Single-Quantum-Dot Tracking Reveals Altered Membrane Dynamics of an Attention-Deficit/Hyperactivity-Disorder-Derived Dopamine Transporter Coding Variant

Abstract

The presynaptic, cocaine- and amphetamine-sensitive dopamine (DA) transporter (DAT, SLC6A3) controls the intensity and duration of synaptic dopamine signals by rapid clearance of DA back into presynaptic nerve terminals. Abnormalities in DAT-mediated DA clearance have been linked to a variety of neuropsychiatric disorders, including addiction, autism, and attention deficit/hyperactivity disorder (ADHD). Membrane trafficking of DAT appears to be an important, albeit incompletely understood, post-translational regulatory mechanism; its dysregulation has been recently proposed as a potential risk determinant of these disorders. In this study, we demonstrate a link between an ADHD-associated DAT mutation (Arg615Cys, R615C) and variation on DAT transporter cell surface dynamics, a combination only previously studied with ensemble biochemical and optical approaches that featured limited spatiotemporal resolution. Here, we utilize high-affinity, DAT-specific antagonist-conjugated quantum dot (QD) probes to establish the dynamic mobility of wild-type and mutant DATs at the plasma membrane of living cells. Single DAT-QD complex trajectory analysis revealed that the DAT 615C variant exhibited increased membrane mobility relative to DAT 615R, with diffusion rates comparable to those observed after lipid raft disruption. This phenomenon was accompanied by a loss of transporter mobilization triggered by amphetamine, a common component of ADHD medications. Together, our data provides the first dynamic imaging of single DAT proteins, providing new insights into the relationship between surface dynamics and trafficking of both wild-type and disease-associated transporters. Our approach should be generalizable to future studies that explore the possibilities of perturbed surface DAT dynamics that may arise as a consequence of genetic alterations, regulatory changes, and drug use that contribute to the etiology or treatment of neuropsychiatric disorders.

Ack1 is a dopamine transporter endocytic brake that rescues a trafficking-dysregulated ADHD coding variant

“…However, it is unclear whether antibody-bound DAT traffics similar to native DAT, as we investigate here. Multiple studies also demonstrate that DAT partitions into cholesterol-rich membrane microdomains (36,(39)(40)(41)(42)(43)(44) and that the membrane raft protein flotillin-1 is required for PKC-and AMPH-mediated DAT internalization (42), consistent with a clathrin-independent endocytic mechanism. However, a separate study reported that flotillin-1 contributes to DAT membrane mobility rather than PKC-stimulated DAT internalization (45).…”

Neuropsychiatric disease–associated genetic variants of the dopamine transporter display heterogeneous molecular phenotypes

“…Importantly, A559V knock in mice were recently found to display both behavioral and functional alteration, supporting a significant impact of ADE in vivo (66). It is therefore also highly interesting that the R615C variant, has been found to undergo dysregulated trafficking with enhanced constitutive internalization and altered microdomain distribution (28,67). This finding further supports that the potential pathological impact of altered DAT function is not necessarily recapitulated in direct assessment of dopamine uptake, and that there might be facets of DAT function that require a native context to uncover.…”

Endosomal system genetics and autism spectrum disorders: A literature review

“…The authors postulated that there is increased PKC signaling due to D2R activation in the mutant causing the redistribution and trafficking perturbation (Bowton et al, 2014). Additionally, using a novel experimental approach that employed quantum dot labeling of DAT, Kovtun et al showed that neuropsychiatric-associated variants in DAT caused an increase in membrane diffusion and constitutive endocytosis and recycling (Kovtun et al, 2015). These studies linking DAT to ASDs show how disturbances in its function and its membrane trafficking have the ability to directly affect neurological phenotypes, but they also intimately connect its surface trafficking to its function.…”