Functional Dynamics of Neurotransmitter:Sodium Symporters (NSSs)

Neurotransmitter:sodium symporters (NSS), the target of therapeutic and illicit drugs, share a common structural core, e.g., the LeuT fold, with secondary transporters that harness the energy from (electro)chemical ion and/or substrate gradients to power concentrative substrate symport and/or antiport. This multidisciplinary proposal, involving iterative functional, spectroscopic and computational methods, seeks to identify commonalities and divergences in the modes of transport of archetypes of LeuT-fold transporters in the context of differences in the regulatory role of ions on the driving mechanism and the conformational changes associated with substrate binding and translocation with special emphasis on how psychoactive drugs modulate the activity of human serotonin transporter (hSERT). By taking advantage of recent and preliminary findings and state-of-the-art technologies established in the participating labs, the major goals of the research described in this grant proposal are the identification of the conformational dynamics associated with the different modes of transport, the binding sites and permeation pathways of substrates, ions, and drugs within the transporters, and how therapeutic and illicit psychostimulants modulate the transport process in hSERT. We are particularly interested in testing our central hypothesis that the mechanistic differences associated with discernable local structural changes and dynamics are specific to the respective LeuT-fold families, culminating with the remarkable complexity of the transport mechanism of hSERT, representative for mammalian NSSs, with the overall goal to close the gap in our understanding of their role in health, disease, and pharmacotherapy. To reach this goal, our line of attack follows three specific aims, 1) to map the conformational dynamics associated with concentrative substrate transport by testing our hypothesis that functional specialization entails, in addition to common elements of alternating access, divergent structural changes between transporter families using double electron-electron resonance (DEER) spectroscopy to measure distance distributions between pairs of strategically selected positions in representative LeuT-fold proteins, 2) to delineate the ion-dependent regulation of concentrative substrate transport by interrogating our hypothesis that concentrative substrate transport is regulated by transporter-specific discernable ion binding events using binding and flux studies in conjunction with DEER experiments, and 3) to test the model that amphetamine-induced hSERT reverse transport entails an antiport mode by testing the hypothesis that amphetamine-elicited serotonin efflux entails in part a switching of the transport mode that is associated to unique conformational changes in the N-terminal region, whereas the binding of cocaine, another psychoactive drug, inhibits serotonin transport by blunting the forward transport mode-associated transitions. Thus, obtaining a holistic understanding of transport mechanism and implementing a new paradigm for illuminating drug action and providing guidelines for improved therapy design are the long- term goals of this proposal.