The dopamine transporter (DAT) is a transmembrane protein that terminates dopamine (DA) signaling in the brain by driving rapid DA reuptake into the presynaptic nerve terminals. Several lines of evidence indicate that DAT dysfunction is linked to neuropsychiatric disorders such as attention-deficit/hyperactivity disorder (ADHD), bipolar disorder, and autism spectrum disorder (ASD). Since membrane trafficking is an important mechanism of DAT regulation, our group pioneered the use of antagonist-conjugated quantum dots (QDs) to monitor individual membrane DAT molecules in live cells. In a new research effort, we implemented our QD labeling approach and total internal reflection fluorescence (TIRF) microscopy to characterize the membrane diffusion dynamics of a recently discovered ADHD/ASD/bipolar disorder-associated hDAT A559V coding variant. Published studies have demonstrated that the A559V mutant is hyper-phosphorylated at N-terminus serine residues and demonstrates anomalous DAT-mediated DA efflux. We hypothesize that the A559V substitution and the consequent DAT hyper-phosphorylation will destabilize the transient interactions between the transporter and its binding partners provoking an increase in DAT membrane mobility. Our mean square displacement analysis of WT DAT and DAT Val559 transiently transfected HEK-293 cells shows the mutant coding variant exhibits significantly higher diffusion coefficients compared to the wild type, supporting the empirical hypothesis. The altered diffusion dynamics therefore suggest a possible relationship between lateral membrane mobility and hDAT A559V dysfunction. Subsequent investigation on the effect of various stimuli on DAT diffusion will also be discussed.

Tracking single quantum dots to unravel membrane diffusion dynamics of the ADHD/autism/bipolar disorder-associated Ala559Val dopamine transporter coding variant

253rd National Meeting (2017)

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