# Probing mechanisms of amphetamine action at plasma membrane and vesicular transporters in vitro and in vivo

> **NIH NIH R01** · COLUMBIA UNIVERSITY HEALTH SCIENCES · 2020 · $539,654

## Abstract

Amphetamines (AMPHs) are potent psychostimulants that are widely used and abused, with profound medical
and societal impact. They are known to cause mobilization of cytoplasmic dopamine (DA) to the cell exterior
via DA transporter (DAT)-mediated efflux, yet the mechanisms that mediate these actions remain poorly
defined and are a focus of this proposal. Using heterologous expression systems and a Drosophila behavioral
model, we have shown that AMPH-induced DA efflux and consequent behaviors, but not DA uptake, are
dependent on N-terminal phosphorylation of DAT. Our team has also made critical advances in understanding
the molecular mechanisms of substrate uptake by studying the bacterial transporter LeuT as a prototype, using
state-of-the-art single-molecule approaches and computational analyses. Although the N-terminal region is
essentially absent in LeuT and was truncated in the Drosophila DAT (dDAT) structures, our team has reported
a computational model of the N terminus of the human DAT (hDAT) from ab initio structure prediction in
combination with extensive atomistic molecular dynamics simulations. The analysis shows the N terminus to
be highly dynamic, to contain secondary structure elements, and to interact with lipid membranes through
electrostatic interactions. Here we aim to probe these structural elements to gain insight into the physiology of
DAT and its regulation by AMPHs, using our team's synergistic behavioral, biochemical, biophysical, and
computational tools. In parallel studies we aim to explore the mechanisms that regulate AMPH-induced release
of DA from synaptic vesicles into the cytoplasm. Using multiphoton imaging of living Drosophila brain we have
shown that at pharmacologically relevant concentrations, AMPHs must be actively transported both by DAT
and by the vesicular monoamine transporter VMAT in order to diminish the vesicular pH gradient and
redistribute vesicular contents. Still, how these events lead to redistribution of DA to the cytoplasm remains
unknown. Recent data suggest that VMAT N-terminal phosphorylation is essential for AMPH-induced DA efflux
from vesicles, and we propose to explore this hypothesis mechanistically and test it in vivo. Our established
multi-scale approach integrates biochemistry and biophysics of purified proteins, single-molecule FRET and
computational analysis, with cell-based assays, Drosophila brain imaging, analysis of in vivo phosphorylation,
and behavioral studies in living flies to probe the role of DAT and VMAT in the actions of AMPHs in the
appropriate physiological and structural contexts, in the following SPECIFIC AIMs: AIM 1. To elucidate the role of
membrane interactions in modulating phosphorylation of the N terminus of DAT and its ability to mediate
AMPH-induced DA efflux and behaviors. AIM 2. To determine how N-terminal phosphorylation alters DAT
function and dynamics. AIM 3. To determine the role of VMAT and its putative N-terminal phosphorylation in
AMPH-induced DA efflu...

## Key facts

- **NIH application ID:** 9851377
- **Project number:** 5R01DA041510-04
- **Recipient organization:** COLUMBIA UNIVERSITY HEALTH SCIENCES
- **Principal Investigator:** Jonathan A Javitch
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $539,654
- **Award type:** 5
- **Project period:** 2017-04-01 → 2022-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9851377

## Citation

> US National Institutes of Health, RePORTER application 9851377, Probing mechanisms of amphetamine action at plasma membrane and vesicular transporters in vitro and in vivo (5R01DA041510-04). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9851377. Licensed CC0.

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