Aarhus Universitet

Christian Bjerggaard Vægter

Membrane mobility and microdomain association of the dopamine transporter studied with fluorescence correlation spectroscopy and fluorescence recovery after photobleaching:

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  • Erika M. Adkins, Panum Institute
  • ,
  • Devadoss J. Samuvel, Medical University of South Carolina
  • ,
  • Jacob U. Fog, Panum Institute
  • ,
  • Jacob Eriksen, Department of Neuroscience and Pharmacology, Panum Institute
  • ,
  • Lankupalle D. Jayanthi, Medical University of South Carolina
  • ,
  • Christian Bjerggaard Vaegter
  • Sammanda Ramamoorthy, Medical University of South Carolina, Panum Institute
  • ,
  • Ulrik Gether, Department of Neuroscience and Pharmacology, Panum Institute
To investigate microdomain association of the dopamine transporter (DAT), we employed FCS (fluorescence correlation spectroscopy) and FRAP (fluorescence recovery after photobleaching). In non-neuronal cells (HEK293), FCS measurements revealed for the YFP-DAT (DAT tagged with yellow fluorescent protein) a diffusion coefficient (D) of ~3.6 × 10-9 cm2/s, consistent with a relatively freely diffusible protein. In neuronally derived cells (N2a), we were unable to perform FCS measurements on plasma membrane-associated protein due to photobleaching, suggesting partial immobilization. This was supported by FRAP measurements that revealed a lower D and a mobile fraction of the YFP-DAT in N2a cells compared to HEK293 cells. Comparison with the EGFP-EGFR (epidermal growth factor receptor) and the EGFP-beta2AR (beta2 adrenergic receptor) demonstrated that this observation was DAT specific. Both the cytoskeleton-disrupting agent cytochalasin D and the cholesterol-depleting agent methyl-beta-cyclodextrin (mbCD) increased the lateral mobility of the YFP-DAT but not that of the EGFP-EGFR. The DAT associated in part with membrane raft markers both in the N2a cells and in rat striatal synaptosomes as assessed by sucrose density gradient centrifugation. Raft association was further confirmed in the N2a cells by cholera toxin B patching. It was, moreover, observed that cholesterol depletion, and thereby membrane raft disruption, decreased both the Vmax and KM values for [3H]dopamine uptake without altering DAT surface expression. In summary, we propose that association of the DAT with lipid microdomains in the plasma membrane and/or the cytoskeleton serves to regulate both the lateral mobility of the transporter and its transport capacity
Sider (fra-til)10484 -10497
Antal sider14
StatusUdgivet - 2007
Eksternt udgivetJa

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