The plasma membrane transporters for the biogenic amine neurotransmitters dopamine, norepinephrine and serotonin are of particular therapeutic interest because these molecular pumps are the primary sites of action of psychostimulant (e.g., cocaine, amphetamine) and antidepressant drugs (e.g., fluoxetine, imipramine bupropion). Although it is poorly understood how the transporters work or how drugs interact with them, recent electrophysiological studies of cloned transporters have advanced our understanding by demonstrating that these transporter mediate several ionic currents that provide sensitive readouts of their functional states. Currents identified in the human dopamine transporter (hDAT) include a transport current reflecting substrate movement, transient currents associated with sodium binding, and a tonic leak conductance which can be blocked by both cocaine-like drugs and by substrates. We have embarked on a study of how small ligands affect the currents affect the currents and transport activity of the hDAT in order to learn about transporter mechanisms and drug- transporter interactions at the molecular level. The sites of ligand interaction associated with specific structural states will be modeled in conjunction with other components of the PPG. The electrophysiological and pharmacological tools we identified will be useful for probing the functional consequences of structural mutations of hDAT generated in this and other components of the PPG. The resulting models will be further tested by direct biochemical and biophysical analysis using a yeast hDAT expression system to generate quantities of protein sufficient for such studies. The long term goals of structural defining functionally distinct states of transporter-ligand interaction will provide insight into the molecular mechanisms of transport and will guide future development of novel therapeutic agents.
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