Mechanisms of Mechanical and Chemical Gating in Mechanosensitive Piezo1 Channels

摘要 PiezO1和Piezo2是哺乳动物阳离子选择性机械敏感离子通道同系物，它们打开它们的 毛孔对各种机械刺激的反应。机械转导信号通过Piezo通道发挥作用 在一系列令人困惑的重要生理过程中扮演中心角色，包括红细胞渗透 动态平衡，躯体和内脏的机械感觉，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 干细胞症和淋巴水肿直接与Piezo通道的基因突变有关，许多研究 进一步表明Piezo介导的信号在痛觉过敏和痛觉过敏中的作用以及Piezo的可能作用 睡眠呼吸暂停中的通道。选择性激活或抑制Piezo通道药物的研究进展 为治疗其中一些与压电有关的疾病提供了一个很有希望的治疗机会。至 Date，Yoda1，一种能够选择性激活具有微摩尔亲和力的Piezo1的合成小分子激动剂Yoda1， 代表了扩展Piezo通道药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的情况下激活的基本机制 尤达1号目前仍不得而知。在本提案中，我们将使用多学科来解决这两个悬而未决的问题 将分子动力学(MD)刺激和实验分析相结合的方法。在我们的第一个目标中，我们将 通过模拟Piezo1中的通道分子，识别Piezo1中力诱导的快速结构重排 膜处于张力状态。另一方面，利用力钳荧光法，我们将探索局部构象 使用光谱测量的变化。这将通过将构象探针插入到Strategic中来实现 在实时监测蛋白质功能的同时，通道的位置在细胞中表达。这种组合 的计算和实验将使我们能够捕获发生在 时间窗口跨越几个数量级，从微秒到分钟。在我们的第二个目标，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 使用预测性MD模拟和实验验证相结合的方法。我们将描述结构性的 转变自由能的变化和变构残基-残基相互作用的修饰 发生在Yoda1绑定时。这一目标将有助于阐明Piezo通道的化学激活机制 对开发具有临床价值的药理制剂具有重要意义。

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.