The dopamine transporter (DAT) mediates the inactivation of released dopamine (DA) through its reuptake. The long-term goals of this research are to understand how DA accumulation and efflux are regulated by post-translatlonal modification of DAT, by association of DAT with other proteins, and by localization of DAT to specific membrane microdomains. During the previous project period we demonstrated that phosphorylation ofthe DAT N-terminus is essential for AMPH-induced DA efflux, CaMKIIa binds to the distal C-termlnus of DAT, and CaMKIIa phosphorylates serines In the distal N-terminus of DAT in vitro. The CaMKll Inhibitor KN93 reduces AMPH-induced DA efflux in cells as well as in vivo in mouse striatum. In order to develop a model system for mechanistic examination, we have established a behavioral assay for AMPH-induced DAT-mediated DA efflux in Drosophila melanogaster larvae. In larvae, inhibition of CaMKll only in DA neurons inhibits the AMPH-induced behavior, whereas expression of constitutively active CaMKll enhances AMPH-induced behavior. We have shown that the membrane raft-associated protein Flotillini (Floti) is necessary for localization of DAT in membrane rafts. Floti knockdown blunts AMPH-induced DA efflux in mouse DA neurons in primary culture and AMPH-induced behavior in D. melanogaster larvae. The precise mechanisms by which Floti modulates DAT localization and function remain unknown. Our working hypothesis is that Floti traffics DAT to a membrane raft compartment containing the necessary signaling machinery to phosphorylate the DAT N-terminus and thereby allow AMPH-induced DA efflux. We propose to: 1) characterize the relationship between Floti, DAT, and DAT-interacting proteins in membrane rafts, 2) determine the role of Floti in AMPH-induced DA efflux and behavio, and 3) determine the role of N-terminal phosphorylation of DAT and its raft localization in AMPH-induced DA efflux and behavior.
These aims will be pursued in heterologous cells, in intact behaving D. melanogaster larvae, and in genetically modified mice, in a collaborative and synergistic interaction with the other PPG projects and the electrophysiological expertise of the core.

Public Health Relevance

The dopamine transporter is responsible for the rewarding properties and abuse potential of cocaine, amphetamine, and related psychostimulants. The related transporters for serotonin and norepinephrine are targets for antidepressant medications and secondary targets for psychostimulants. We strive to understand how these transporters are regulated in health and In disease.

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Research Program Projects (P01)
Project #
5P01DA012408-14
Application #
8435543
Study Section
Special Emphasis Panel (ZRG1-MDCN-G)
Project Start
Project End
Budget Start
2013-03-01
Budget End
2014-02-28
Support Year
14
Fiscal Year
2013
Total Cost
$368,428
Indirect Cost
$32,160
Name
Weill Medical College of Cornell University
Department
Type
DUNS #
060217502
City
New York
State
NY
Country
United States
Zip Code
10065
Mayer, Felix P; Schmid, Diethart; Owens, W Anthony et al. (2018) An unsuspected role for organic cation transporter 3 in the actions of amphetamine. Neuropsychopharmacology 43:2408-2417
Quick, Matthias; Abramyan, Ara M; Wiriyasermkul, Pattama et al. (2018) The LeuT-fold neurotransmitter:sodium symporter MhsT has two substrate sites. Proc Natl Acad Sci U S A 115:E7924-E7931
Herborg, Freja; Andreassen, Thorvald F; Berlin, Frida et al. (2018) Neuropsychiatric disease-associated genetic variants of the dopamine transporter display heterogeneous molecular phenotypes. J Biol Chem 293:7250-7262
Razavi, Asghar M; Khelashvili, George; Weinstein, Harel (2018) How structural elements evolving from bacterial to human SLC6 transporters enabled new functional properties. BMC Biol 16:31
Doktorova, Milka; Weinstein, Harel (2018) Accurate In Silico Modeling of Asymmetric Bilayers Based on Biophysical Principles. Biophys J 115:1638-1643
LeVine, Michael V; Cuendet, Michel A; Razavi, Asghar M et al. (2018) Thermodynamic Coupling Function Analysis of Allosteric Mechanisms in the Human Dopamine Transporter. Biophys J 114:10-14
Li, Xue; Maretzky, Thorsten; Perez-Aguilar, Jose Manuel et al. (2017) Structural modeling defines transmembrane residues in ADAM17 that are crucial for Rhbdf2-ADAM17-dependent proteolysis. J Cell Sci 130:868-878
Wragg, Rachel T; Parisotto, Daniel A; Li, Zhenlong et al. (2017) Evolutionary Divergence of the C-terminal Domain of Complexin Accounts for Functional Disparities between Vertebrate and Invertebrate Complexins. Front Mol Neurosci 10:146
Doktorova, M; Harries, D; Khelashvili, G (2017) Determination of bending rigidity and tilt modulus of lipid membranes from real-space fluctuation analysis of molecular dynamics simulations. Phys Chem Chem Phys 19:16806-16818
Runegaard, Annika H; Jensen, Kathrine L; Fitzpatrick, CiarĂ¡n M et al. (2017) Preserved dopaminergic homeostasis and dopamine-related behaviour in hemizygous TH-Cre mice. Eur J Neurosci 45:121-128

Showing the most recent 10 out of 146 publications