Presynaptic reuptake is the primary means to limit extracellular monoamine levels and terminate synaptic signaling. Biogenic amine transporters in SLC6 carrier gene family facilitate reuptake and are the major targets for addictive and therapeutic psychostimulants, as well as for antidepressants. These drugs potently inhibit monoamine reuptake, and thereby increase extracellular monoamine concentrations, enhance neuronal signaling and significantly modulate monoamine-related behaviors. Thus, transporter activity and availability are critical determinants of normal biogenic amine neurotransmission and psychoactive drug efficacy. A wealth of data supports that biogenic amine transporter surface presentation is not static. Rather, transporters are subject to robust constitutive endocytic trafficking. The dopamine transporter (DAT) is acutely modulated by protein kinase C (PKC) activation and exposure to psychostimulants cocaine and amphetamine (AMPH), which modulate DAT internalization and recycling rates, ultimately decreasing DAT surface availability. The major goal of these studies is to elucidate the mechanisms that control both basal and regulated biogenic amine trafficking. In this renewal application we build on our previous strengths in cellular and molecular approaches and our previous findings that Rin GTPase binds to DAT and is required for PKC-mediated DAT internalization. Specifically, we aim to (1) Elucidate the molecular determinants governing Rin-dependent DAT endocytosis and determine whether Rin GTPase is required for AMPH-mediated DAT trafficking in neuronal cell lines, (2) Determine whether PKC- and AMPH-mediated DAT trafficking are region-dependent and whether Rin GTPase is required for PKC- and AMPH-mediated DAT trafficking in situ, and (3) test whether Rin-dependent DAT trafficking is required for psychostimulant reward. These hypotheses are based on strong preliminary data that demonstrate specific Rin interactions with DAT, but not other SLC6 transporters, and a potential role for calmodulin in Rin downstream signaling. We will use chimeric proteins to define the DAT domains that confer specificity of the DAT/Rin interaction. We will use biochemical and pharmacological approaches in combination with GTPase mutants and shRNA-mediated Rin knockdown to determine the signaling pathways downstream of Rin that are necessary and sufficient for regulated DAT internalization in neuronal cell lines and mouse striatum. We will further test whether PKC- and AMPH-mediated DAT trafficking occur in a region-specific manner in the striatum, and use in vivo Rin knockdown to test the role Rin in regulated DAT endocytosis in situ. Finally, we will use in vivo shRNA approaches to knockdown Rin GTPase in mouse VTA to test whether DAT trafficking is required for psychostimulant reward. The results obtained from these endeavors will provide a clearer understanding of the mechanisms controlling transporter surface expression and the role of DAT trafficking in rewarding behaviors. We anticipate that our findings will greatly impact future strategies aimed at treating affective disorders and drug addiction. Moreover, the results will undoubtedly enhance our understanding of the molecular factors influencing monoamine availability in the brain.

Public Health Relevance

Psychostimulant addiction and affective disorders are growing health concerns in the United States, reaching near epidemic proportions over the past several years. Despite numerous investigations into the underlying causes of these conditions, our knowledge is still rather limited. The current project will investigate the major targets for both psychostimulants and antidepressants, and how intrinsic mechanisms in the brain control their availability. Our expectation is that the forthcoming information may lead to novel therapeutic approaches for treating these disorders.

National Institute of Health (NIH)
National Institute on Drug Abuse (NIDA)
Research Project (R01)
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Molecular Neuropharmacology and Signaling Study Section (MNPS)
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Pilotte, Nancy S
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University of Massachusetts Medical School Worcester
Schools of Medicine
United States
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Wu, Sijia; Fagan, Rita R; Uttamapinant, Chayasith et al. (2017) The Dopamine Transporter Recycles via a Retromer-Dependent Postendocytic Mechanism: Tracking Studies Using a Novel Fluorophore-Coupling Approach. J Neurosci 37:9438-9452
Sweeney, Carolyn G; Tremblay, Bradford P; Stockner, Thomas et al. (2017) Dopamine Transporter Amino and Carboxyl Termini Synergistically Contribute to Substrate and Inhibitor Affinities. J Biol Chem 292:1302-1309
Gabriel, Luke R; Wu, Sijia; Melikian, Haley E (2014) Brain slice biotinylation: an ex vivo approach to measure region-specific plasma membrane protein trafficking in adult neurons. J Vis Exp :
Gabriel, Luke R; Wu, Sijia; Kearney, Patrick et al. (2013) Dopamine transporter endocytic trafficking in striatal dopaminergic neurons: differential dependence on dynamin and the actin cytoskeleton. J Neurosci 33:17836-46
Gabriel, Luke; Lvov, Anatoli; Orthodoxou, Demetra et al. (2012) The acid-sensitive, anesthetic-activated potassium leak channel, KCNK3, is regulated by 14-3-3?-dependent, protein kinase C (PKC)-mediated endocytic trafficking. J Biol Chem 287:32354-66
Navaroli, Deanna M; Stevens, Zachary H; Uzelac, Zeljko et al. (2011) The plasma membrane-associated GTPase Rin interacts with the dopamine transporter and is required for protein kinase C-regulated dopamine transporter trafficking. J Neurosci 31:13758-70
Navaroli, Deanna M; Melikian, Haley E (2010) Insertion of tetracysteine motifs into dopamine transporter extracellular domains. PLoS One 5:e9113
Gabriel, Luke; Stevens, Zachary; Melikian, Haley (2009) Measuring plasma membrane protein endocytic rates by reversible biotinylation. J Vis Exp :
Boudanova, Ekaterina; Navaroli, Deanna M; Melikian, Haley E (2008) Amphetamine-induced decreases in dopamine transporter surface expression are protein kinase C-independent. Neuropharmacology 54:605-12
Boudanova, Ekaterina; Navaroli, Deanna M; Stevens, Zachary et al. (2008) Dopamine transporter endocytic determinants: carboxy terminal residues critical for basal and PKC-stimulated internalization. Mol Cell Neurosci 39:211-7

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