Exploring the coupling between PIEZO1 subunits gating motions using TIRF

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Abstract The ability of cells to rapidly sense and respond to mechanical stimuli is vital for all living beings. In vertebrates, this task is mainly achieved by mechanosensitive ion channels PIEZO1 and PIEZO2. Indeed, a growing number of studies have outlined the central role played by these channels to numerous biological processes including sensory physiology, osmotic homeostasis, and organ development. Not surprisingly, abnormal PIEZO channel activity is associated with many clinical conditions such as lymphedema, anemia, arthrogryposis, cancer, inflammation, and pain. These proteins are formed by three long polypeptide chains (subunits) assembled around a central ion conduction pathway (pore). The first specific Aim of our awarded project consists of identifying and characterizing specific conformational rearrangements taking place in these subunits as the channel opens (gates) its pore upon mechanical stimulation. Recent studies from our team and others suggest that the gating motion of one subunit may cooperatively influence that of the others. The purpose of the requested administrative supplement is to test this hypothesis. To this aim, we intend to purchase a Total Internal Reflection Fluorescence (TIRF) upgrade for our epifluroescence microscope. TIRF measurements will enable single-molecule fluorescence measurements of PIEZO1 channels in which each subunit is genetically-tagged with a shear-stress sensitive fluorescence probe. Our new epifluorescence data indicate that these probes emit large fluorescence signals that correlate with pore opening when channels are stimulated by fluid shear stress. We anticipate that the flow-mediated gating motion of each subunit will be accompanied by a discrete, jump-like increase of pixel brightness in TIRF images. If this gating motion is coupled, these discrete jumps are predicted to be temporally correlated. If not, these fluorescence jumps are predicted to occur independently. If successful, these experiments will deepen our fundamental understanding of how these essential ion channels open their pore in response to a physiological stimulus.