A New Approach to Study Mechanically Activated Ion Channels

研究机械激活离子通道的新方法

• 批准号: 10242489
• 负责人: Swetha Murthy
• 金额: $ 130.5万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-23 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10242489
• 关键词: Atherosclerosis; Biological; Biological Assay; Biophysics; Blood flow; Calcium; Cell membrane; Cells; Disease; Ear; Electrophysiology (science); Environment; Event; Family; Feathers; Glass; Hearing; Human; Image; In Vitro; Ion Channel; Lead; Light; Lipids; Measurement; Mechanical Stimulation; Mechanics; Membrane; Methods; Molecular; Muscle Contraction; Muscular Dystrophies; Optics; Organelles; Pathologic; Pharmacology; Pharmacology Study; Physiological; Physiological Processes; Process; Property; Proteins; Role; Signal Transduction; Skin; Stimulus; Stretching; Structure; Techniques; Tissues; Touch sensation; Work; azobenzene; blood pressure regulation; chronic pain; cis trans isomerization; in vivo; innovation; mechanical force; mechanotransduction; novel; novel strategies; pain sensation; pressure; sound; vibration

Project summary. Some proteins have the unique ability to sense and respond to mechanical force, a process called mechanotransduction, and can confer mechanosensitivity to cells, tissue, or organelles that express them. Sound waves in the ear, caress of a feather on the skin, or blood flow in arterial vessels are some instances where a force-inducing stimulus such as vibration, pressure, or stretch activates mechanically activated (MA) ion channels that initiate a cascade of events allowing the body to hear, sense touch, or regulate blood pressure. In the past decade, identification of novel MA ion channels like PIEZOs, K2Ps, TMCs, and OSCA/TMEM63s has revealed their importance in many physiological processes, but mechanistic details of how these channels sense force is, strikingly, incomplete. A major challenge impeding the field in comprehending MA channel gating mechanisms is the lack of a channel activation method that faithfully replicates the transduction of force, within a physiological environment. This proposal aims to apply photoswitchable lipids as a new and innovative method to assay mechanically activated (MA) ion channels, which will facilitate the study of these channels with greater ease, precision, and detail. In vivo, mechanical stimulation exerts force, which alters tension within the cell membrane where mechanosensitive proteins reside. MA ion channels detect this change in membrane tension leading to channel activation. Traditional in vitro techniques to activate MA channels are rather crude including pushing on membrane with a blunt glass probe or stretching the membrane by applying pressure. Although these techniques to alter membrane tension have enabled measurement of MA channel activity, they are indirect, low-throughput, and poorly mimic physiological stimuli. Here I propose to modulate membrane tension by directly targeting lipids that encompass the channel using photoswitchable lipids (or photolipids). Incorporation of the photochromic molecule azobenzene into fatty acyl chains provides optical control over lipids as they undergo cis-trans isomerization when irradiated with UV-A and blue light. Therefore, azobenzene-modified lipids can be used to reversibly manipulate membrane structure with light, which can either directly activate or modulate MA ion channel activity. Using the bona-fide MA ion channel family OSCAs we will screen and optimize photolipids to selectively change membrane properties on a cellular scale and assay MA channel activity with electrophysiology and/or calcium imaging. This unique strategy will be combined with structural, functional, and pharmacological studies to gain a better perspective on the cellular and molecular underpinnings of MA ion channel functions. Ultimately, this work will also lead to a deeper mechanistic understanding of mechanotransduction processes that drive vital physiological and pathological states in humans.

项目总结。 一些蛋白质具有感知机械力并对其做出反应的独特能力，这一过程称为 机械转导，并能赋予细胞、组织或表达它们的细胞器机械敏感性。 例如，耳朵里的声波，皮肤上的羽毛抚摸，或者动脉血管中的血液流动 力诱导刺激，如振动、压力或拉伸，会激活机械激活(MA)离子 启动一系列事件的通道，使身体能够听到、感觉到触摸或调节血压。在……里面 在过去的十年中，发现了新的MA离子通道，如PIEZO，K2Ps，TMCs和OSCA/TMEM63s 揭示了它们在许多生理过程中的重要性，但这些通道如何意义的机械性细节 令人惊讶的是，武力是不完整的。阻碍该领域理解移动通信信道门控的主要挑战 机制是缺乏一种忠实地复制内力传递的通道激活方法 生理环境。这项提议旨在应用可光开关脂质作为一种新的创新方法。 测定机械激活(MA)离子通道，这将有助于对这些通道进行更多的研究 轻松、精确和细节。 在体内，机械刺激施加力，改变细胞膜内的张力， 对机械敏感的蛋白质存在。MA离子通道检测到导致通道的膜张力的这种变化 激活。传统的激活MA通道的体外技术相当粗糙，包括推动 用钝的玻璃探头或通过施加压力拉伸膜。尽管这些技术 改变膜张力使得能够测量MA通道活性，它们是间接、低通量、 对生理刺激的模仿能力很差。在这里，我建议通过直接靶向脂类来调节膜张力 使用可光切换的脂类(或光脂)包围通道。光致变色剂的掺入 脂肪酰链上的偶氮苯分子在脂类发生顺式反式反应时提供光学控制 在UV-A和蓝光照射下发生异构化。因此，偶氮苯修饰的脂类可用于 用光可逆操纵膜结构，可直接激活或调制MA离子 通道活动。使用真正的MA离子通道家族OSCA，我们将筛选和优化光脂以 在细胞尺度上有选择地改变膜的性质，并用电生理学方法测定MA通道的活性 和/或钙成像。这一独特的策略将与结构、功能和药理学相结合 研究以更好地了解MA离子通道功能的细胞和分子基础。 最终，这项工作还将导致对机械转导过程的更深层次的机械理解 会导致人类的重要生理和病理状态。