Single Particle Investigation of Dopamine Transporters: Transitioning from Cultured Cells to Living Brain Tissue

Abstract

The dopamine transporter (DAT) protein determines strength and duration of neurotransmission by dopamine reuptake from extracellular space into presynaptic nerve cells. Variation of the DAT gene (DAT1, SLC6A3) has been implicated in several neuropsychiatric disorders, though the exact mechanism of the consequent DAT dysfunction remains unclear. The coding variant, DAT Val559, was previously identified in subjects diagnosed with bipolar disorder, autism spectrum disorder, or attention-deficit/hyperactive disorder, and harbors an anomalous dopamine efflux phenotype. In this dissertation, the cell surface diffusion of DAT Val559 was monitored by single particle tracking with ligand-conjugated quantum dots (QDs) to elucidate the impact of the DAT Val559 variant on membrane diffusion dynamics. DAT Val559 exhibited faster surface diffusion rates than the wild-type in transfected HEK-293 and dopaminergic SK-N-MC cells. Faster diffusion rates were found to be dependent on PKCβ-mediated phosphorylation and could be normalized by a PKCβ inhibitor. Furthermore, DAT Val559 also displayed reduced clustering at the cell surface. To extend the superior optical properties of QDs to more physiologically relevant cell platforms, such as acute brain slices, the photophysics of compact ligand-conjugated CdSe/CdS QDs were examined using both ensemble and single particle analysis in brain tissue media. It was found that symmetric core passivation is critical for both photostability in oxygenated media and for prolonged single particle imaging in brain slices. The utility of these QDs was demonstrated by imaging single DAT proteins in acute brain slices, achieving 20 nm localization precision at 10 Hz frame rates. These findings detail design requirements needed for new QD probes in complex living environments, and open the door to physiologically relevant studies that capture the utility of QD probes in acute brain slices.