Cardiovascular drug target, TRPV2

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The cation channel TRPV2 is important in development and function of the cardiovascular system. Multiple studies suggest it is gated in response to mechanical stimulus, but the mechanism appears to be indirect and is not yet known. Drugs targeting either activation or inhibition of TRPV2 have been reported to have beneficial effects on pathological cardiovascular symptoms in both animal models and clinical studies, but the available drugs have poor specificity and potency. The goal of this project is to develop a better understanding of the structural and cellular basis of TRPV2 regulation, and to begin the process of developing more specific and efficacious inhibitors and activators. Specific Aim 1 is to determine the effects of phosphoinositides and mechanical stress on TRPV2 activity in a purified and reconstituted state. Phosphoinositide composition will be manipulated and channel activity with and without added agonists and inhibitors, or in response to mechanical stimulus, will be measured using Ca2+-indicator dyes or with patch-clamp electrophysiology. These experiments will test the hypothesis that TRPV2 is a constitutively active channel and subject to negative regulation by phosphatidylinositol (4,5) bisphosphate (PIP2), as well as the counter hypotheses that PIP2 is stimulatory or alters channel selectivity or that effects of phosphoinositides are indirect. They will also test the hypothesis that TRPV2 is directly mechanosensitive. Specific Aim 2 is to determine the effects of phosphoinositides and mechanical stress on TRPV2 activity in the context of cellular membranes. TRPV2 will be expressed in cultured cells and assayed using Ca2+-flux assays and patch clamp experiments. Expression of recombinant enzymes engineered to respond to extracellular ligands, or to membrane voltage, as well as fluorescently tagged phosphoinositide binding domains and inhibitors, will be used to manipulate and monitor phosphoinositides on the cytoplasmic face of the membrane. These experiments will test the hypothesis that TRPV2 is regulated by intracellular phosphoinositides and membrane tension in a cellular context. Specific Aim 3: Determine the structural basis for regulation of TRPV2. The structure of TRPV2 in lipid nanodics will be determined in the presence of phosphoinositides as well as agonists and inhibitors using cryo-electron microscopy. Mutagenesis experiments will target residues proposed to play important roles in regulation by phosphoinositides or other effectors. Mutations will be tested for their effects on function and structure. These experiments will further test the hypothesis that direct interactions mediate the effects of these regulatory molecules and identify the structural basis for those which do so. Accomplishment of these aims will make a major contribution to our understanding of the general principles governing the regulation of ion channels of the TRPV family, clarify the mechanistic basis for the roles of TRPV2 in normal physiology and disease, and help to guide future therapeutic efforts relying on TRPV2 regulation as a target.