Project Summary Many cardiovascular and neurological disorders, and oncogenesis result from changes in cell mechanics. Assessment of human pathophysiology in this context reveals that these diseases share a common root cause: abnormal mechanotransduction – the process by which cells respond to physical stress and forces. Mechanosensitive ion channels, the molecular machines by which cells convert external forces into electrical response, are therefore emerging targets of interest, for understanding biological processes and for therapeutic development. Piezo family (Piezo1 and Piezo2) was discovered in 2010 as the first excitatory mechanosensitive ion channels in vertebrates. Piezo channels are now known to be critical sensors of touch and pain (somatosensation), volume regulation (osmosensation), shear stress (cardiovascular tone), baroreception, proprioception and respiratory physiology, and may have other important functions yet to be discovered. Substantial efforts are made in the last decade to identify Piezo related diseases and incidents within the United State population. So far, Piezo dysfunction is linked to diverse pathologies including hypertension, lymphatic disease and anemias, somatosensory and neurological disorders, cancer and metastasis, amongst others. Despite their biological and medical relevance, the mechanism behind Piezo-dependent mechanotransduction remains elusive. Therefore, our lab’s goal is to understand how physical forces such as pressure and membrane tension control Piezo1 function in health and diseased state. This research proposal focuses on ion permeation and force-dependent gating mechanisms of Piezo1 channels, in cells, as well as in reconstituted lipid bilayer systems. We will employ biochemical and biophysical techniques in efforts to understand how lipid bilayer control the gating of Piezo1 and subsequent ion conduction across the membrane. Moreover, we have identified robust expression and protein purification protocols to examine the function of Piezo1 channels. Droplet lipid bilayers will be used to study the single channel conductance and open probability of the purified protein in biologically relevant lipid compositions. Structurally identified pore domain of Piezo1 will be used as a template to understand the pressure sensitivity and voltage-dependent inactivation - hallmark of Piezo channels - by constructing various deletion mutants- heterologous expression in HEK cells. The preliminary data is striking, and shows that the droplet bilayer approach coupled with traditional cellular patch clamp assays are ideally suited to study mammalian Piezo1 channel function. We are convinced that a comprehensive understanding of Piezo’s function is a timely contribution to the field of mammalian mechanotransduction. Our unique proposal represents the application of single molecule investigation of Piezos. Completion of this proposal will provide a path to dissect and kick-start the development of effective thera...