DESCRIPTION (provided by applicant): The TMC1 and TMC2 membrane proteins are critical components of the transduction complex in vertebrate hair cells, which converts the vibration of sound to an electrical signal, and it is likely they are central subunits of the long-sought transduction channel. However little is known about these proteins at a molecular level, other than their primary amino acid sequence. In this project we will determine the structure of TMC channels at atomic resolution to determine their topology, stoichiometry, permeation pathway, gating, and interaction with other subunits of the complex. We will first use biochemical methods to determine both the stoichiometry of the native channel, and the interaction of TMC1 and TMC2 with each other and with the other six members of the TMC family. We will then synthesize and purify a TMC channel, and determine its structure with electron microscopy. Low-resolution EM structures will confirm stoichiometry and tertiary structure, and may reveal a pore domain. High-resolution cryo-EM images will enable the solution of atomic structures of TMCs in different states. This will reveal a putative pore domain and possible gating movements. Finally, we will test the structure with cysteine mutagenesis of TMC1 and physiological recording. Individual amino acids will be replaced with cysteine and the cys-mutant TMC1 will be expressed in hair cells lacking wild-type TMC1 and TMC2. Cysteine-modifying reagents will be applied during physiological recording; their effects on permeation and gating will be correlated with the predicted structure. Solution of the TMC structure will answer questions about the mechanism of hearing that have stood for over 50 years. It will accelerate our understanding of how the entire transduction complex assembles and functions, and it will lead to molecular understanding of inherited deafness.