# Multicolor Quantum Dot Tracking Uncovers Phenotypic Rescue of DAT A559V Aberrant Diffusion Upon D2R Antagonism

## TL;DR

This study uses quantum dot tracking to show how blocking D2S receptors rescues abnormal dopamine transporter movement in a disease-linked mutant.

## Contribution

First live-cell measurement of DAT-D2S colocalization lifetime and evidence that D2S antagonism rescues DAT A559V mobility.

## Key findings

- D2S antagonism rescues DAT A559V mobility but not CaMKII-dependent ADE.
- DAT and DAT A559V both colocalize with D2S without affecting D2S diffusion.
- D2S downregulation stabilizes DAT confinement in D2S microdomains.

## Abstract

The human dopamine
transporter (DAT) is a presynaptic transmembrane
protein that facilitates the reuptake of synaptically released dopamine.
Several lines of evidence indicate that DAT dysfunction is linked
to neuropsychiatric disorders. Moreover, the lateral membrane diffusion
and clustering propensity of DAT are emergent properties that may
factor into functional dopamine signaling. The disorder-associated
DAT missense mutant A559V undergoes anomalous dopamine efflux (ADE)
and increased lateral mobility and diffuse localization. The D2 dopamine
autoreceptor short isoform (D2S), a popular antipsychotic target,
signaling augments ADE in DAT A559V and may form stable DAT-D2S complexes.
Using quantum dot (Qdot)-based single-molecule localization microscopy,
we investigated the effect of D2S antagonism on DAT and DAT A559V
membrane mobility in transfected HEK-293 cells. Single-color Qdot-DAT
tracking shows phenotypic rescue of DAT A559V mobility upon D2S antagonism,
while aberrant DAT A559V mobility is insensitive to ADE-linked CaMKII
activity. Using two-color Qdot tracking of both the transporter and
receptor, we report the first DAT-D2S colocalization lifetime in live
cells. We show an increased propensity for both transporter types
to colocalize with D2S, without impacting D2S diffusion speed under
D2S antagonism. Downregulating D2S activity may stabilize DAT coconfinement
in D2S microdomains on the cell surface.