# Regulation of Dopamine Transporter by Trafficking

> **NIH NIH R01** · UNIVERSITY OF PITTSBURGH AT PITTSBURGH · 2024 · $424,572

## Abstract

Plasma membrane dopamine (DA) transporter (DAT) is expressed exclusively in dopaminergic neurons and
serves for the reuptake of extracellular DA into these neurons to limit the duration and amplitude of DA signaling
in the brain. DAT is a primary target of addictive psychostimulants such as cocaine and amphetamines. DAT
expression levels at the plasma membrane of synaptic areas of striatal DA axons is the major determinant of
extracellular DA concentrations, and these levels are defined by membrane trafficking. However, mechanisms
by which proper distribution of DAT within the neuron and its membrane compartments is controlled by trafficking,
how intracellular signaling regulates this trafficking and how abused psychostimulants affect DAT traffic in vivo
remain poorly understood.
 The fundamental goal of our research is to define how intracellular trafficking regulates DAT function.
During the last cycle of this program, we described the kinetics and mechanisms of the “long-distance” DAT
traffic between midbrain and striatum through the medial forebrain bundle axons using HA-epitope tagged DAT
(HA-DAT) knock-in mice that we have previously generated. Analysis of HA-DAT trafficking in intact mouse brain
was enabled by the development of a novel HA-DAT endocytosis assay using stereotactic injections of HA
antibodies. We have also discovered dramatic down-regulation of DAT in the striatum of amphetamine-sensitized
male but not female mice, with the strongest amphetamine effects observed in nucleus accumbens. We identified
small-molecule compounds that stabilize DAT oligomers and cause robust DAT endocytosis with remarkable
specificity to DAT, which led to the demonstration of the coupling of DAT molecular mechanics and substrate-
transport activity with its oligomerization and endocytosis. Based on these studies, we hypothesize that an
inward-facing (IF) conformation of DAT induced by amphetamine favors DAT endocytosis. Generation of new
DAT mutants led to hypothesis that a PDZ domain protein mediates protein kinase C dependent ubiquitination
and endocytosis of DAT. Development of the DAT fusion protein with ascorbate peroxidase (APEX2) and
demonstration of the feasibility of tracking the temporally resolved proximity proteome of APEX2-DAT opened
new avenues for defining DAT interactome in specific brain regions and in animals treated with amphetamine,
which will be indispensable for studying mechanisms involved in DAT regulation in vivo. We will pursue our
comprehensive analysis of DAT trafficking and function by: 1) defining the molecular mechanisms of drug- and
stimuli-induced endocytosis of DAT using heterologous cells and DA neurons; 2) elucidating the mechanisms
underlying down-regulation of DAT in response to amphetamine challenge of amphetamine-sensitized mice and
sex-dependence of this response; 3) defining the brain-region-specific proximity proteome of DAT in mouse brain
using APEX2-based biotin labeling combined with quantitative mass-sp...

## Key facts

- **NIH application ID:** 10896413
- **Project number:** 5R01DA014204-21
- **Recipient organization:** UNIVERSITY OF PITTSBURGH AT PITTSBURGH
- **Principal Investigator:** ALEXANDER D SORKIN
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $424,572
- **Award type:** 5
- **Project period:** 2001-04-01 → 2028-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10896413

## Citation

> US National Institutes of Health, RePORTER application 10896413, Regulation of Dopamine Transporter by Trafficking (5R01DA014204-21). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10896413. Licensed CC0.

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