Molecular understanding of membrane sensors

项目总结/摘要 整合膜蛋白作为关键的传感器，响应细胞内和细胞外的刺激。这些 蛋白质参与许多稳态细胞功能，例如张力/机械感觉， 这些传感器的突变可能导致病理生理状态。在本提案中，我们将 研究机械敏感离子通道Piezo1的结构和功能，以及渗透敏感体积- 调节阴离子通道（VRAC）。这两个通道最近在一个合作PI的实验室中被发现，并且是活跃的 结构研究的目标。机械激活的离子通道被认为是负责听力， 感测触摸/疼痛，而且还感测动脉血压以及肺和膀胱充气。Piezos是 机械敏感离子通道对于触觉、本体感觉、血管生物学、红细胞形态学， 和呼吸生理学。Piezo1传感脂质双层中的机械力;然而，膜张力如何 被这些蛋白质感知并被传输到离子通道门控中是未知的。最近，我们和其他人 Piezo1的结构解析度<4 × 10 - 5，但在所有结构中，Piezo1的关键部分解析度不高 解决，阻碍机械感觉和离子通道活动如何耦合的机械理解。 我们提出了几种方法来建立我们的初步成功，并使用新的结构测试假设， 解决关于Piezo1的剩余结构和机械问题。 细胞对超出稳态范围的渗透压的反应对生存至关重要， 显著促进由脑缺血、中风、创伤和低钠血症引起的损伤。细胞肿胀 由低渗透压应激引起的低渗透压激活离子通道，包括体积调节阴离子通道（VRAC）。 VRAC是由必需的LRRC8A组成的一组复杂性不确定的异聚体通道 （"SWELL1"）亚基和4个其他LRRC8家族成员中的任一个。尽管最近的高分辨率结构， 同源六聚体LRRC8A从我们的小组和其他人，亚基的数量，确切的组成和 VRAC化学计量仍然未知。异源表达揭示了重要的差异表达， VRAC的生理功能依赖于相关亚基的特性。我们的主要重点是 建议的研究是使用高分辨率的VRAC的结构和亚基排列的阐明 冷冻电子显微镜（cryo-EM），以及各种组件中的每一个如何实现不同的功能。 我们相信，针对这些重要离子通道的这一提议将显著影响我们对 响应于环境压力的细胞体积稳态，以及响应于膜张力的细胞， 由于压电和VRAC几乎无处不在，因此它们会影响所有脊椎动物的器官系统。

Project Summary/Abstract Integral membrane proteins act as critical sensors that respond to intra- and extra-cellular stimuli. These proteins are involved in many homeostatic cellular functions such as tension/mechanosensation and osmosensation, and mutations in these sensors can cause pathophysiological states. In this proposal, we will study the structure and function of the mechanosensitive ion channel, Piezo1, and the osmotic sensing volume- regulated anion channels (VRACs). Both channels were recently identified in one of the co-PI’s lab and are active targets for structural studies. Mechanically activated ion channels are thought to be responsible for hearing, sensing touch/pain, but also sensing arterial blood pressure, and lung and bladder inflation. Piezos are mechanosensitive ion channels essential for touch, proprioception, vascular biology, red blood cell morphology, and respiratory physiology. Piezo1 senses mechanical force in lipid bilayers; however, how membrane tension is sensed by these proteins and transmitted into ion channel gating is not known. Recently, we and others have solved <4Å resolution structures of Piezo1, however in all structures key portions of Piezo1 were not well resolved, hindering mechanistic understanding of how mechanosensation and ion channel activity are coupled. We propose several approaches to build off our initial success and using new structures test hypotheses to address the remaining structural and mechanistic questions about Piezo1. The cellular response to osmotic pressures beyond the homeostatic range is critical for survival and yet significantly contributes to damage caused by cerebral ischemia, stroke, trauma, and hyponatremia . Cell swelling caused by hypo-osmotic stress activates ion channels including volume-regulated anion channels (VRAC). VRAC is a diverse set of heteromeric channels of undefined complexity composed of the essential LRRC8A (“SWELL1”) subunit and any of 4 other LRRC8 family members. Despite recent high-resolution structures of homo-hexameric LRRC8A from our group and others, the number of subunits, exact composition and stoichiometry of VRAC are still unknown. Heterologous expression has revealed that important differential physiological functions of VRAC are dependent on the identity of associating subunits. The primary focus of our proposed studies is the elucidation of the structure and subunit arrangement of VRACs using high-resolution cryo-electron microscopy (cryo-EM), and how each of the various assemblies accomplishes different functions. We believe this proposal targeting these important ion channels will significantly impact our knowledge of cell volume homeostasis in response to environmental stresses, as well as cell response to membrane tension, impinging on all vertebrate organ systems since Piezos and VRACs are nearly ubiquitous.