PROJECT SUMMARY As a member of the class of neurotransmitter:sodium symporters (NSSs), the serotonin transporter (SERT) regulates levels of serotonin in the brain through reuptake or forward transport. Dysfunction of SERT has been associated with neuropsychiatric disorders and autism in humans. As such, SERT is a major target for therapeutic development as well as sites of action of drugs of abuse. Transport of serotonin from the synapse into the presynaptic cell is energetically coupled to symport of sodium and chloride ions. However, in the presence of amphetamine derivatives, this mechanism is altered. Molecules in the amphetamine class induce reverse transport by NSSs, leading to increased synaptic levels of serotonin, dopamine, and norepinephrine. This reverse transport mechanism is mediated by the N-terminus of the transporter, a hub for protein-protein interactions and signaling, and is modulated by phosphatidylinositol-4,5-bisphosphate (PIP2), a lipid enriched in the plasma membrane of neurons. The central objective of this proposal is to examine the conformational dynamics underlying transport for human SERT (hSERT), illuminating the role of its N-terminus in both forward and reverse transport. Aim 1 will use double electron-electron resonance (DEER) spectroscopy to probe conformational equilibria of full-length hSERT under different conditions to benchmark structural transitions across the transport cycle. Aim 2 will characterize the conformation of the N-terminus through surveying the mobility and solvent accessibility of singly labeled sites and define its structural reorganization in the presence of PIP2. The N-terminus and PIP2 are hypothesized to impose structural changes to the hSERT transmembrane domain, which prompts reverse transport in the presence of amphetamine. The proposed experiments are expected to reveal insights into the transport mechanism of eukaryotic NSSs and could be foundational for understanding how the presence of disease-linked mutations or exogenous molecules such as amphetamine could disturb this mechanism. The laboratory of Dr. Hassane Mchaourab specializes in revealing the conformational dynamics of transporters, including bacterial homologs of NSS, utilizing the tools of electron paramagnetic resonance (EPR) Our collaborator, Dr. Eric Gouaux has expertise in the field of eukaryotic NSS proteins and has determined several high-resolution structures of hSERT. Together, the mentorship of Dr. Mchaourab and the guidance of Dr. Gouaux will enable the success of experiments outlined in my aims from mammalian protein expression and purification through EPR data acquisition and analysis and culminating in mechanistic models that integrate high- resolution structures with conformational dynamics.