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.