TMC gene function in sensory hair cells

Boston Children'S Hospital, Boston MA

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Abstract

PROJECT SUMMARY Mutations in transmembrane channel-like gene 1 (TMC1) underlie dominant, progressive hearing loss (DFNA36) and recessive nonsyndromic hearing loss (DFNB7/B11) in humans (Kurima et al., 2002). Similarly, semidominant and recessive alleles of Tmc1 cause hearing loss in Beethoven (Bth) and deafness (dn) mutant mice (Vreugde et al.,2002; Kurima et al., 2002). Tmc1 is a member of the Tmc gene family that includes seven other paralogs in mammals (Keresztes et al., 2003). Tmc1 and closely related Tmc2 are expressed in auditory and vestibular hair cells of the mouse inner ear and are necessary for mechanosensory transduction. In prior work, funded by this RO1, we demonstrated that TMC1 is a pore-forming subunit of the hair cell transduction channel and contains four transmembrane domains (S4-S7) that line the ion channel pore (Pan et al., 2018). With compelling evidence in hand demonstrating that TMC1 is a major component of the channel, we can now use this information to tackle both basic science and translational research questions that were previously intractable. This project is organized around five independent, but related specific aims focused on understanding the structure and function of TMC1 in isolated systems and in native hair cells. In addition, we will explore novel techniques for restoration of auditory function in mouse models of the human genetic inner ear disorders DFNA36 and DFNB7/11. We have optimized protocols for expression and purification of high-quality mammalian TMC1 protein. For aims 1 and 2, we will use purified protein to generate the cryo-EM structures of mammalian TMC1 and will examine the function of isolated TMC1 channels in heterologous systems. For aim 3, we will characterize the N- and C- termini of TMC1 and TMC2 and identify motifs of these regions that confer unique properties to these proteins. We will express TMC1 and TMC2 proteins bearing N-terminal mutations in hair cells of Tmc1/Tmc2 double mutant mice and assay for changes in the properties of hair cell sensory transduction. Likewise, for aim 4, we will examine the interaction between TMC1 and its binding partner TMIE using Tmie-null mice. We will express WT or mutant TMIE constructs in hair cells of Tmie-null mice and assay for functional differences in hair cell transduction. For aim 5 we will explore a novel therapeutic approach for targeting all dominant and recessive Tmc1 mutations in auditory and vestibular hair cells using a single therapeutic strategy. Based on new information about the structure and function of TMC1, projects included in this proposal will allow us to expand our understanding of sensory transduction in auditory hair cells and develop a cutting-edge translational approach suitable for treating both dominant and recessive TMC1 mutations that cause genetic hearing loss in humans.