Cryo-EM structures of a serotonin gated ion channel

血清素门控离子通道的冷冻电镜结构

• 批准号: 9011376
• 负责人: David Bulkley
• 金额: $ 5.61万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-02-01 至 2017-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9011376
• 关键词: Acetylcholine; Acids; Address; Adverse effects; Affinity; Agonist; Antiemetics; Architecture; Binding; Binding Sites; Biochemistry; Biological Response Modifier Therapy; Biophysics; Collaborations; Complex; Cryoelectron Microscopy; Crystallization; Drug usage; Emetics; Environment; Family; Family member; Gated Ion Channel; Goals; Health; Homo; In Vitro; Ion Channel; Label; Laboratories; Ligands; Light; Lipids; Mediating; Membrane; Membrane Proteins; Modeling; Molecular Conformation; Nervous system structure; Neurodegenerative Disorders; Neurotransmitters; Nootropic Agents; Pharmaceutical Preparations; Pharmacology; Play; Property; Resolution; Role; Sampling; Series; Serotonin; Serotonin Receptors 5-HT-3; Signal Transduction; Structure; Synapses; System; TRPV1 gene; Training; Variant; Work; cancer therapy; chemotherapy; design; gamma-Aminobutyric Acid; hypnotic; in vivo; insight; macromolecule; member; novel; particle; receptor; receptor function; receptor structure function; reconstitution; research study; response; sedative; serotonin 5 receptor; serotonin receptor

 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.

 描述（由申请人提供）：五聚体配体门控离子通道（pLGIC），即cys环受体，代表神经系统中介导突触中快速兴奋或抑制反应的主要受体家族。Cys环受体是临床上重要药物的靶标，例如镇静催眠药、用于治疗神经退行性疾病的认知增强剂和用于癌症治疗的止吐药。尽管它们具有生物学和治疗重要性，但该家族的许多成员（包括5-羟色胺受体（5-HT 3））的原子结构仍是未知的，并且pLGIC激活的结构机制仍未解决。我的目标是通过使用单粒子冷冻电子显微镜（cryo-EM）确定5-HT 3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UCSF的Yifan Cheng和大卫朱利叶斯实验室之间的密切合作，最近使用单粒子冷冻EM在近原子分辨率（3-4 μ m）下确定了TRPV 1离子通道的一系列结构。在建立了类似的合作之后，大卫朱利叶斯的实验室将提供膜蛋白生物物理学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特性的5-HT 3变体。利用Cheng实验室在单粒子cryo-EM方面的专业知识，我们将确定各种功能状态下5-HT 3受体的高分辨率结构，包括载脂蛋白，激动剂和拮抗剂结合状态，并解决各种5-HT 3激动剂和拮抗剂的结合位点和作用机制。最后，将进行实验，以确定异聚体5-HT 3内的亚基的排列，探测脂质组成对5-HT 3结构和功能的影响，并确定这些复合物的高分辨率结构。该项目中提出的实验将显示通道门控中涉及的结构转变，提供5-HT 3激活的原子分辨率模型，并揭示Cys环受体的一般机制。由于5-HT 3受体的激活导致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具有降低副作用的新型止吐药物和化疗药物提供结构框架。