Conformational Dynamics of Neurotransmitter:Sodium Symporters: Insights from MhsT and hSERT

Abstract

Neurotransmitter:sodium symporters (NSSs) play critical roles in neural signaling by regulating neurotransmitter uptake into cells powered by sodium electrochemical gradients. Due to their significance in disease and as drug targets, extensive research has focused on understanding the underlying functional mechanisms of NSSs and their bacterial homologs, with a focus on the alternating access model. In this dissertation, we investigated the dynamics of two NSS family members, MhsT from Bacillus halodurans and the human serotonin transporter (hSERT), using a combination of electron paramagnetic resonance (EPR) spectroscopy and AlphaFold2 (AF2) techniques. The core of this work centers on comprehensive double electron-electron resonance (DEER) spectroscopy experiments on MhsT in both detergent and lipid nanodisc environments. We developed a novel computational/experimental methodology to illuminate the conformational landscape of MhsT alternating access, which can be broadly applied across a diverse spectrum of transporters. While our model of MhsT transport highlights conserved features of alternating access shared with hSERT, the N-terminal regions of mammalian NSSs introduce more complex functions not present in their bacterial counterparts. Therefore, we focused on evaluating hSERT’s N-terminal structural dynamics. However, much of our effort involved optimizing procedures to facilitate EPR studies. This work culminated in preliminary EPR data for five sites along the N-terminus, providing insights into its structural flexibility. Additionally, our data reveal differences in N-terminal dynamics between detergent and proteoliposome environments. Our research on both transporters emphasizes the importance of the lipid environment in modulating NSS dynamics. Collectively, these findings contribute to a deeper understanding of NSS function, highlighting the conserved features that bridge bacterial and mammalian systems as well as their distinct differences, showcasing the broad spectrum of neurotransmitter transport systems.