Reconstituting Purified Rat TRPV4 in Lipid Bilayer to Test Its Mechanosensitivity

描述(申请人提供)：与基于GPCR的视觉、嗅觉和味觉不同，听觉、触觉和其他机械感官的分子机制仍然神秘。离子通道是如何接收机械力的，目前还不清楚。作用力可能来自邻近的蛋白质(如细胞骨架)或来自脂双层。我们已经将纯细菌机械敏感(MS)通道功能重组为纯脂双层，毫无疑问，在这些情况下通道门控力来自双分子层。虽然有间接证据表明这一原则也适用于动物通道，但我们计划进行一次直接测试。瞬时受体电位通道TRPV4在HEK细胞和酵母中表达时对渗透肿胀有反应。它显然可以通过施加压力直接激活，以表达酵母膜斑块。我们计划从酵母细胞中纯化rTRPV4蛋白，并通过几种方法将其重组为人工脂质体。我们将通过膜片钳采样来检测生物物理，特别是外加压力的激活。如果实验成功，将证明TRPV4的S是一种力传感器，并为机械传感的物理化学原理提供了坚实的基础。为了进行比较，我们计划重建TRPV1，以测试力对这样的Trp蛋白可能产生的一般影响。 与公共健康相关：离子通道的机械敏感性是听觉、触觉、血压感觉、全身渗透压等生理和病理的基础。了解通道蛋白如何受力是基础。机械敏感的TRPV4通道的分子机制直接影响人类健康：TRPV4突变导致人类常染色体显性遗传性短冻症。

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.