Molecular understanding of membrane sensors

项目摘要/摘要 整合膜蛋白是对细胞内外刺激作出反应的关键感受器。这些 蛋白质参与许多动态平衡的细胞功能，如张力/机械感觉和 这些感受器的突变会导致病理生理状态。在这项提案中，我们将 研究机械敏感离子通道Piezo1的结构和功能，以及渗透压敏感量 调节阴离子通道(VRAC)。这两个通道都是最近在联合PI的一个实验室中发现的，并且都很活跃 结构研究的目标。机械激活的离子通道被认为负责听力， 不仅可以感知触摸/疼痛，还可以感知动脉血压、肺和膀胱充气。Piezo是 机械敏感离子通道对触摸、本体感觉、血管生物学、红细胞形态、 和呼吸生理学。PIEZO1感觉到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递到离子通道门控尚不清楚。最近，我们和其他人 解决了Piezo1的分辨率结构，但在所有结构中，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建立我们最初的成功，并使用新的结构测试假设来 解决关于Piezo1的剩余的结构和机械问题。 细胞对超出动态平衡范围的渗透压的反应是生存的关键，但 对脑缺血、中风、创伤和低钠血症造成的损害有显著影响。细胞肿胀 由低渗应激引起的离子通道激活，包括容量调节阴离子通道(Vrac)。 Vrac是由基本的LRRC8A组成的一组未知复杂性的不同的异构体通道 (“SWELL1”)亚基和其他4个LRRC8家族成员中的任何一个。尽管最近的高分辨率结构 LRRC8A，亚基的数量，确切的组成和 Vrac的化学计量仍不清楚。异源表达揭示了重要的差异 Vrac的生理功能依赖于相关亚基的同一性。我们的主要重点是 建议的研究是利用高分辨率阐明VRAC的结构和亚基排列 冷冻电子显微镜(Cryo-EM)，以及每个不同组件如何实现不同的功能。 我们相信，这项针对这些重要离子通道的提议将显著影响我们对 细胞体积对环境压力的动态平衡，以及细胞对膜张力的反应， 自从Piezos和VRAC几乎无处不在以来，冲击到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.