Insertion of Tetracysteine Motifs into Dopamine Transporter Extracellular DomainsReportar como inadecuado




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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. 3HDA 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.



Autor: Deanna M. Navaroli, Haley E. Melikian

Fuente: http://plos.srce.hr/



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