Modified Project Summary/Abstract Section The plasma-membrane monoamine transporters (MATs), including the serotonin (SERT), norepinephrine (NET) and dopamine (DAT) transporters, serve a pivotal role in limiting monoamine-mediated neurotransmission through the reuptake of their respective neurotransmitters. The transporters are the main target of clinically used psychostimulants and antidepressants. The continuing need for therapeutic drugs to treat brain disorders involving aberrant monoamine signaling provides a compelling reason to further our understanding of transporter function and to identify novel ways of targeting them. In this project we will pursue the novel idea that compounds that specifically engage a novel allosteric site we have identified in DAT will provide useful mechanistic information regarding allosteric transporter modulation. Of further significance we believe these compounds could have therapeutic potential. Previous experiments in our group targeted the equivalent allosteric site in SERT in a virtual screening of chemical libraries that identified molecules that interact with this site and display remarkable transporter-modulating activities. These compounds have revealed that engaging this site modulate MAT activity in entirely novel ways, including stimulating transporter function and affecting the interaction with transporter ligands such as the selective serotonin reuptake inhibitors (SSRIs) and psychostimulants. In corresponding experiments on DAT, we have now identified compounds, KM822 among others, that modifies DAT function and its interaction with exogenous ligands and modifies psychostimulant-elicited behaviors. The overarching hypothesis of this project is that the specific engagement of the allosteric site in DAT will provide valuable information regarding mechanisms of the dopamine transport process and could provide novel therapeutic avenues for developing DAT-based medications. We propose to pursue this idea by further developing our tool compounds to study different types of allosteric modulation employing computational, functional, and biochemical assays to characterize how the compounds modulate transport mechanisms. Consequently, the successful completion of this project will result in the development of novel ligands of DAT that can be employed as experimental tools to further our understanding of this important neurotransmitter transporter and importantly, it could open new therapeutic avenues.