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Title page for ETD etd-11232013-194613

Type of Document Dissertation
Author Kovtun, Oleg
URN etd-11232013-194613
Title Probing ensemble and single-molecule behavior of cocaine-sensitive dopamine transporter with antagonist-conjugated quantum dots
Degree PhD
Department Chemistry
Advisory Committee
Advisor Name Title
Sandra J. Rosenthal Committee Chair
David W. Wright Committee Member
Eva M. Harth Committee Member
Randy D. Blakely Committee Member
  • flow cytometry
  • mental illness
  • fluorescence
  • attention deficit/hyperactivity disorder
  • amphetamine
  • cocaine
  • dopamine transporter
  • single quantum dot tracking
  • membrane dynamics
  • quantum dot
  • depression
Date of Defense 2013-11-15
Availability unrestricted
The presynaptic, cocaine- and amphetamine-sensitive dopamine (DA) transporter (DAT, SLC6A3) controls the intensity and duration of synaptic dopamine signals by rapid clearance of the neurotransmitter back into presynaptic nerve terminals. Abnormalities in DAT-mediated dopamine clearance have been linked to a variety of neuropsychiatric disorders, including addiction and attention deficit/hyperactivity disorder (ADHD). Despite its increasing clinical importance, little is known about DAT dynamic regulation due to the poor spatiotemporal resolution of conventional biochemical and optical techniques. To this end, we developed and validated a high-resolution labeling approach to detect DAT molecules at ensemble and single-molecule level. The labeling strategy utilizes a high-affinity, DAT-specific cocaine analogue conjugated to the surface of quantum dot (QD) probes. As a result, we were able to, for the first time, monitor lateral mobility of single wild-type and mutant DAT molecules at the plasma membrane of living cells. Single DAT-QD tracking analyses provided real-time trajectory data on a millisecond-to-second timescale which revealed that the lateral diffusion of DAT molecules in multiple cell hosts is constrained by membrane confinement domains of ~200 nm in diameter. Parallel analyses of membrane mobility of the lipid raft constituent GM-1 ganglioside revealed identical diffusion patterns, supporting previous findings of a DAT/lipid raft association. In addition, the ADHD-associated DAT R615C variant exhibited increased membrane mobility relative to wild-type DAT, with diffusion rates comparable to that achieved after lipid raft disruption, accompanied by a loss of transporter mobilization triggered by amphetamine, a component of widely prescribed ADHD medications. Together, our data report the first dynamic imaging of DAT molecules, providing new insights into the surface 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.
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