Interdisciplinary Graduate Program
Department of Psychiatry, Brudnick Neuropsychiatric Research Institute
Life Sciences | Medicine and Health Sciences | Neuroscience and Neurobiology
The neuronal dopamine transporter (DAT) is a major determinant of extracellular dopamine (DA) levels and is the primary target for a variety of addictive and therapeutic psychoactive drugs. DAT is acutely regulated by protein kinase C (PKC) activation and amphetamine exposure, both of which modulate DAT surface expression by endocytic trafficking. In order to use live imaging approaches to study DAT endocytosis, methods are needed to exclusively label the DAT surface pool. The use of membrane impermeant, sulfonated biarsenic dyes holds potential as one such approach, and requires introduction of an extracellular tetracysteine motif (tetraCys; CCPGCC) to facilitate dye binding. In the current study, we took advantage of intrinsic proline-glycine (Pro-Gly) dipeptides encoded in predicted DAT extracellular domains to introduce tetraCys motifs into DAT extracellular loops 2, 3, and 4. [(3)H]DA uptake studies, surface biotinylation and fluorescence microscopy in PC12 cells indicate that tetraCys insertion into the DAT second extracellular loop results in a functional transporter that maintains PKC-mediated downregulation. Introduction of tetraCys into extracellular loops 3 and 4 yielded DATs with severely compromised function that failed to mature and traffic to the cell surface. This is the first demonstration of successful introduction of a tetracysteine motif into a DAT extracellular domain, and may hold promise for use of biarsenic dyes in live DAT imaging studies.
Rights and Permissions
Copyright: 2010 Navaroli, Melikian. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
DOI of Published Version
Navaroli DM, Melikian HE (2010) Insertion of Tetracysteine Motifs into Dopamine Transporter Extracellular Domains. PLoS ONE 5(2): e9113. Link to article on publisher's website
Navaroli, Deanna M. and Melikian, Haley E., "Insertion of tetracysteine motifs into dopamine transporter extracellular domains" (2010). GSBS Student Publications. 1637.