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

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.