Cryo-EM structures of a serotonin gated ion channel

 描述(申请人提供)：五聚体配体门控离子通道(PLGICs)，即环状受体，代表神经系统中的一个主要受体家族，在突触中介导快速兴奋或抑制反应。Cys-loop受体是临床重要药物的靶点，如镇静催眠药，用于治疗神经退行性疾病的认知增强剂，以及用于癌症治疗的止吐药物。尽管它们具有重要的生物学和治疗作用，但该家族许多成员的原子结构，包括5-羟色胺受体(5-HT3)，仍然未知，激活pLGIC的结构机制也仍未解决。我的目标是通过使用单粒子冷冻电子显微镜(Cryo-EM)确定5-HT3受体的一系列原子结构来打破这一障碍。我的项目将依赖于加州大学旧金山分校的程一帆和David Julius的实验室之间的密切合作，最近他们利用单粒子冷冻EM在近原子分辨率(3-4？)下确定了TRPV1离子通道的一系列结构。在建立了类似的合作之后，David Julius的实验室将提供膜蛋白生物物理和生物化学方面的培训，使我能够识别具有适合高分辨率结构工作的特性的5-HT3变体。利用郑实验室在单粒子低温EM方面的专业知识，我们将确定5-HT3受体在各种功能状态下的高分辨结构，包括脱氧核糖核酸、激动剂和拮抗剂结合状态，并解析各种5-HT3激动剂和拮抗剂的结合部位和作用机制。最后，将进行实验以确定5-HT3异构体中的亚基排列，探索脂质组成对5-HT3结构和功能的影响，并确定这些配合物的高分辨结构。本项目中提出的实验将展示涉及通道门控的结构转变，提供5-HT3激活的原子分辨率模型，并揭示Cys-loop受体的一般机制。由于5-HT3受体的激活会引起许多与化疗相关的呕吐副作用，我们的研究也将为合理设计副作用减少的新型止吐药物和化疗药物提供一个结构框架。

 DESCRIPTION (provided by applicant): Pentameric ligand-gated ion channels (pLGICs), i.e. cys-loop receptors, represent a major family of receptors in the nervous system that mediate rapid excitatory or inhibitory responses in synapses. Cys-loop receptors are targets of clinically important drugs such as sedative hypnotics, cognitive enhancers used in the treatment of neurodegenerative diseases, and anti-emetics used in cancer treatment. Despite their biological and therapeutic importance, atomic structures of many members of this family, including the serotonin receptor (5-HT3), are unknown and the structural mechanisms underlying pLGIC activation remain unresolved. My goal is to break this barrier by determining a series of atomic structures of the 5-HT3 receptor using single-particle cryo-electron microscopy (cryo-EM). My project will rely on the close collaboration between the labs of Yifan Cheng and David Julius here at UCSF, which recently led to the determination of a series of structures of the TRPV1 ion channel at near- atomic resolution (3-4 Å) using single particle cryo-EM. Having established a similar collaboration, David Julius' lab will provide training in membrane protein biophysics and biochemistry, enabling me to identify 5-HT3 variants with properties suitable for high resolution structural work. Making use of the Cheng lab's expertise in single particle cryo-EM, we will then determine high resolution structures of the 5-HT3 receptor in various functional states, including apo-, agonist- and antagonist-bound states, and resolve the binding sites and mechanisms of action of various 5-HT3 agonists and antagonists. Finally, experiments will be carried out to determine the arrangement of subunits within heteromeric 5-HT3, probe the consequences of lipid composition on 5-HT3 structure and function, and determine high resolution structures of these complexes. The experiments proposed in this project will show the structural transitions involved in channel gating, providing an atomic resolution model of 5-HT3 activation and also shedding light on the mechanism of Cys-loop receptors in general. Because activation of the 5-HT3 receptor causes many of the emetic side effects associated with chemotherapy, our studies will also provide a structural framework for the rational design of novel antiemetic drugs and chemotherapeutics with reduced side effects.