Project Summary The most fundamental event in the initiation of hearing is the conversion of sound into an electrical signal by the opening of ion channels in hair cells. We know that TMC1 and TMC2 are the pore-forming subunits of the mechanotransduction complex, we know the general tertiary structure of TMCs, and we know which transmembrane domains constitute the pore. But we now need to know specifically how the sound-evoked increase in tip-link tension leads to channel opening: how does PCDH15 in the tip link attach to TMC1, what moves in TMC1 to open the pore, and how does tension propagate through the TMC channel to cause that opening? In this project we will first identify the parts of PCDH15 that bind to TMC1, using a combination of co- immunoprecipitation, biolayer interferometry, protein biochemistry, site-directed mutagenesis, and single-cell electrophysiology. Mutagenesis will be guided by cryo-electron microscopy of a related TMC channel and by new atomic structures generated by structure-prediction software such as AlphaFold2. We will then identify the parts of TMC1 that bind to PCDH15, using the same methods. In parallel, we will evaluate recent data from other laboratories on regions of LHFPL5 that bind to TMC1. To understand the gating of TMC1, we will use additional protein structure predictions from AlphaFold2 to identify different conformational states of TMC1 that may represent open and closed states of the channel. Combining knowledge of the binding interface and the predicted gating transitions, we can use molecular dynamics to determine whether and how the application of force—through either PCDH15 or LHFPL5—tends to produce channel opening. We will then test the models generated by molecular dynamics, by introducing cysteine residues at key locations in TMC and transiently linking them with heavy metal ions or bivalent MTS reagents. These bridges will lock TMC in one or another conformation. By expressing different cysteine mutants in hair cells and applying these reagents while recording transduction currents, we can determine whether open or closed states are favored, and thereby test the predicted gating movements. We will also test whether predicted movement of an intracellular loop drives the gating movement, by inserting flexible peptide sequences. This will generate an atomic-level understanding of mechanotransduction, unprecedented for hair cells or for any eukaryotic force-gated ion channel. It will also provide deep insight into the function of TMC1 and PCDH15, and how mutations in these proteins cause hereditary deafness.