Cooperative Modulation of Piezo Channels - SUMMARY Mechanosensitive PIEZO channels sense various forms of mechanical deformations within cells and tissues. In the cardiovascular system, these proteins are essential to detect blood and lymph flow, blood pressure, and cardiac contractions. PIEZO1 dysfunctions are associated with various cardiovascular pathologies including hypertension, xerocytosis, dilated cardiomyopathy, and lymphedema. Understanding how these proteins activate in the native cellular context is needed to better understand how mechanical sensations are perceived, and how these diseases emerge and could be treated more effectively. At the molecular level, PIEZO channels possess a non- planar bowl-like structure that creates a curvature in the surrounding membrane, known as “PIEZO membrane footprint”. Mechanical forces that stretch or flatten the PIEZO footprint promote a flattened channel conformation thought to be associated with an open channel. When multiple channels diffuse at a high density in a membrane at rest, their large footprints are predicted to frequently collide. This proposal tests the hypothesis that such collisions remodel adjacent PIEZO footprints, promoting cooperative interactions that favor channel clustering and modulation of channel activity. In Aim1, we use multi-scale molecular dynamics simulations to study the energetics and dynamics of footprint-footprint interactions in large membranes. In Aim2, we use high-speed single molecule Ca2+ imaging to map the mobility and activity of endogenous PIEZO1 channels expressed at the surface of endothelial cells derived from human induced pluripotent stem cells. This will allow us to examine cooperativity between neighboring channels under physiological conditions. In Aim3, we use high-resolution patch clamp electrophysiology to precisely quantify cooperativity by comparing the distributions of multi-channel open probabilities to binomial distributions expected for independent channels. If successful, this project will establish PIEZO1 cooperativity as a potential mechanism to contextually regulate mechanotransduction signaling, enhancing our understanding of its role in cardiovascular health and disease.
