This work is designed to understand how hair cells, the sensory cells of the inner ear, use their mechanically- sensitive hair bundle to convert sound to electrical signals. Among other approaches, we identify and quantify proteins in purified bundles using mass spectrometry. Focusing on molecular complexes that contribute to bundle function, our experiments address two fundamental questions: First, how is the hair bundle formed? Second, how does the mechanotransduction complex work at a molecular level? In Aim 1, we will identify protein complexes including radixin, an action-to-membrane crosslinker that is essential for hair-bundle cytoskeleton structure, and SLC9A3R2, a PDZ-domain adaptor protein that binds to radixin. We will also express dominant-negative radixin and SLC9A3R2 constructs to determine how complexes with these proteins control bundle structure.
In Aims 2 and 3, we will continue our efforts to identify and characterize the transduction channel itself. We have a pair of strong candidates for the channel, members of the transient receptor potential (TRP) channel family. We propose to locate the channels within stereocilia, determine their interactions with other known members of the transduction complex, and examine mechanotransduction in mice missing one or both of the channels. Ongoing experiments could prove that these TRP channels are not the transduction channel, however, so Aim 3 proposes to improve our ongoing biochemical preparation of the transduction complex, as well as to identify transmembrane proteins in purified stereocilia membranes. Research proposed here will show how mechanotransduction operates in the normal inner ear. As stated in the most recent strategic plan, one of the major goals of the NIDCD is to use """"""""genomic, proteomic, informatic, bioinformatic, and expression...approaches...to understand the molecular bases of normal and disordered [hearing and balance]."""""""" Understanding how the bundle is assembled and how its transduction machinery normally operates, the focus of the proposed research, is essential for rational design of therapies for hearing loss and balance disorders.

Public Health Relevance

These experiments will allow us to understand how the hair bundle, the component of the inner ear that converts sound to neural signals, operates. Our study will reveal how several molecules of known importance to the hair bundle, which mediate its assembly and mechanical-to-electrical conversion activity, carry out their roles. More significantly, these experiments will allow us to design rational approaches to detecting and ameliorating hearing loss and disrupted balance.

Agency
National Institute of Health (NIH)
Institute
National Institute on Deafness and Other Communication Disorders (NIDCD)
Type
Research Project (R01)
Project #
5R01DC002368-20
Application #
8691779
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Freeman, Nancy
Project Start
1994-07-01
Project End
2018-06-30
Budget Start
2014-07-01
Budget End
2015-06-30
Support Year
20
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Oregon Health and Science University
Department
Otolaryngology
Type
Schools of Medicine
DUNS #
City
Portland
State
OR
Country
United States
Zip Code
97239
Ren, Tianying; He, Wenxuan; Barr-Gillespie, Peter G (2016) Reverse transduction measured in the living cochlea by low-coherence heterodyne interferometry. Nat Commun 7:10282
Ebrahim, Seham; Avenarius, Matthew R; Grati, M'hamed et al. (2016) Stereocilia-staircase spacing is influenced by myosin III motors and their cargos espin-1 and espin-like. Nat Commun 7:10833
Krey, Jocelyn F; Drummond, Meghan; Foster, Sarah et al. (2016) Annexin A5 is the Most Abundant Membrane-Associated Protein in Stereocilia but is Dispensable for Hair-Bundle Development and Function. Sci Rep 6:27221
Geszvain, Kati; Smesrud, Logan; Tebo, Bradley M (2016) Identification of a Third Mn(II) Oxidase Enzyme in Pseudomonas putida GB-1. Appl Environ Microbiol 82:3774-82
Barr-Gillespie, Peter-G (2015) Assembly of hair bundles, an amazing problem for cell biology. Mol Biol Cell 26:2727-32
Francis, Shimon P; Krey, Jocelyn F; Krystofiak, Evan S et al. (2015) A short splice form of Xin-actin binding repeat containing 2 (XIRP2) lacking the Xin repeats is required for maintenance of stereocilia morphology and hearing function. J Neurosci 35:1999-2014
Wilmarth, Phillip A; Krey, Jocelyn F; Shin, Jung-Bum et al. (2015) Hair-bundle proteomes of avian and mammalian inner-ear utricles. Sci Data 2:150074
Müller, Ulrich; Barr-Gillespie, Peter G (2015) New treatment options for hearing loss. Nat Rev Drug Discov 14:346-65
Krey, Jocelyn F; Sherman, Nicholas E; Jeffery, Erin D et al. (2015) The proteome of mouse vestibular hair bundles over development. Sci Data 2:150047
Cugusi, Simona; Kallappagoudar, Satish; Ling, Huiping et al. (2015) The Drosophila Helicase Maleless (MLE) is Implicated in Functions Distinct From its Role in Dosage Compensation. Mol Cell Proteomics 14:1478-88

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