Mechanisms of Mechanical and Chemical Gating in Mechanosensitive Piezo1 Channels

机械敏感 Piezo1 通道中的机械和化学门控机制

• 批准号: 10166873
• 负责人: YUN LUO
• 金额: $ 32.43万
• 依托单位: WESTERN UNIVERSITY OF HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-05 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10166873
• 关键词: Address; Affinity; Agonist; Agreement; Amino Acids; Architecture; Automobile Driving; Binding; Binding Sites; Biological; Biological Assay; Calcium; Cations; Cells; Chemicals; Clinical; Closure by clamp; Coupled; Coupling; Cryoelectron Microscopy; DNA Sequence Alteration; Data; Development; Distal; Electrophysiology (science); Engineering; Erythrocytes; Exhibits; Free Energy; Goals; Hand; Homeostasis; Hyperalgesia; Image; Ion Channel; Light; Link; Lymphedema; Mammals; Measurement; Mechanical Stimulation; Mechanics; Mediating; Membrane; Methods; Modality; Modeling; Modification; Molecular; Molecular Conformation; Monitor; Motion; Mutagenesis; Mutation; Pathology; Pathway Analysis; Peripheral; Pharmacology; Physiological; Physiological Processes; Piezo 1 ion channel; Piezo 2 ion channel; Piezo ion channels; Play; Positioning Attribute; Process; Proprioception; Protein Region; Proteins; Reporter; Role; Shapes; Signal Transduction; Sleep Apnea Syndromes; Stimulus; Structure; Swelling; System; Testing; Therapeutic; Thermodynamics; Time; Tissues; Validation; Visceral; allodynia; blood pressure regulation; body system; drug development; experimental study; fluorescence imaging; human disease; interdisciplinary approach; mechanical force; mechanotransduction; microscopic imaging; millisecond; multidisciplinary; protein function; proteoliposomes; response; shear stress; simulation; small molecule; tool

Abstract Piezo1 and Piezo2 are mammalian cation-selective mechanosensitive ion channels homologs which open their pore in response to various mechanical stimuli. Mechanotransduction signaling through Piezo channels plays a central role in a bewildering variety of important physiological processes including red blood cell osmotic homeostasis, somatic and visceral mechanosensation, proprioception, blood pressure regulation and development and differentiation of many tissues and organ systems. Several human diseases including xerocytosis and lymphedema have been directly linked to genetic mutations in Piezo channels and many studies further indicate a role of Piezo-mediated signaling in allodynia and hyperalgesia and a possible role of Piezo channels in sleep apnea. The development of drugs capable of selectively activating or inhibiting Piezo channels represent a promising therapeutic opportunity for the treatment of some of these Piezo-related pathologies. To date, Yoda1, a synthetic small molecule agonist capable of selectively activating Piezo1 with micromolar affinity, represents the best small molecule candidate to expand the pharmacome of Piezo channels. Unfortunately, the fundamental mechanisms by which Piezo channel sense mechanical forces and activates in the presence of Yoda1 are still unknown. In this proposal we will address these two unsolved questions using a multidisciplinary approach combining molecular dynamic (MD) stimulations and experimental assays. In our first aim, we will identify rapid, force-induced structural rearrangements in Piezo1 by simulating the channel molecule in a membrane under tension. On another hand, using force-clamp fluorimetry, we will probe local conformational changes using spectroscopic measurements. This will be done by inserting conformational probes into strategic positions of the channel expressed in cells while protein function is being monitored in real-time. This combination of computations and experiments will allow us to capture structural dynamic information that happens in a temporal window spanning several orders of magnitude, from microsecond to minutes. In our second Aim, we will identify how Yoda1 interacts with and activates Piezo1. We have already identified a Yoda1 binding site using a combination of predictive MD simulations and experimental validations. We will characterize structural changes, changes in transition free energy, and modifications of allosteric residue-residue interactions that happen upon Yoda1 binding. This aim will shed light on the mechanism of chemical activation of a Piezo channel and will be invaluable to develop pharmacological agents with clinical value.

抽象的 压电1和压电2是哺乳动物阳离子选择机械敏感的离子通道，可打开其 孔响应各种机械刺激。通过压电通道的机械转导信号传导播放 在包括红细胞渗透在内的各种重要生理过程中的令人困惑的多种多样的中心作用 稳态，体细胞和内脏机械敏化，本体感受，血压调节和 许多组织和器官系统的开发和区分。几种人类疾病，包括 骨细胞增多症和淋巴水肿已与压电通道中的遗传突变直接相关，许多研究 进一步表明压电介导的信号传导在异常性和痛觉过敏中的作用以及压电的可能作用 睡眠呼吸暂停中的通道。能够选择性激活或抑制压电通道的药物的开发 代表了治疗其中一些与压电相关的病理的有前途的治疗机会。到 日期，Yoda1，一种合成的小分子激动剂，能够以微摩尔亲和力选择性激活压电1 代表了扩展压电通道的药物组的最佳小分子候选者。不幸的是， 压电通道感知机械力并在存在下激活的基本机制 Yoda1仍然未知。在此提案中，我们将使用多学科解决这两个未解决的问题 结合分子动力学（MD）刺激和实验测定的方法。在我们的第一个目标中，我们将 通过模拟A中的通道分子来确定压电1中力诱导的快速，力诱导的结构重排 膜下的膜。另一方面，使用力钳氟化法，我们将探测局部构象 使用光谱测量的变化。这将通过将构型探针插入战略性来完成 在实时监测蛋白质功能时，在细胞中表达的通道的位置。这种组合 计算和实验将使我们能够捕获发生在 时间窗口跨越了几个数量级，从微秒到几分钟。在我们的第二个目标中，我们 将确定Yoda1如何与Piezo1相互作用并激活Piezo1。我们已经确定了Yoda1结合位点 结合预测MD模拟和实验验证。我们将表征结构 变化，过渡能能的变化以及变构残基残基相互作用的修改， 发生在Yoda1结合上。这个目标将阐明压电通道的化学激活机理 并且将具有临床价值的药理学剂是无价的。