Biophysical, Structural and Functional Analysis of Mechanosensitive Channels

机械敏感通道的生物物理、结构和功能分析

• 批准号: 7441162
• 负责人: DOUGLAS CHARLES REES
• 金额: $ 39.09万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-15 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7441162
• 关键词: Address; Advisory Committees; Animals; Arabidopsis; Bacteria; Biological Assay; Biological Process; Biology; Cell membrane; Cell physiology; Cells; Characteristics; Chemistry; Classification; Collaborations; Complex; Coupling; Development; Electrophysiology (science); Environment; Equilibrium; Escherichia coli; Family; Force of Gravity; Growth and Development function; Health; Homologous Gene; Ion Channel; Laboratories; Lead; Lipid Bilayers; Liquid substance; Measurement; Measures; Mechanics; Membrane; Membrane Proteins; Molecular; Molecular Conformation; Morphology; Mutagenesis; Organelles; Organism; Osmotic Pressure; Osmotic Shocks; Perception; Physics; Physiological; Plants; Property; Proteins; Public Health; Reporter; Research; Role; Series; Stretching; Structure; Surveys; Theoretical model; Thick; Touch sensation; Vesicle; Water; Width; Work; cell growth; improved; interest; protein structure; research study; response

DESCRIPTION (provided by applicant): All organisms, from single-celled bacteria to multi-cellular animals and plants, must sense and respond to mechanical force in their external environment (shear force, gravity, touch) and in their internal environment (osmotic pressure, membrane deformation) for proper growth, development, and health. Our research focuses on two families of mechanosensitive channels, the prokaryotic channels MscL and MscS and their eukaryotic homologs in Arabidopsis. MscL and MscS are intrinsically stretch-activated channels that open and close in response to tension applied directly to the bilayer and consequently are sensitive reporters of protein- membrane energetics. Elucidating how these mechanosensitive channels function in the context of the membrane will help us understand how mechanical force can generate biophysical alterations that in turn lead to adaptive changes in cell physiology. Aim 1: Investigate the crystal structures of MscL and MscS in multiple conformational states. MscL and MscS are among the few gated channels that have been crystallographically determined. Our highest priority is to improve the structures of the E. coli channels, but we will also systematically survey prokaryotic homologs and the use of molecular doorstops to trap channels in alternate conformational states to define the gating transition in structural detail. Aim 2: Analyze the biophysical interactions between mechanosensitive channels and the lipid bilayer. Working within the context of a theoretical model, the coupling between gating tension, bilayer thickness, and width of the hydrophobic region of MscL will be explored through mutagenesis and single channel electrophysiology. These studies will dissect the energetic contributions of different membrane deformation terms to the conformational equilibrium between channel states. The physiologically crucial permeation of water through MscL and MscS in giant unilamellar vesicles will be measured volumetrically and compared to the fluid transport properties anticipated from conductance measurements. Aim 3: Characterize functional and structural aspects of eukaryotic MscS-Like channels. The MscS-Like (MSL) channels of Arabidopsis provide an opportunity to investigate the structure and function of mechanosensitive channels in the context of multi-cellular eukaryotic organisms. The oligomeric state, channel characteristics, and structure of these proteins will be investigated. We will also use a series of new and established assays to characterize their biological function in osmotic shock protection, intramembrane localization, electrophysiology and organelle morphology control. Our proposed experiments on the MSLs, together with the experiments proposed above for MscL and MscS, are the start towards a systematic approach to revealing how MS channels function in the context of the membrane and the cell. PUBLIC HEALTH RELEVANCE Force-sensing is a critical aspect of healthy cell growth, morphology and development. We will study in molecular detail how force-sensing is achieved by two families of stretch-activated membrane channels.

描述（由申请人提供）：所有生物，从单细胞细菌到多细胞动物和植物，必须在其外部环境（剪切力，重力，触摸）和内部环境（渗透压，膜变形）中感知和响应机械力，以实现正常的生长，发育和健康。我们主要研究拟南芥中两个机械敏感通道家族，即原核通道MscL和MscS及其真核同源物。间充质干细胞和间充质干细胞本质上是拉伸激活的通道，它们在直接施加于双分子层的张力下打开和关闭，因此是蛋白质膜能量学的敏感报告者。阐明这些机械敏感通道在膜环境下的功能将有助于我们理解机械力如何产生生物物理改变，从而导致细胞生理学的适应性变化。目的1：研究MscS和MscS在多种构象状态下的晶体结构。MscL和MscS是已经被晶体学确定的少数门控通道之一。我们的首要任务是改善大肠杆菌通道的结构，但我们也将系统地调查原核同源物和使用分子门挡来捕获处于交替构象状态的通道，以确定结构细节的门挡转变。目的2：分析机械敏感通道与脂质双分子层之间的生物物理相互作用。在理论模型的背景下，将通过诱变和单通道电生理学来探索门控张力、双层厚度和MscL疏水区域宽度之间的耦合。这些研究将剖析不同膜变形项对通道态之间构象平衡的能量贡献。生理上至关重要的水通过间充质干细胞和巨大单层囊泡中的间充质干细胞的渗透将进行体积测量，并与电导测量预测的流体传输特性进行比较。目的3：表征真核msc样通道的功能和结构方面。拟南芥的msc - like （MSL）通道为研究多细胞真核生物中机械敏感通道的结构和功能提供了机会。这些蛋白质的低聚状态、通道特性和结构将被研究。我们还将使用一系列新的和已建立的分析来表征它们在渗透休克保护，膜内定位，电生理和细胞器形态控制方面的生物学功能。我们提出的关于MscS的实验，以及上述针对MscS和MscS提出的实验，是朝着揭示膜和细胞背景下MS通道功能的系统方法的开始。力传感是健康细胞生长、形态和发育的一个关键方面。我们将在分子细节上研究如何通过两个拉伸激活膜通道家族实现力传感。