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 have shown that DAT N-terminus phosphorylation at the five most distal Ser is required for AMPH-induced DA efflux, but does not regulate DA uptake. Furthermore, our preliminary studies suggest that the DAT N-terminus interacts electrostatically with phosphatidylinositol-4,5-bisphosphate PIP2 (a key phospholipid enriched at the inner leaflet of the plasma membrane), and that this interaction impairs DA efflux, but not uptake. Our mechanistic hypothesis is that upon phosphorylation, the DAT N-terminus uncouples from PIP2 to disengage from the membrane. Both of these events are required to elicit the DA efflux produced by AMPH. We propose to test our hypothesis and its implications for the behavioral effects of AMPH in vivo, through the following specific aims: 1) To determine the nature of the interaction between DAT N-terminus and PIP2, and describe it in a structural context provided by computational modeling; 2) To determine the role of N-terminus phosphorylation in regulating how DAT and PIP2 interact, and its effect on DAT function; 3) To determine the role of DAT/PIP2 interactions in AMPH-induced behaviors in Drosophila melanogaster. In this animal model, we have established that locomotion is a DAT-dependent 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 findings from specific aims 1 and 2. This will allow us to understand how hDAT N-terminus phosphorylation and associations with PIP2 determine AMPH-induced behaviors. The long-term goals of this research are to understand how AMPH-induced DA efflux and its associated behaviors are dictated by the interactions of hDAT N-terminus with PIP2, and/or by phosphorylation of the N- terminus. By specifically impairing DA efflux, but not uptake, we will determine the contribution of DA efflux in AMPH behaviors. The intent is to uncover novel targets for the treatment of AMPH abuse. From a broad perspective of transporter biology, we will reveal how a DAT structural domain (the N-terminus) via its interactions with the plasma membrane dictates different aspects of transport function.
The dopamine transporter is responsible for clearing extracellular dopamine once it has been released upon action potential depolarization. The dopamine transporters and related family members are targets for medications, and secondary targets for psychostimulants. Our effort is to understand how these transporters are regulated in health as well as in disease, with the intent to identify novel therapeutic targets.
|Garcia-Olivares, J; Baust, T; Harris, S et al. (2017) G?? subunit activation promotes dopamine efflux through the dopamine transporter. Mol Psychiatry 22:1673-1679|
|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|
|LeVine, Michael V; Cuendet, Michel A; Khelashvili, George et al. (2016) Allosteric Mechanisms of Molecular Machines at the Membrane: Transport by Sodium-Coupled Symporters. Chem Rev 116:6552-87|
|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|
|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|
|Hamilton, Peter J; Shekar, Aparna; Belovich, Andrea N et al. (2015) Zn(2+) reverses functional deficits in a de novo dopamine transporter variant associated with autism spectrum disorder. Mol Autism 6:8|
|Khelashvili, George; Weinstein, Harel (2015) Functional mechanisms of neurotransmitter transporters regulated by lipid-protein interactions of their terminal loops. Biochim Biophys Acta 1848:1765-74|
|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|
|Hamilton, Peter J; Belovich, Andrea N; Khelashvili, George et al. (2014) PIP2 regulates psychostimulant behaviors through its interaction with a membrane protein. Nat Chem Biol 10:582-589|
|Hansen, Freja H; Skjørringe, Tina; Yasmeen, Saiqa et al. (2014) Missense dopamine transporter mutations associate with adult parkinsonism and ADHD. J Clin Invest 124:3107-20|
Showing the most recent 10 out of 11 publications