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和响应机械力的独特能力，这一过程称为 机械转导，并且可以赋予表达它们的细胞、组织或细胞器机械敏感性。 耳朵中的声波、皮肤上羽毛的爱抚或动脉血管中的血流都是一些例子 其中振动、压力或拉伸等力诱导刺激会激活机械激活 (MA) 离子 启动一系列事件的通道，使身体能够听到、感知触觉或调节血压。在 在过去的十年中，新型 MA 离子通道（如 PIEZO、K2P、TMC 和 OSCA/TMEM63）的鉴定已取得进展。 揭示了它们在许多生理过程中的重要性，但这些通道如何感知的机制细节 令人惊讶的是，力量是不完整的。阻碍该领域理解 MA 通道门控的主要挑战 机制是缺乏忠实地复制力的传导的通道激活方法， 生理环境。该提案旨在将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，这将有助于更深入地研究这些通道 轻松、精确和细节。 在体内，机械刺激会施加力，从而改变细胞膜内的张力，其中 存在机械敏感性蛋白质。 MA 离子通道检测到膜张力的这种变化，导致通道 激活。激活 MA 通道的传统体外技术相当粗糙，包括推动 使用钝的玻璃探针或通过施加压力拉伸膜。虽然这些技术 改变膜张力已能够测量 MA 通道活性，它们是间接的、低通量的、 并且对生理刺激的模仿能力较差。在这里我建议通过直接靶向脂质来调节膜张力 其包含使用光可切换脂质（或光脂质）的通道。光致变色剂的加入 偶氮苯分子进入脂肪酰基链，在脂质经历顺反式时提供对脂质的光学控制 经UV-A和蓝光照射时发生异构化。因此，偶氮苯修饰的脂质可用于 用光可逆地操纵膜结构，可以直接激活或调节MA离子 渠道活动。使用真正的 MA 离子通道系列 OSCA，我们将筛选和优化光脂质，以 在细胞尺度上选择性改变膜特性并通过电生理学测定 MA 通道活性 和/或钙成像。这种独特的策略将与结构、功能和药理学相结合 研究以获得更好的视角来了解 MA 离子通道功能的细胞和分子基础。 最终，这项工作还将带来对机械传导过程更深入的机械理解 驱动人类重要的生理和病理状态。