters: Vulnerable and vital doorkeepers. Progress in Molecular Biology and Translational Science, 98, 1–46. doi: 10.1016/B978‐0‐12‐385506‐0.00001‐6.
  Lundholt, B. K., Scudder, K. M., & Pagliaro, S. (2003). A simple technique for reducing edge effects in cell‐based assays. Journal of Biomolecular Screening, 8(5), 566–570. doi: 10.1177/1087057103256465.
  Mortensen, O. V., & Kortagere, S. (2015). Designing modulators of monoamine transporters using virtual screening techniques. Frontiers in Pharmacology, 6(223), 1–8. doi: 10.3389/fphar.2015.00223.
  Partilla, J. S., Baumann, M. H., Decker, A. M., Blough, B. E., & Rothman, R. B. (2016). Interrogating the activity of ligands at monoamine transporters in rat brain synaptosomes. In H. Bönisch & H. H. Sitte (Eds.), Neurotransmitter Transporters: Investigative Methods (pp. 41–52). New York, NY: Springer.
  Phelan, K., and May, K. M. (2016). Mammalian cell tissue culture techniques. Current Protocols in Pharmacology, 73, 12.1.1–12.1.23. doi: 10.1002/cpph.1.
  Ramamoorthy, S., Shippenberg, T. S., & Jayanthi, L. D. (2011). Regulation of monoamine transporters: Role of transporter phosphorylation. Pharmacology & Therapeutics, 129(2), 220–238. doi: 10.1016/j.pharmthera.2010.09.009.
  Rives, M. L., Javitch, J. A., & Wickenden, A. D. (2017). Potentiating SLC transporter activity: Emerging drug discovery opportunities. Biochemical Pharmacology, 135, 1–11. doi: 10.1016/j.bcp.2017.02.010.
  Rothman, R. B., Ananthan, S., Partilla, J. S., Saini, S. K., Moukha‐Chafiq, O., Pathak, V., & Baumann, M. H. (2015). Studies of the biogenic amine transporters 15. Identification of novel allosteric dopamine transporter ligands with nanomolar potency. The Journal of Pharmacology and Experimental Therapeutics, 353, 529–538. doi: 10.1124/jpet.114.222299.
  Steinkellner, T., Mayer, F. P., Hofmaier, T., Holy, M., Montgomery, T., Eisenrauch, B., … Sitte, H. H. (2016). Tracer flux measurements to study outward transport by monoamine neurotransmitter transporters. In H. Bönisch & H. H. Sitte (Eds.), Neurotransmitter Transporters: Investigative Methods (pp. 23–40). New York, NY: Springer.
  Sucic, S., & Bönisch, H. (2016). Classical radioligand uptake and binding methods in transporter research: An emphasis on the monoamine neurotransmitter transporters. In H. Bönisch & H. H. Sitte (Eds.), Neurotransmitter Transporters: Investigative Methods (pp. 1–21). New York, NY: Springer New York.
  Volpe, D. A. (2016). Transporter assays as useful in vitro tools in drug discovery and development. Expert Opinion on Drug Discovery, 11(1), 91–103. doi: 10.1517/17460441.2016.1101064.
Key References
  Kenakin, T. P. (Ed.) (2014). A Pharmacology Primer: Techniques for more effective and strategic drug discovery, (4th edn.). Elsevier: Academic Press.
  Describes basic concepts underlying pharmacological assays.
  Bonisch, H., Sitte, H. (Eds.). (2016). Neurotransmitter Transporters: Investigative methods, (1st edn.). Totowa, NJ: Humana Press.
  Describes methods and protocols used in the field of neuroscience.
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