Aarhus Universitet

Christian Bjerggaard Vægter

Visualization of dopamine transporter trafficking in live neurons by use of fluorescent cocaine analogs

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisTidsskriftartikelForskningpeer review

  • Jacob Eriksen, Department of Neuroscience and Pharmacology, Panum Institute
  • ,
  • Søren G.F. Rasmussen, Department of Neuroscience and Pharmacology, Panum Institute
  • ,
  • Trine Nygaard Rasmussen
  • Christian Bjerggaard Vaegter
  • Hwan Cha Joo, National Institute on Drug Abuse
  • ,
  • Mu Fa Zou, National Institute on Drug Abuse
  • ,
  • Amy Hauck Newman, National Institute on Drug Abuse
  • ,
  • Ulrik Gether, Department of Neuroscience and Pharmacology, Panum Institute

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.

OriginalsprogEngelsk
TidsskriftJournal of Neuroscience
Vol/bind29
Nummer21
Sider (fra-til)6794-6808
Antal sider15
ISSN0270-6474
DOI
StatusUdgivet - 27 maj 2009
Eksternt udgivetJa

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