Dynamics and heteromerization of PIEZO channel subunits - SUMMARY The rapid detection of mechanical stress - and adaptation to it - is essential to all organisms. In vertebrates, this task is mainly achieved by two homotrimeric ion channels called PIEZO1 and PIEZO2. Together, these mechanosensitive channels transduce a rich diversity of mechanical forces, such as compression, vibration, stretch, torque, and shear stress, into cellular signals that play essential roles across all physiological systems. To date, it remains unclear how such a complex landscape of mechanical stimuli can be effectively transduced by the two PIEZO mechanotransducers. In Aim1 of this exploratory proposal, we address this question by testing the hypothesis that PIEZO1 and PIEZO2 subunits interact to form heterotrimeric channels, potentially expanding the functional properties of PIEZO channels. To fulfill this aim, we will use bimolecular fluorescence complementation (BFC), co-immunoprecipitation and electrophysiology. Our preliminary data reveals that, when co-expressed in the same cells, the PIEZO1 and PIEZO2 subunits position their C-termini within nanometric proximity, consistent with both subunits co-existing within the same channel. Co- immunoprecipitation with validated antibodies will allow us to test whether wild-type PIEZO1/2 subunits also interact. To test whether these heteromers consist of functional channels, we will use a novel functional BFC approach (based on a split fluorescent calcium indicator) that we have created and validated. In Aim2, we will test the hypothesis that different mechanical stimuli (including indentations, fluid shear stress, and osmotic shocks) activate PIEZO channels through distinct conformational rearrangements of mechanosensory blade- like domains. To this aim, we will measure intramolecular fluorescence resonance energy transfer (FRET) from donor/acceptor probes genetically-inserted at strategic positions of adjacent PIEZO1 blades. Our preliminary data shows that FRET efficiency decreases as a response to osmotic shocks. FRET will be measured using epifluorescence, total internal reflection fluorescence and confocal imaging. We will determine if a correlation exists between changes in FRET efficiency and the nature of the mechanical stimulus. If successful, this exploratory R21 proposal will advance our fundamental understanding of mechanotransduction by mammalian PIEZO channels, provide innovative research tools, and contribute to elucidating the molecular bases of force- based therapeutic interventions.
