Project Summary/Abstract The body’s ability to sense and respond to mechanical forces, or mechanosensation, is critical to a variety of physiological processes from macroscopic processes like regulation of blood pressure and breathing to small- scale processes like cell migration, cell fate determination, and cell alignment during tissue development. Despite the importance of mechanosensation in human heath, the details governing pressure sensing in different tissues and organs remain unknown. Mechanosensitive ion channels called PIEZO channels that respond to stretching or compression of the plasma membrane are now known to play an important role in mechanical signaling within cells, but the ability of PIEZO channels to tune their response over a vast dynamic range of forces in different cell types is still a mystery. Evidence points to the existence of distinct PIEZO interaction networks in different cell types (e.g., attachments to the cytoskeleton, extracellular matrix, and/or interactions with other protein auxiliary subunits) that enable calibration of PIEZO mechanotransduction. Reliable identification of such interactions has faced significant obstacles due to gaps in technology for studying ion channels. This proposal will aim to address these gaps by developing a new proteomic mass spectrometry-based approach for mapping PIEZO protein networks that sense cellular force. Success of this project will provide insight into many mechanically mediated processes that are poorly understood in human health and disease and provide a generalizable new technique for defining of ion channel interaction networks on the cell surface. The research training for this project will be conducted with joint mentorship in the Patapoutian and Cravatt labs at the Scripps Research Institute, two well-equipped laboratories with expertise in ion channel physiology and chemical biology/proteomic mass spectrometry, respectively. Regularly occurring joint meetings with the sponsor and co-sponsor will provide an adequate training environment to learn new techniques and facilitate career development goals for the duration of the fellowship.