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: 10408005
• 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 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10408005
• 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; mechanical stimulus; 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.

摘要 Piezo 1和Piezo 2是哺乳动物阳离子选择性机械敏感性离子通道同系物，其打开它们的 毛孔对各种机械刺激的反应。通过压电通道的机械传导信号在细胞内起着重要作用。 在各种重要的生理过程中起着重要的作用，包括红细胞渗透压 稳态、躯体和内脏机械感觉、本体感觉、血压调节和 许多组织和器官系统的发育和分化。几种人类疾病，包括 干细胞症和水肿与压电通道的基因突变直接相关， 进一步表明压电介导的信号传导在异常性疼痛和痛觉过敏中的作用，以及压电介导的信号传导在异常性疼痛和痛觉过敏中的可能作用。 睡眠呼吸暂停综合征能够选择性激活或抑制压电通道的药物的开发 代表了治疗这些压电相关病理中的一些的有希望的治疗机会。到 Date，Yoda 1，一种能够以微摩尔亲和力选择性激活Piezo 1的合成小分子激动剂， 代表了扩大压电通道药物组的最佳小分子候选物。可惜 压电通道感测机械力并在存在下激活的基本机制 Yoda 1仍然未知。在本提案中，我们将使用多学科方法解决这两个未解决的问题。 方法结合分子动力学（MD）刺激和实验测定。在我们的第一个目标中，我们将 识别快速，力诱导的结构重排Piezo 1通过模拟通道分子在一个 膜张力。另一方面，利用力钳荧光法，我们将探测局部构象 使用光谱测量的变化。这将通过将构象探针插入到策略性的 在细胞中表达的通道的位置，同时实时监测蛋白质功能。这种组合 计算和实验的结合将使我们能够捕捉到发生在一个 时间窗口跨越几个数量级，从微秒到分钟。在第二个目标中，我们 将识别Yoda 1如何与Piezo 1相互作用并激活Piezo 1。我们已经确定了一个Yoda 1结合位点， 使用预测MD模拟和实验验证的组合。我们将描述结构 改变，转变自由能的改变，以及变构残基-残基相互作用的修饰， 在Yoda 1绑定时发生。这一目标将揭示压电通道的化学激活机制 对开发具有临床应用价值的药物具有重要意义。