Hearing loss affects tens of millions of Americans. For many of these individuals, the deficit arises from damage to hair cells, the sensory cells of the internal ear. By transducing acoustic and mechanical energy into electrical signals that propagate to the brain, hair cells are essential for our appreciation of sound and sense of balance. Damage to hair cells can result in irreversible loss of hearing or vestibular function. By determining fundamental properties of mechanoelectrical transduction, the proposed experiments aim to ascertain what cellular constituents are required to produce an intact, functioning hair cell. A better understanding of a hair cell's normal functions should facilitate design of rational strategies for amelioration of hearing deficits. Experiments proposed in this application are designed to characterize motor molecules utilized by hair cells. An active motor controls the responsiveness of a hair cell, allowing the cell to adapt to sustained stimuli. This motor could also play an important role in the assembly of a hair bundle during development. The hair bundle's adaptation motor is thus an essential component of the mechanoelectrical transduction apparatus. Myosin molecules are excellent candidates for the active component of this adaptation motor; indeed, a myosin isozyme resides in the hair cell's mechanically sensitive organelle, the hair bundle, and appears to be properly situated to carry out adaptation. We will address four principal issues related to hair-bundle myosin molecules. First, how many myosin isozymes are there in a hair bundle, and where are they located? We expect that determination of the subcellular location of a myosin molecule will suggest its role in the cell. Second, what hair- bundle proteins bind myosin molecules? At lest two important molecules should bind an adaptation motor: a Ca2+-binding element, perhaps calmodulin, and a crosslinking protein that ties together enough motor molecules to produce the force necessary for adaptation. Third, how are myosin molecules regulated? Adaptation is controlled by intracellular Ca2+, and may also be regulated by protein phosphorylation. We will determine whether hair-bundle myosin molecules are phosphorylated, then measure the modulation of myosin's ATPase activity by phosphorylation and Ca2+. The final set of experiments will test whether a specific myosin isozyme carries out adaptation. We will first develop an assay for the adaptation motor that relies on Ca2+-modulated hair-bundle movement in permeabilized hair cells. We will then allow dye-labeled antibodies to enter the permeabilized bundles and to bind to their target myosin molecules. Directing intense laser irradiation upon the bundle, we will then specifically denature those antibodies and any bound myosin molecules. Elimination of Ca2+-dependent bundle movement by this treatment would confirm myosin's role in adaptation.
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