Molecular understanding of membrane sensors

膜传感器的分子理解

• 批准号: 9899317
• 负责人: Ardem Patapoutian
• 金额: $ 79.61万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9899317
• 关键词: 3-Dimensional; Address; Anions; Architecture; Biochemical; Biological Models; Biology; Biosensor; Bladder; Blood Pressure; Blood Vessels; Cell Volumes; Cell membrane; Cell physiology; Cells; Cellular Metabolic Process; Cellular Morphology; Cerebral Ischemia; Cerebral Ischemia-Hypoxia; Chlorides; Classification; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Congestive Heart Failure; Coupled; Cryoelectron Microscopy; Data Set; Detergents; Disease; Environment; Erythrocytes; Face; Family member; Hearing; Homeostasis; Homo; Homologous Gene; Hydrophobicity; Hyponatremia; Integral Membrane Protein; Ion Channel; Ion Channel Gating; Ionic Strengths; Ions; Knock-out; Knowledge; Ligands; Lipid Bilayers; Lipids; Lung; Mechanics; Membrane; Modeling; Molecular; Molecular Conformation; Mus; Mutagenesis; Mutation; Osmolar Concentration; Osmotic Pressure; Osmotic Shocks; Pain; Physiological; Piezo 1 ion channel; Piezo 2 ion channel; Pliability; Population; Preparation; Property; Proprioception; Proteins; Regulation; Resolution; Respiratory physiology; Role; Sampling; Shapes; Stimulus; Stress; Stroke; Structure; Structure-Activity Relationship; Swelling; System; Testing; Touch sensation; Trauma; Vesicle; base; body system; disease-causing mutation; drinking water; experimental study; extracellular; insight; mechanical force; molecular modeling; mutant; nanodisk; pressure; response; sensor; stoichiometry; success

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.

项目摘要/摘要 积分膜蛋白充当临界传感器，对细胞内和细胞外刺激有反应。这些 蛋白质参与许多稳态细胞功能，例如张力/机械敏感和 这些传感器中的渗透压和突变会引起病理生理状态。在此提案中，我们将 研究机械敏感离子通道的结构和功能 调节的阴离子通道（VRACS）。最近在Co-Pi的一个实验室中确定了两个渠道，并且很活跃 结构研究的目标。机械激活的离子通道被认为是造成听力的原因， 感应触摸/疼痛，但还感应动脉血压以及肺和膀胱膨胀。压电是 机械敏感的离子通道对于触摸，本体感受，血管生物学，红细胞形态至关重要， 和呼吸生理学。压电在脂质双层中感应机械力；但是，膜张力如何 这些蛋白质会感受到并传播到离子通道门控尚不清楚。最近，我们和其他人有 解决的<4Å分辨率结构的压电结构，但是在所有结构中，Piezo1的关键部分都不好 解决机械性和离子通道活性如何耦合的解决，阻碍的机械理解。 我们提出了几种方法来建立我们的最初成功，并使用新的结构测试假设 解决有关Piezo1的其余结构和机械问题。 超出稳态范围以外的渗透压的细胞反应对于生存至关重要，但是 细胞吞咽 由低渗透应力引起的激活离子通道，包括体积调节的阴离子通道（VRAC）。 VRAC是由不确定复杂性的异源通道的潜水员集 （“ Swell1”）亚基和其他4个LRRC8家庭成员中的任何一个。尽管最近的高分辨率结构 来自我们小组和其他人的同性杂志LRRC8A，亚基的数量，确切的组成和 VRAC的化学计量仍未知。异源表达揭示了重要的差异 VRAC的生理功能取决于关联亚基的身份。我们的主要重点 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），以及各个组件中的每个组件如何完成不同的功能。 我们认为，针对这些重要离子渠道的该建议将极大地影响我们对 细胞体积稳态响应环境应力以及细胞对膜张力的反应， 由于压电和VRAC几乎无处不在，因此影响所有脊椎动物器官系统。