Amphetamines (AMPHs) are psychostimulants commonly used for the treatment of neuropsychiatric 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), leading to an increase in extracellular DA levels. This increase is mediated, at least in part, by the reversal of the DA transporter (DAT) function, which causes non-vesicular DA release (DA efflux). This DA efflux is thought to be essential for the psychomotor stimulant properties of AMPHs, a notion supported by evidence that specific inhibition of DA efflux impairs the ability of AMPH to cause locomotor behaviors. To date, no pharmacotherapies are available for the treatment of AMPHs abuse. Therefore, it is essential to understand: a) the molecular mechanisms targeted by AMPH to promote DA efflux; b) whether DA efflux disrupts DA functions in brain, and whether these disruptions support AMPH-induced behaviors; and c) how we can target these mechanisms to impair DA efflux to regulate AMPH behaviors. Previously, using a combination of biochemistry, electrophysiology, as well as behavioral assays, we and others have shown that the DAT N-Terminus (NT) is a critical structural domain for the ability of AMPH to promote specific behaviors. This is because AMPH stimulates DAT NT phosphorylation, which is vital for its ability to cause DA efflux. Furthermore, we provided the first evidence that the human DAT (hDAT) NT engages in direct associations with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein, syntaxin 1 (Stx1), and that this interaction, in addition to hDAT phosphorylation, regulates AMPH-induced DA efflux and behaviors. This was the first demonstration that the interaction of a plasma membrane protein with Stx1 is essential for psychostimulant behaviors. Noteworthy is that our data suggest that the strength of this interaction is regulated by AMPH-induced phosphorylation of Stx1. We hypothesize that the functional and behavioral role of the hDAT NT phosphorylation is dictated by its interactions with the plasma membrane protein, Stx1, a process regulated by the phosphorylation status of Stx1. We propose to test this hypothesis through the following specific aims: 1) to determine how hDAT interacts with Stx1; 2) to determine the involvement of Stx1 in AMPH-induced DA efflux. The molecular discoveries of S.A. #1 and S.A. #2 will be evaluated in neurons, in isolated Drosophila brains, and behaviorally, by using Drosophila as an animal model to study AMPH actions. We are now able to ?humanize? flies by expressing hDAT in DA neurons of Drosophila lacking the endogenous DAT (KO). In this system, we have established that AMPH- associated behaviors, such as locomotion and grooming, are DAT-dependent. Also, in this animal model, we can now determine preference (reward) and avoidance for AMPH. Thus, specific aim 3) is to determine whether hDAT NT-Stx1 interactions are required for AMPH-induced behaviors.
The dopamine transporters and related family members are targets for medications, and secondary targets for psychostimulants such as amphetamine. In this proposal, we will combine biochemistry, biophysics, and behavioral studies with the intent of understanding the transporter domains, the plasma membrane proteins, and the mechanisms underlying the functional modifications of the dopamine transporter that support dysregulated dopamine neurotransmission.
