DOI
https://doi.org/10.25772/5PHB-VA29
Defense Date
2012
Document Type
Thesis
Degree Name
Master of Science
Department
Physiology
First Advisor
Louis De Felice
Abstract
The human dopamine transporter (hDAT) provides the primary mechanism for dopamine clearance in synapses and thus facilitates the regulation of dopaminergic functions in cognition and reward. It is the molecular target of many centrally-active agents including amphetamines and cocaine. Therefore, an understanding of hDAT function and its modulation by these therapeutic drugs and drugs of abuse can provide insight into the mechanisms of abuse and addiction. In the presented studies, hDAT is tagged with a hexahistidine construct and heterologously expressed in Xenopus laevis oocytes. The plasma membranes are isolated, solubilized, and applied to a Nickel affinity column to obtain purified hDAT with preserved functionality. Purified hDAT reconstituted in planar lipid bilayers exhibited channel behaviors at physiological membrane potentials. We observed that the current mediated by single hDAT molecules is (1) induced by dopamine, (2) dependent on the sodium electrochemical gradient, and (3) blocked by cocaine. Our data support hDAT channel activity that is associated with dopamine uptake and presents a novel electrophysiological approach to studying monoamine transporter function and modulation by drugs.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
August 2012