Single-quantum-dot tracking reveals altered membrane dynamics of an attention-deficit/hyperactivity-disorder-derived dopamine transporter coding variant.
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.
Oleg
O
Kovtun
Dhananjay
D
Sakrikar
Ian D
ID
Tomlinson
∥Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10065, United States.
Jerry C
JC
Chang
Xochitl
X
Arzeta-Ferrer
Randy D
RD
Blakely
◆Materials Science and Technology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, United States.
Sandra J
SJ
Rosenthal
D000661:Amphetamine / Q000494:pharmacology; D001289:Attention Deficit Disorder with Hyperactivity / Q000235:genetics; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D000697:Central Nervous System Stimulants / Q000494:pharmacology; D004058:Diffusion; D050483:Dopamine Plasma Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009154:Mutation; D045663:Quantum Dots; D059008:Time-Lapse Imaging
D000697:Central Nervous System Stimulants; D050483:Dopamine Plasma Membrane Transport Proteins; C493214:SLC6A3 protein, human; D000661:Amphetamine
Attention deficit/hyperactivity disorder; amphetamine; cocaine; dopamine transporter; membrane dynamics; single quantum dot tracking