Amphetamines (AMPHs) are psychostimulants commonly used for the treatment of neuropsychiatric disorders (e.g. attention deficit disorders). They are also abused, with devastating outcomes. The abuse potential of AMPHs has been associated with their ability to cause mobilization of cytoplasmic dopamine (DA), which leads to an increase in extracellular DA levels. This increase is mediated by the reversal of the DA transporter (DAT) function that causes non-vesicular DA release, herein defined as DA efflux. However, the molecular events underlying DA efflux and how these events translate to specific AMPH behaviors is not well understood and is the focus of this proposal. We have shown that the DAT N-terminus (NT) is a structural domain that upon phosphorylation supports AMPH-induced DA efflux, but does not regulate DA uptake. Also, our preliminary data indicate that this phosphorylation event regulates DA-associated behaviors. Previously, using a combination of biochemistry, electrophysiology, and atomistic molecular dynamics simulations, as well as behavioral assays, we have shown that the DAT NT contains structural elements (Lys) that interact with plasma membrane lipids, specifically, phosphatidylinositol (4,5)-bisphosphate (PIP2). Impairing the interaction of the DAT NT with PIP2, either pharmacologically or molecularly, inhibits both DA efflux and AMPH hyperlocomotion. This was the first demonstration that the interaction of a plasma membrane protein with PIP2 is essential for psychostimulant behaviors. It also raised the possibility, that this interaction is essential for AMPH to cause DAT NT phosphorylation. DA efflux also requires the NT to be present and highly dynamic, since either anchoring the DAT NT to the plasma membrane or deleting the NT impairs DA efflux, but not DA uptake. Our mechanistic hypothesis is that the interaction between the NT and PIP2 is pivotal for AMPH to cause NT phosphorylation. Upon phosphorylation, the DAT NT uncouples from PIP2 and disengages from the membrane, forming new interactions with a specific motif of intracellular loop 4 (IL4) as predicted by our preliminary data. These new interactions, facilitated by NT phosphorylation, are essential for AMPH actions. We propose to test this hypothesis through the following specific aims: 1) To determine the role of hDAT-plasma membrane interactions in regulating NT phosphorylation; 2) To determine how hDAT NT phosphorylation supports DA efflux and the involvement of IL4. Our molecular discoveries will be then translated in vivo using Drosophila melanogaster as an animal model in which we express the human DAT (hDAT) in DA neurons of flies lacking the Drosophila DAT (“humanized flies”). In this animal model, we developed the ability to study hDAT function in isolated brains, both biochemically and biophysically, and to determine whether molecular manipulations of hDAT impairing DA efflux, but not uptake, impair complex behaviors associated with AMPH, including reward/...