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.

Public Health Relevance

The molecular core of human hearing is the transduction channel complex that is opened by the mechanical stimulus of sound and that enables electrical current flow to initiate a neural signal. This complex is composed of several proteins including TMC1 and TMC2. Understanding their atomic structure will solve one of the fundamental questions in auditory science, and will both reveal how inherited mutations in these proteins cause deafness and suggest how such deafness might be treated.

Agency
National Institute of Health (NIH)
Institute
National Institute on Deafness and Other Communication Disorders (NIDCD)
Type
Research Project (R01)
Project #
5R01DC000304-33
Application #
9414012
Study Section
Auditory System Study Section (AUD)
Program Officer
Freeman, Nancy
Project Start
1984-09-01
Project End
2021-01-31
Budget Start
2018-02-01
Budget End
2019-01-31
Support Year
33
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Harvard Medical School
Department
Biology
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
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