Structural Dynamics of the Human Serotonin Transporter

人类血清素转运蛋白的结构动力学

• 批准号: 10386296
• 负责人: Alexandra Corinne Schwartz
• 金额: $ 3.21万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-12-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10386296
• 关键词: 3-Dimensional; American; Amphetamine Addiction; Amphetamines; Benchmarking; Biochemical; Brain; Cell membrane; Cells; Chloride Ion; Collaborations; Coupled; Cryoelectron Microscopy; Crystallization; Crystallography; Data; Data Analyses; Dimensions; Disease; Dopamine; Drosophila melanogaster; Drug Design; Electron Microscopy; Electron Spin Resonance Spectroscopy; Electrons; Elements; Engineering; Equilibrium; Fingerprint; Foundations; Functional disorder; Future; Goals; Human; Ion Cotransport; Knowledge; Label; Laboratories; Length; Ligands; Light; Link; Lipids; Mediating; Mental Depression; Mental disorders; Mentorship; Methodology; Modeling; Molecular; Molecular Conformation; Mutation; N-terminal; Neurons; Neurotransmitters; Norepinephrine; Phosphatidylinositol 4,5-Diphosphate; Physiological; Proteins; Research; Resolution; Role; Sampling; Serotonin; Signal Transduction; Site; Sodium; Solvents; Spectrum Analysis; Spin Labels; Stimulant; Structure; Surveys; Synapses; Testing; Transmembrane Domain; abuse liability; addiction; autism spectrum disorder; computer studies; conformational conversion; cryogenics; data acquisition; dopamine transporter; drug action; drug of abuse; ecstasy; experimental study; in vivo; insight; member; neuropsychiatric disorder; neurotransmitter transport; novel therapeutics; presynaptic; protein expression; protein protein interaction; protein purification; reuptake; serotonin transporter; skills; sodium ion; success; symporter; targeted treatment; therapeutic development; tool

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.

项目总结