Rational design of treatments directed at amelioration of hearing or vestibular deficits must arise from a basic understanding of hair-cell function. The integrity of our hair cells' mechanoelectrical transduction apparatus, a molecular structure that converts acoustic or accelerational energy into electrical signals, is essential for our senses of hearing and balance. At minimum, this apparatus consists of a mechanically gated ion channel, a gating spring through which forces are conveyed to the channel, and an adaptation motor that ensures that transduction channels are optimally poised to detect the most minute forces. Although considerable progress has been made towards the biophysical characterization of the hair-cell transduction apparatus, biochemical identification of the molecules responsible for transduction has proved more difficult. Our recent biochemical and biophysical experiments have provided strong evidence that a particular isozyme of myosin, a member of a class of enzymes that generate force by hydrolyzing ATP, is a major component of the hair cell's adaptation motor. This isozyme - myosin I- beta - has been purified and cloned. Useful tools such as monoclonal antibodies are thus available for its study. In the proposed research, we will develop an in vitro assay that will allow detailed molecular dissection of the properties and identity of the adaptation motor. Because adaptation has formerly been assayed by measuring changes in the hair cell's transduction current using microelectrodes, it has been difficult to fully characterize this motor. As an alternative approach, we will permeabilize hair cells and elicit hair-bundle movement - produced by the adaptation motor - that is under our control. This assay should greatly assist biochemical characterization of the motor. This permeabilized hair-cell preparation will be employed to determine the cellular constituents, like ATP and Ca2+, that are required for adaptation, and to determine the motor's force and velocity dependence on the concentrations of those constituents. These studies will allow us a more complete understanding of the biophysical properties of the motor, and should aid in understanding adaptation in the intact hair cell. We will also directly test whether myosin I-beta carries out adaptation. Using the permeabilized hair cell preparation, we will allow dye-labeled antibodies to enter 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 adaptation-motor-dependent bundle movement by this treatment would confirm myosin's role in adaptation.
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