Reconstituting Purified Rat TRPV4 in Lipid Bilayer to Test Its Mechanosensitivity

描述（由申请人提供）：与基于gpcr的视觉、嗅觉和味觉不同，听觉、触觉和其他机械感官的分子机制仍然是神秘的。离子通道如何接受机械力尚不清楚。力可能来自邻近的蛋白质（如细胞骨架）或脂质双分子层。我们已经在功能上将纯细菌机械敏感（MS）通道重组为纯脂质双分子层，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可能适用于动物通道，但我们计划进行直接测试。瞬时受体电位通道TRPV4在HEK细胞和酵母中表达时响应渗透性肿胀。显然，它可以通过施加压力来直接激活表达酵母膜斑块。我们计划从酵母细胞中纯化rTRPV4蛋白，并通过多种方法将其重组为人工脂质体。我们将通过膜片钳取样来研究生物物理学，特别是施加压力的激活。如果实验成功，将证明TRPV4是一种力传感器，并为机械感觉的理化原理提供坚实的基础。为了比较，我们计划重建TRPV1，在生理上不知道是机械敏感的，以测试力对这种TRP蛋白可能的一般影响。

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.