PROJECT SUMMARY Vibrations from sound and mechanical stimuli from head movements are transformed into electrochemical signals for brain processing by inner-ear hair-cell mechanoreceptors mediating our senses of hearing and balance. Essential to hair-cell function are the proteins that form its mechanotransduction apparatus comprised of a fine tip-link filament that pulls on an ion channel complex to trigger sensory perception. The tip-link filament is formed by cadherin-23 (CDH23) and protocadherin-15 (PCDH15) proteins while the ion channel complex is thought to be formed by members of the transmembrane channel-like protein family TMC1 and TMC2, the transmembrane inner ear protein TMIE, and the tetraspan membrane protein of hair-cell stereocilia TMHS (also known as LHFPL5). In addition, the calcium and integrin binding protein CIB2 binds to TMC channels to regulate mechanotransduction. All these proteins are important for hearing and balance and are involved in inherited deafness, yet their molecular structures and the functional architecture of the transduction complex they form are poorly understood. The overall long-term goal of this project is to reveal the structural determinants of function for the proteins forming the inner-ear tip link and transduction ion channel complex. In Aim 1, we will use cryo-electron microscopy, high-speed atomic force microscopy, and molecular dynamics simulations to study the full-length extracellular domains of CDH23 and PCDH15 and thereby establish the structural determinants of tip-link function in inner ear mechanotransduction. In Aim 2, we will generate testable predictions using microsecond-long molecular dynamics simulations with biasing membrane potentials to characterize permeation of ions and ototoxic aminoglycosides through experimentally validated structural models of TMC protein pores. In Aim 3, we will use various computational and biophysical techniques, including nuclear magnetic resonance and native mass spectrometry, to explore regulatory mechanisms of transduction by CIB proteins. Results obtained from the proposed experiments and simulations will provide an initial and dynamic molecular view of the protein components of the inner ear mechanotransduction apparatus as we advance to understand its architecture and function in normal and impaired hearing and balance.