Molecular understanding of membrane sensors

项目概要/摘要 整合膜蛋白充当对细胞内和细胞外刺激做出反应的关键传感器。这些 蛋白质参与许多稳态细胞功能，例如张力/机械感觉和 渗透感觉和这些传感器的突变可以引起病理生理状态。在本提案中，我们将 研究机械敏感离子通道 Piezo1 和渗透传感体积的结构和功能 - 调节阴离子通道（VRAC）。这两个通道最近在联合 PI 的实验室之一中被识别出来并且处于活跃状态 结构研究的目标。机械激活的离子通道被认为负责听力、 感应触摸/疼痛，还感应动脉血压、肺和膀胱充气。压电是 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、 和呼吸生理学。 Piezo1 感知脂质双层中的机械力；然而，膜张力如何 是否被这些蛋白质感知并传输到离子通道门控尚不清楚。最近，我们和其他人 解决了 Piezo1 的 <4Å 分辨率结构，但是在所有结构中 Piezo1 的关键部分都不是很好 解决了这一问题，阻碍了对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巩固我们最初的成功，并使用新的结构测试假设来 解决有关 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于生存至关重要，但 显着促进脑缺血、中风、创伤和低钠血症引起的损伤。细胞肿胀 低渗透压引起的离子通道激活，包括容量调节阴离子通道 (VRAC)。 VRAC 是一组具有不确定复杂性的多样化异聚通道，由必需的 LRRC8A 组成 （“SWELL1”）亚基和 4 个其他 LRRC8 家族成员中的任何一个。尽管最近的高分辨率结构 来自我们组和其他人的同源六聚体 LRRC8A，亚基的数量，确切的组成和 VRAC 的化学计量仍然未知。异源表达揭示了重要的差异 VRAC 的生理功能取决于相关亚基的特性。我们的首要关注点 拟议的研究是使用高分辨率阐明 VRAC 的结构和亚基排列 冷冻电子显微镜 (cryo-EM)，以及各个组件如何完成不同的功能。 我们相信这项针对这些重要离子通道的提案将极大地影响我们的知识 响应环境压力的细胞体积稳态，以及细胞对膜张力的反应， 由于压电和 VRAC 几乎无处不在，因此它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.