Insertion of Tetracysteine Motifs into Dopamine Transporter Extracellular Domains

Deanna M Navaroli,Haley E Melikian

doi:10.1371/journal.pone.0009113

Abstract

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. [3H]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.

Highlights

DA reuptake mediated by dopamine transporter (DAT) is the primary means for clearing synaptic DA and terminating dopaminergic neurotransmission [1], [2]
We took advantage of these intrinsic Pro-Gly residues and mutated the 2 upstream and downstream residues to cysteines to generate three DAT constructs with CCPGCC sequences in either extracellular loops 2, 3 or 4
DAT-extracellular loop 2 (EL2)-CCPGCC retained 63.5% of wildtype activity, which was significantly greater than both DAT-EL3CCPGCC and DAT-EL4-CCPGCC (p,.001)

Summary

Introduction

DA reuptake mediated by DAT is the primary means for clearing synaptic DA and terminating dopaminergic neurotransmission [1], [2]. DAT is acutely downregulated by PKC activation [8], [9] and amphetamine exposure [10], [11], [12], which markedly decrease DAT surface levels via endosomal trafficking. While it is well established DAT surface levels are modulated by endocytic trafficking, the cellular mechanisms that mediate DAT trafficking are not well defined. Recent studies using GFP-tagged DAT [15] and fluorescently-labeled cocaine analogs [16] have proved successful in achieving live DAT trafficking images; each approach has inherent limitations. Use of cocaine analogs allows for exclusive labeling of the DAT surface pool, but may not reflect native DAT trafficking as cocaine binding has itself been reported to alter DAT surface expression [17], [18]

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Conclusion