A New Approach to Study Mechanically Activated Ion Channels

项目摘要。 一些蛋白质具有独特的能力，可以感知和响应机械力，这一过程被称为 这些细胞可以是机械转导，并且可以赋予表达它们的细胞、组织或细胞器机械敏感性。 耳朵里的声波、羽毛在皮肤上的抚摸或动脉血管中的血流都是一些例子 其中诸如振动、压力或拉伸的力诱导刺激激活机械激活（MA）离子 启动级联事件的通道，使身体能够听到，感觉触摸或调节血压。在 在过去的十年中，新的MA离子通道如PIEZO、K2 Ps、TMC和OSCA/TMEM 63的鉴定 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 力量是不完整的阻碍该领域理解MA通道门控的主要挑战 机制是缺乏一个通道激活方法，忠实地复制力的转导， 生理环境。该提案旨在应用光开关脂质作为一种新的创新方法 测定机械活化（MA）离子通道，这将有助于这些通道的研究， 轻松、精确和细节。 在体内，机械刺激施加力，这改变了细胞膜内的张力， 机械敏感蛋白质存在。MA离子通道检测膜张力的这种变化，导致通道 activation.传统的激活MA通道的体外技术相当粗糙， 用钝的玻璃探针或通过施加压力拉伸膜。虽然这些技术 改变膜张力已经能够测量MA通道活性，它们是间接的，低通量的， 并且很难模仿生理刺激。在这里，我建议通过直接靶向脂质来调节膜张力 其使用可光转换的脂质（或光脂质）包围通道。加入光致变色 分子偶氮苯进入脂肪酰基链提供了光学控制脂质，因为它们经历顺-反 当用UV-A和蓝光照射时异构化。因此，偶氮苯修饰的脂质可用于 用光可逆地操纵膜结构，其可以直接激活或调节MA离子 渠道活动。使用真正的MA离子通道家族OSCA，我们将筛选和优化光脂， 选择性地改变细胞膜的性质，用电生理学方法测定MA通道的活性 和/或钙成像。这种独特的策略将结合结构，功能和药理学 研究，以获得对MA离子通道功能的细胞和分子基础的更好的观点。 最终，这项工作也将导致机械转导过程的更深层次的机械理解 驱动着人类重要的生理和病理状态。

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.