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The Journal of Neuroscience, May 27, 2009, 29(21):6794-6808; doi:10.1523/JNEUROSCI.4177-08.2009

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Cellular/Molecular
Visualization of Dopamine Transporter Trafficking in Live Neurons by Use of Fluorescent Cocaine Analogs

Jacob Eriksen,^1 * Søren G. F. Rasmussen,^1 * Trine Nygaard Rasmussen,¹ Christian Bjerggaard Vaegter,¹ Joo Hwan Cha,² Mu-Fa Zou,² Amy Hauck Newman,² and Ulrik Gether¹

¹Molecular Neuropharmacology Group and Center for Pharmacogenomics, Department of Neuroscience and Pharmacology, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark, and ²Medicinal Chemistry Section, National Institute on Drug Abuse–Intramural Research Program, National Institutes of Health, Baltimore, Maryland 21224

Correspondence should be addressed to Dr. Ulrik Gether, Molecular Neuropharmacology Group and Center for Pharmacogenomics, Department of Neuroscience and Pharmacology, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark. Email: gether{at}sund.ku.dk

The dopamine transporter (DAT) mediates reuptake of dopamine from the synaptic cleft and is a target for widely abused psychostimulants such as cocaine and amphetamine. Nonetheless, little is known about the cellular distribution and trafficking of natively expressed DAT. Here we use novel fluorescently tagged cocaine analogs to visualize DAT and DAT trafficking in cultured live midbrain dopaminergic neurons. The fluorescent tags were extended from the tropane N-position of 2β-carbomethoxy-3β-(3,4-dichlorophenyl)tropane using an ethylamino-linker. The rhodamine-, OR Green-, or Cy3-labeled ligands had high binding affinity for DAT and enabled specific labeling of DAT in live neurons and visualization by confocal imaging. In the dopaminergic neurons, DAT was uniformly distributed in the plasma membrane of the soma, the neuronal extensions, and varicosities along these extensions. FRAP (fluorescence recovery after photobleaching) experiments demonstrated bidirectional movement of DAT in the extensions and indicated that DAT is highly mobile both in the extensions and in the varicosities (immobile fraction less than 30%). DAT was constitutively internalized into vesicular structures likely representing intracellular transporter pools. The internalization was blocked by lentiviral-mediated expression of dominant-negative dynamin and internalized DAT displayed partial colocalization with the early endosomal marker EGFP-Rab5 and with the transferrin receptor. DAT internalization and function was not affected by activation of protein kinase C (PKC) with phorbol-12-myristate-13-acetate (PMA) or by inhibition with staurosporine or GF109203X. These data are in contrast to findings for DAT in transfected heterologous cells and challenge the paradigm that trafficking and cellular distribution of endogenous DAT is subject to regulation by PKC.

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Received Sept. 2, 2008; revised April 6, 2009; accepted April 15, 2009.

Correspondence should be addressed to Dr. Ulrik Gether, Molecular Neuropharmacology Group and Center for Pharmacogenomics, Department of Neuroscience and Pharmacology, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark. Email: gether{at}sund.ku.dk

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