Project Summary/Abstract: 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.