Project Summary Many cardiovascular and neurological disorders result from changes in cell mechanics. Assessment of human pathophysiology in this context reveals that these diseases share a root cause: abnormal mechanotransduction – the process by which cells respond to physical stress and forces. Mechanosensitive ion channels convert external forces into electrical response and are emerging targets of interest for understanding biological processes and therapeutic development. The PIEZO family (PIEZO1 and PIEZO2) was discovered in 2010 as the first excitatory mechanosensitive ion channels in vertebrates. PIEZO channels are 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. PIEZO dysfunction has been 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. Our lab’s goal is to understand how physical forces such as pressure and membrane tension control PIEZO1 function. The parent research proposal focuses on ion permeation and force-dependent gating mechanisms of PIEZO1 channels in cells and reconstituted lipid bilayer systems. We will employ biochemical and biophysical techniques to understand how lipid bilayers 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 and functionally identified pore domain of PIEZO1 will be used as a template to understand the pressure sensitivity and voltage-dependent inactivation, hallmarks of PIEZO channels. To study global effect of PIEZO in cellular context, calcium flux assay will be used. The preliminary data is striking and shows the droplet bilayer approach coupled with traditional cellular patch clamp assays are ideally suited to study mammalian PIEZO1 channel function. Our unique proposal represents the application of single molecule investigation of PIEZOs. Completion of this proposal will provide a path to development of effective therapeutics targeting neuropathic pain, brain ischemia and gliomas, amongst others.