Reconstituting Purified Rat TRPV4 in Lipid Bilayer to Test Its Mechanosensitivity

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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.