The dopamine (DA) transporter (DAT) controls DA homeostasis and neurotransmission by the active reuptake of synaptically released DA. The DAT is the major molecular target responsible for the rewarding properties and the abuse potential of amphetamine (AMPH) and cocaine. AMPH acts as a DAT substrate, promoting the reversal of DA transport, thereby resulting in DA efflux via DAT. This efflux leads to increased extracellular DA levels, an event of importance for the psychomotor stimulant properties of AMPHs. The N-terminus of the DAT is a structural domain that is critical for AMPH to cause DA efflux. We demonstrated that DAT N-terminus phosphorylation at the five most distal Ser is required for AMPH-induced DA efflux. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein syntaxin1 (Stx1) interacts with the DAT N-terminus and this interaction supports the ability of AMPH to cause DA efflux. Stx1 is phosphorylated at Ser14 by casein kinase 2 (CK2)1, an event we hypothesize is promoted by AMPH and regulates DAT-Stx1 association. Finally, we discovered that phosphatidylinositol-4,5-bisphosphate (PIP2) (a cofactor we show interacts with Stx1) directly interacts with the DAT N-terminus, and here we hypothesize coordinates Stx1 phosphorylation, leading to DA efflux. Our mechanistic hypothesis is that AMPH-induced Stx1 phosphorylation mediated by CK2 leads to DAT N-terminus phosphorylation. These events are coordinated by DAT-PIP2 interaction and are required to transmit the actions of AMPH. We propose to test our hypothesis through the following specific aims: 1) To define how DAT-Stx1 association is coordinated under basal and AMPH conditions; and 2) To determine the role of Stx1 phosphorylation in AMPH-induced DA efflux. To test our molecular discoveries in vivo, we have developed a behavioral model in Drosophila melanogaster. In this system, we have established that locomotion is a DAT-regulated behavior, and is stimulated by AMPH. Deletion of Drosophila DAT (dDAT) in DA neurons of flies inhibits AMPH-induced locomotion, an effect that is restored by the expression of the human DAT (hDAT) in these dDAT-deficient DA neurons. Using this strategy, we will translate in vivo our molecular observations. We will elucidate how CK2-mediated Stx1 phosphorylation and Stx1 interaction with the DAT N-terminus determine AMPH behaviors. Thus, our last specific aim is: 3) To determine the role of DAT-Stx1 interactions in AMPH-induced behaviors. The long-term goal of this research is to understand how AMPH-induced DA efflux and its associated behaviors are dictated and coordinated by Stx1 phosphorylation. Supported by our preliminary data, we hypothesize that inhibiting CK2 function impairs specifically DA efflux, but not uptake. Thus, we will learn how to selectively manipulate different aspects of the DAT transport cycle and determine the contribution of DA efflux in AMPH behaviors. This will uncover a new druggable target (CK2) for the treatment of AMPH abuse.

Public Health Relevance

The dopamine transporter is responsible for the abuse potential and the rewarding properties of amphetamine, cocaine, and related psychostimulants. The serotonin and norepinephrine transporters (related family members) are also targets for antidepressant medications, and secondary targets for psychostimulants. Our effort is 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 Project (R01)
Project #
5R01DA038058-02
Application #
8862445
Study Section
Neurotransporters, Receptors, and Calcium Signaling Study Section (NTRC)
Program Officer
Sorensen, Roger
Project Start
2014-07-01
Project End
2019-06-30
Budget Start
2015-07-01
Budget End
2016-06-30
Support Year
2
Fiscal Year
2015
Total Cost
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Physiology
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37240
Toki, Shinji; Goleniewska, Kasia; Reiss, Sara et al. (2018) Glucagon-like peptide 1 signaling inhibits allergen-induced lung IL-33 release and reduces group 2 innate lymphoid cell cytokine production in vivo. J Allergy Clin Immunol 142:1515-1528.e8
Shekar, Aparna; Aguilar, Jenny I; Galli, Greta et al. (2017) Atypical dopamine efflux caused by 3,4-methylenedioxypyrovalerone (MDPV) via the human dopamine transporter. J Chem Neuroanat 83-84:69-74
Reddy, I A; Pino, J A; Weikop, P et al. (2016) Glucagon-like peptide 1 receptor activation regulates cocaine actions and dopamine homeostasis in the lateral septum by decreasing arachidonic acid levels. Transl Psychiatry 6:e809
Dadalko, Olga I; Niswender, Kevin; Galli, Aurelio (2015) Impaired mTORC2 signaling in catecholaminergic neurons exaggerates high fat diet-induced hyperphagia. Heliyon 1:e00025
Dadalko, Olga I; Siuta, Michael; Poe, Amanda et al. (2015) mTORC2/rictor signaling disrupts dopamine-dependent behaviors via defects in striatal dopamine neurotransmission. J Neurosci 35:8843-54
Bellot, Morgane; Galandrin, Ségolène; Boularan, Cédric et al. (2015) Dual agonist occupancy of AT1-R-?2C-AR heterodimers results in atypical Gs-PKA signaling. Nat Chem Biol 11:271-9
Cartier, Etienne; Hamilton, Peter J; Belovich, Andrea N et al. (2015) Rare autism-associated variants implicate syntaxin 1 (STX1 R26Q) phosphorylation and the dopamine transporter (hDAT R51W) in dopamine neurotransmission and behaviors. EBioMedicine 2:135-146