Purification and Reconstitution into Planar Bilayers of the Human Dopamine Transporter

• Main Author: Kim, YooRi
• Format: Others
• Published: VCU Scholars Compass 2012
• Subjects: Life Sciences; Physiology
• Online Access: http://scholarscompass.vcu.edu/etd/2850; http://scholarscompass.vcu.edu/cgi/viewcontent.cgi?article=3849&context=etd

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.