The neurotransmitter: sodium symporter (NSS) family includes the transporters for serotonin, dopamine, and norepinephrine, which are targeted by antidepressants, methylphenidate (Ritalin), and the widely abused psychostimulants cocaine and amphetamine. In addition, NSS for GABA and glycine are promising targets for the treatment of epilepsy and anxiety disorders or schizophrenia, respectively. NSS are regulated by a complex interplay of substrates, ions, post-translational modifications and interacting proteins. Elucidating the molecular mechanism of NSS function is a necessary foundation for understanding NSS regulation in sufficient detail to design improved means of therapeutic intervention. While the purification and crystallization of human NSS proteins has yet to be achieved, prokaryotic homologues have proven powerful model systems for understanding the NSS mechanism in molecular detail. LeuT is a sodium-dependent amino acid transporter that has been captured crystallographically in distinct conformations. These data have greatly advanced our understanding of the NSS transport mechanism. However, these static "snapshots" of LeuT fall short of revealing dynamic aspects of the gating mechanisms that are critical to understanding NSS regulation. To address this shortcoming, we have developed single-molecule Forster resonance energy transfer (smFRET) methods that enable us to directly measure conformational dynamics in LeuT in real time. In our published work, we have investigated substrate- and inhibitor-dependent modulation of gating dynamics at the intracellular face of LeuT. These data have provided critical hypotheses about the transport mechanism, which we now aim to test and explore by imaging the dynamics at the extracellular face of LeuT for the first time. These investigations will enable us to gain a deeper understanding of the role of conformational events at the extracellular face, from which substrates and inhibitors enter the transporter, as well as the relationship between these events and the dynamics observed at the intracellular face that regulate substrate release. I propose to characterize the relationship between intracellular and extracellular dynamics in LeuT, and its modulation by ligands. As H+ antiport has been implicated in the transport cycle, I will quantitatively characterize the effect of H+ binding to LeuT on the kinetics of transport. Furthermore, I will examine the modulation of gating by a Na+ gradient, which is the physiological driver of transport. Completion of these aims will contribute to the elucidation of the NSS transport mechanism, potentially informing downstream efforts to screen and/or design novel therapeutics targeting the NSS. Furthermore, the single-molecule methods developed in this proposal will aid in the establishment of a platform for single-molecule imaging of other membrane proteins, including the human NSS, in situ on the cell membrane.
The proposed research aims to advance our understanding of the mechanisms of neurotransmitter: sodium symporters (NSS). These proteins include the transporters for the neurotransmitters serotonin, dopamine, and norepinephrine, which are targeted by antidepressants, methylphenidate (Ritalin), and the widely abused psychostimulants cocaine and amphetamine. Through the implementation of state-of-the-art single-molecule imaging techniques in an NSS homolog, the present study will elucidate the mechanism of transport, potentially enabling the design of more effective therapeutic strategies targeting these transporters.