Molecular mechanisms of polymodal TRP channels in regulation of microvascular endothelial function

Transient receptor potential (TRP) channels, which can integrate diverse hemodynamic and chemical stimuli into cellular signaling, play essential roles in cardiovascular health and disease. As one of the most studied vascular TRP channels, TRPV4 is a Ca2+-permeable cation channel broadly expressed in endothelial and other vascular cells contributing to vascular tone regulation, angiogenesis, and various other functions. Accumulating studies have also implicated TRPV4 in many diseases and pathological conditions such as endothelial dysfunction, hypertension, polycystic kidney disease, and Charcot-Marie-Tooth neuropathy. As a polymodal ion channel, TRPV4 is regulated by a range of physical and chemical stimuli such as shear stress, heat, and chemical agonists, as well as by different protein phosphorylation pathways. However, the molecular mechanisms underlying this polymodal regulation remain poorly understood, and this knowledge is essential for understanding the complex roles of TRPV4 in (patho)physiology and developing novel strategies to treat TRPV4-related diseases. The overall goal of this proposal is to define the molecular basis of TRPV4 activation by arachidonic acid (AA) and derivatives as key mediators of shear stress/flow-induced dilation of coronary arterioles from human subjects with coronary artery disease (CAD), and its allosteric regulation by protein phosphorylation. Our specific aims are: 1) establish the molecular basis of TRPV4 activation by endogenous lipid mediators in vascular endothelial cells and human arterioles, and 2) determine the mechanisms of endothelial TRPV4 regulation by protein phosphorylation. To achieve our objectives, we will use a combination of functional, biochemical, structural, and computational approaches, including vessel function, fura-2 Ca2+ imaging, patch-clamp electrophysiology, structure-guided mutagenesis, photoaffinity labeling (PAL), mass spectrometry (MS) analysis of ligand-labeled TRPV4, modern cryo-electron microscopy (cryo-EM), and molecular dynamics (MD) simulations. The proposed studies will advance knowledge of the fundamental mechanisms of TRPV4 activation and regulation, which will provide critical insights for understanding its complex roles in vascular and other systems and may lead to innovative approaches for a broad range of TRPV4-related pathologies.