Reconstituting Purified Rat TRPV4 in Lipid Bilayer to Test Its Mechanosensitivity

在脂质双层中重建纯化的大鼠 TRPV4 以测试其机械敏感性

• 批准号: 7921363
• 负责人: CHING KUNG
• 金额: $ 7.35万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2011-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7921363
• 关键词: Animals; Biophysics; Blood Pressure; Cells; Chemicals; Cytoskeleton; Foundations; Health; Hearing; Human; Ion Channel; Left; Lipid Bilayers; Liposomes; Mechanics; Membrane; Methods; Molecular; Mutation; Osmolar Concentration; Pathology; Physiology; Procedures; Proteins; Rattus; Sampling; Smell Perception; Solid; Swelling; TRP channel; TRPV1 gene; Taste Perception; Testing; Touch sensation; Uncertainty; Vision; Yeasts; base; patch clamp; pressure; public health relevance; receptor; reconstitution; research study; sensor; yeast protein

DESCRIPTION (provided by applicant): Unlike the GPCR-based vision, smell, and taste, the molecular mechanisms of hearing, touch, and other mechanical senses remain mysterious. How ion channels receive mechanical forces is obscure. The force may come from neighboring proteins (e.g. cytoskeleton) or from the lipid bilayer. We have functionally reconstituted pure bacterial mechanosensitive (MS) channels into pure lipid bilayer, leaving no doubt that the channel-gating force is from the bilayer in these cases. Though there is indirect evidence showing that this principle may also apply to animal channels, we plan a direct test. Transient-receptor-potential channel TRPV4 responds to osmotic swelling when expressed in HEK cell and in yeast. It can apparently be activated directly by pressure applied to expressing yeast-membrane patches. We plan to purify rTRPV4 protein from yeast cells and reconstitute it into artificial liposomes by several methods. We will examine the biophysics through patch-clamp sampling, especially the activation by applied pressure. If successful, this experiment will prove TRPV4's being a force sensor and help provide a solid foundation for the physio-chemical principle of mechanosensation. For comparison, we plan to reconstitute TRPV1, not known to be mechanosensitive physiologically, to test for possible general effects of force on such a TRP protein. PUBLIC HEALTH RELEVANCE: Ion-channel mechanosensitivity underlies the physiology and pathology of hearing, touch, the sensing of blood pressure, systemic osmolarity etc. Understanding how a channel protein receives forces is fundamental. The molecular mechanism of the mechanosensitive TRPV4 channel has direct impact on human health: TRPV4 mutations cause autosomal dominant brachyolmia in humans.

描述（由申请人提供）：与基于GPCR的视觉、嗅觉和味觉不同，听觉、触觉和其他机械感觉的分子机制仍然是神秘的。离子通道如何接受机械力还不清楚。力可能来自邻近的蛋白质（例如细胞骨架）或来自脂质双层。我们已经在功能上将纯细菌机械敏感（MS）通道重建成纯脂质双层，毫无疑问，在这些情况下，通道门控力来自双层。虽然有间接的证据表明，这一原则也可能适用于动物通道，我们计划直接测试。瞬时受体电位通道TRPV4在HEK细胞和酵母中表达时对渗透性肿胀有反应。它显然可以直接激活压力施加到表达酵母膜补丁。我们计划从酵母细胞中纯化rTRPV 4蛋白，并通过几种方法将其重组到人工脂质体中。我们将通过膜片钳采样来研究生物物理学，特别是施加压力的激活。如果成功，这个实验将证明TRPV 4是一个力传感器，并有助于为机械感觉的生理化学原理提供坚实的基础。为了进行比较，我们计划重组TRPV1，不知道是机械敏感的生理，测试可能的一般影响力对这样的TRP蛋白。 公共卫生关系：离子通道机械敏感性是听觉、触觉、血压感测、全身渗透压等生理和病理的基础。了解通道蛋白如何接收力是基础。机械敏感性TRPV4通道的分子机制对人类健康有直接影响：TRPV4突变导致人类常染色体显性遗传性短肢症。