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The Journal of Neuroscience, February 4, 2009, 29(5):1361-1374; doi:10.1523/JNEUROSCI.3250-08.2009

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Cellular/Molecular
Negative Regulation of Dopamine Transporter Endocytosis by Membrane-Proximal N-Terminal Residues

Tatiana Sorkina, Toni L. Richards, Anjali Rao, Nancy R. Zahniser, and Alexander Sorkin

Department of Pharmacology and Neuroscience Program, Anschutz Medical Center, University of Colorado Denver, Aurora, Colorado 80045

Correspondence should be addressed to Alexander Sorkin, Department of Pharmacology, Anschutz Medical Center, University of Colorado Denver, Mail Stop 8303, P.O. Box 6511, Aurora, CO 80045. Email: Alexander.sorkin{at}uchsc.edu

The plasma membrane dopamine transporter (DAT) takes extracellular dopamine back up into dopaminergic neurons. Although the number of DATs at the cell surface is regulated by endocytosis and recycling, the molecular mechanisms that control this endocytic trafficking of DAT are not defined. To map the sequence motifs that are involved in constitutive DAT endocytosis, mutagenesis of human DAT tagged with yellow fluorescent protein (YFP) and an extracellular HA epitope was performed. Removal of the entire N terminus of DAT resulted in accumulation of the resulting DAT mutant (YFP-HA-N-DAT) in early and recycling endosomes in HeLa and PAE cells, and in primary rat mesencephalic-striatal neuronal cocultures. This endosomal accumulation was due to rapid constitutive internalization of YFP-HA-N-DAT by the clathrin-dependent pathway. Small deletions and multialanine substitutions in the N terminus revealed two molecular determinants within the membrane proximal residues 60–65 that are important for preventing rapid internalization of DAT. First, mutations of Arg60 or Trp63, leading to disruption of the "outward facing" DAT conformation, correlated with an increased pool of mobile DATs in the plasma membrane and accelerated constitutive internalization of the DAT mutants. Second, mutation of Lys65 also correlated with elevated endocytosis. While none of these mutations alone recapitulated the marked endocytic phenotype of YFP-HA-N-DAT, simultaneous elimination of both the outward conformation of DAT and Lys65 resulted in DAT mutants that were rapidly internalized. Thus, our studies reveal a new link between DAT endocytosis and conformation-dependent uptake activity that represents a novel mode for regulating DAT function.

Key words: dopamine transporter; endocytosis; recycling; dopaminergic neurons; amphetamine; clathrin; fluorescence microscopy

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Received July 11, 2008; revised Dec. 21, 2008; accepted Dec. 26, 2008.

Correspondence should be addressed to Alexander Sorkin, Department of Pharmacology, Anschutz Medical Center, University of Colorado Denver, Mail Stop 8303, P.O. Box 6511, Aurora, CO 80045. Email: Alexander.sorkin{at}uchsc.edu

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