Project Summary Auditory mechanotransduction channels reside at the tips of hair-cell stereocilia, where they mediate the conversion of sound-induced mechanical stimuli into electrical signals that are transmitted to the brain. These channels open in response to mechanical force and allow a selective influx of cations into hair cells. During gating, a channel component is thought to undergo a particularly large conformational change - with an estimated gating movement of ~ 4 nm. Their molecular identity has been pursued for over two decades. Recently, we provided strong evidence that TMC1 forms the pore of the auditory transduction channels and bears structural similarity to the TMEM16 family of ion channels. But we still don’t know how the channel works at a molecular level. This is a fundamental aspect of hearing, as it underlies the conversion of sound into neural signals by the mechanotransduction complex. The proposed research will integrate protein biochemistry, molecular engineering, and hair cell physiology to probe the gating domains of TMC1. It will pave a path to more extensive studies combining structural and functional biochemistry with single-cell physiology to provide a comprehensive molecular characterization of how mechanotransduction channels open in response to force.