The dopamine transporter (DAT) is the primary target for the abused psychostimulants such as amphetamines (AMPHs) and cocaine. DAT belongs to the family of NeurotransmitterSodium: Symporters (NSS) and Is responsible for rapid clearance of dopamine from the extracellular space. It is our long term goal to understand the molecular and cellular processes governing the activity and availability of DAT in the presynaptic membrane and how these processes are altered in diseased states involving dysfunction of dopaminergic signaling. To perform its function, DAT participates in a network of protein-protein interactions and protein-llpid interactions that ensure proper trafficking, localization and catalytic activity of the transporter. We hypothesize that interaction ofthe DAT C-terminus with Ca2+/calmodulin dependent protein kinase Ila (CaMKIlo) and with PDZ (PSD-95/Discs-large/ZO-1 homology) domain proteins Is of central mportance for these processes. In knock-in mice expressing a DAT mutant incapable of forming PDZ interactions we observe a dramatic decrease in axonal and dendritic localization ofthe transporter. Our data suggest that this phenotype is not caused by disrupting the interaction with PICKI (protein interacting with C kinase-1), the so far only PDZ protein known to Interact with DAT, but rather the interaction with yet unidentified PDZ proteins. Instead we hypothesize that PICKI binding regulates DAT electrogenic properties. An in vivo role of C-terminal protein-protein interactions in mediating AMPH action is supported by the ability of a C-terminal membrane permeable DAT peptide to reduce the locomotor response to acute AMPH in mice. We propose therefore i) to determine the role of PDZ interactions in governing axonal and dendritic localization of DAT, and to examine the physiological consequences of disrupting these interactions;11) to investigate the role of PICKI in regulating DAT ion conductances and AMPH-induced dopamine efflux;and iii) to determine the role, in vivo, that binding of CaMKIIa and PICKI to the DAT C- terminus has in establishing the behavioral effects of AMPH. These investigations should contribute significantly to future development of better therapies for drug abuse and perhaps psychiatric disorders.

Public Health Relevance

The abuse of cocaine and amphetamine causes profound socio-economic problems in the society. However, we know still little about the mechanisms in the brain that are responsible for the development of addiction to these drugs. Insight into the molecular mechanisms governing the activity and availability of their primary target, the dopamine transporter, should prove Instrumental in our search for new therapeutic strategies.

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Research Program Projects (P01)
Project #
5P01DA012408-15
Application #
8610266
Study Section
Special Emphasis Panel (ZRG1-MDCN-G)
Project Start
Project End
Budget Start
2014-03-01
Budget End
2015-02-28
Support Year
15
Fiscal Year
2014
Total Cost
$124,955
Indirect Cost
Name
Weill Medical College of Cornell University
Department
Type
DUNS #
060217502
City
New York
State
NY
Country
United States
Zip Code
10065
Billesbølle, Christian B; Mortensen, Jonas S; Sohail, Azmat et al. (2016) Transition metal ion FRET uncovers K(+) regulation of a neurotransmitter/sodium symporter. Nat Commun 7:12755
Vuorenpää, Anne; Jørgensen, Trine N; Newman, Amy H et al. (2016) Differential Internalization Rates and Postendocytic Sorting of the Norepinephrine and Dopamine Transporters Are Controlled by Structural Elements in the N Termini. J Biol Chem 291:5634-51
Khelashvili, George; Schmidt, Solveig Gaarde; Shi, Lei et al. (2016) Conformational Dynamics on the Extracellular Side of LeuT Controlled by Na+ and K+ Ions and the Protonation State of Glu290. J Biol Chem 291:19786-99
Freyberg, Zachary; Sonders, Mark S; Aguilar, Jenny I et al. (2016) Mechanisms of amphetamine action illuminated through optical monitoring of dopamine synaptic vesicles in Drosophila brain. Nat Commun 7:10652
Stolzenberg, Sebastian; Michino, Mayako; LeVine, Michael V et al. (2016) Computational approaches to detect allosteric pathways in transmembrane molecular machines. Biochim Biophys Acta 1858:1652-62
Zhao, Yiming; Fay, François; Hak, Sjoerd et al. (2016) Augmenting drug-carrier compatibility improves tumour nanotherapy efficacy. Nat Commun 7:11221
Apuschkin, Mia; Stilling, Sara; Rahbek-Clemmensen, Troels et al. (2015) A novel dopamine transporter transgenic mouse line for identification and purification of midbrain dopaminergic neurons reveals midbrain heterogeneity. Eur J Neurosci 42:2438-54
Billesbølle, Christian B; Krüger, Mie B; Shi, Lei et al. (2015) Substrate-induced unlocking of the inner gate determines the catalytic efficiency of a neurotransmitter:sodium symporter. J Biol Chem 290:26725-38
Khelashvili, George; Doktorova, Milka; Sahai, Michelle A et al. (2015) Computational modeling of the N-terminus of the human dopamine transporter and its interaction with PIP2 -containing membranes. Proteins 83:952-69
Stolzenberg, Sebastian; Quick, Matthias; Zhao, Chunfeng et al. (2015) Mechanism of the Association between Na+ Binding and Conformations at the Intracellular Gate in Neurotransmitter:Sodium Symporters. J Biol Chem 290:13992-4003

Showing the most recent 10 out of 130 publications