When the vibration of sound causes the movement of stereo cilia on the cochlea's hair cells, ion channels at the tips of the stereo cilia open. These protein channels are the fundamental elements in the conversion of sound to a neural signal-in von Bekesy's words """"""""the final mechanical transformer"""""""" for sensory transduction. Yet the protein identity of the hair cell transduction channel is not known. This proposal seeks to discover the transduction channel in vertebrate hair cells with a systematic screen of the TRP channel super family, a group of channels that has been implicated in a variety of sensory modalities, including mechanosensation, and a group that is most likely to harbor the transduction channel. The first step will be to identify all the TRP channel genes in the mouse genomes (estimated at 33). In situ hybridization for each gene will be used to find which are expressed in the hair cells of the inner ear. For likely candidates, antibodies will be generated to confirm hair-cell expression, and to determine whether the candidate protein is located near the transduction apparatus at the tips of the stereo cilia. If a candidate is a component of the transduction apparatus, it should leave the stereo cilia when the tip links are cut, as found for the tip-link protein cadherin23, and so candidate channel immunoreactivity should redistribute when tip links are cut. Two methods will be used to disrupt candidate channel expression in order to test hair-cell function with reduced or absent channels. In zebra fish, morpholino oligonucleotides will block expression of candidate channels and fish will be tested for inner ear microphonic potentials and for loading by a dye that enters through the transduction channel. In mice, cultured utricular hair cells will be infected with adenoviruses encoding siRNAs targeting candidate channels, and function will be tested with dye loading and with patchclamp recording of transduction currents. Identifying the transduction channel may lead to identification of additional deafness genes. One transduction component, cadherin23, is defective in a form of Usher Syndrome-an inherited deafness and blindness. The transduction channel may also be a deafness gene, as may other transduction components that will be more easily identified once the channel is known.
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