Date of Award


Degree Type


Degree Name

Master of Science (MS)


Medicinal Chemistry and Molecular Pharmacology

First Advisor

Eric L. Barker

Committee Chair

Eric L. Barker

Committee Member 1

Markus A. Lill

Committee Member 2

Nicholas Noinaj

Committee Member 3

Richard M. Van Rijn


The dopamine transporter (DAT) is a monoamine transporter that regulates dopamine (DA) neurotransmission by clearing DA from the synapse. DAT is acted on by a number of psychostimulant drugs, which block reuptake and potentiate DA signaling. Several inhibitors of DAT, both amphetamines and cathinones, also cause reversal of transport. Previous research has shown the importance of a conserved salt bridge in another monoamine transporter, the serotonin transporter, in amphetamine-induced efflux. In our studies here, we engineered a mutant Drosophila melanogaster DAT (dDAT D475N) designed to interrupt this salt bridge and examine the effects on amphetamine- and cathinone-induced efflux in vitro. Understanding the mechanism of action of structurally similar yet functionally diverse psychostimulants necessitates information about their binding sites at the transporter. A second mutant D. melanogaster DAT (dDAT D121G) was engineered to more closely mimic the substrate binding site of human DAT. By using already existing crystal structure data, compounds were computationally docked to the identified binding sites, allowing prediction of binding poses and binding free energies. dDAT D475N was found to have similar inhibition of uptake by synthetic cathinones relative to wild-type. No significant drug-induced efflux was observed in dDAT wild-type or dDAT D475N, while the D121G mutant restored efflux activity beyond that of human DAT. These results show that the presence of a salt bridge at the external gate of DAT, as well as a hydrophobic environment within the DA binding site, are important for drug-induced efflux.