The long-term objectives are to understand the mechanisms of mechanotransduction in auditory hair cells and delineate the factors underlying the cochlea's tonotopic organization. Experiments will focus on the attributes and molecular composition of the mechanotransducer (MT) channel and its modulation by Ca2+. Hair cell responses will be measured in the isolated cochleas of both mouse and chicken. A feature of the work is the use of mouse mutants combined with transfection of cultured auditory epithelia. Specific aims are: (1) to record MT currents in cochlear hair cells from mice with mutations in the transmembrane channel-like (Tmc) proteins Tmc1 and Tmc2, which are molecular candidates for the MT channel. Available mouse mutants will be studied as well as those with engineered point mutations which will be transfected into cochlear cultures; (2) to examine the Ca2+ dependence and mechanism of MT channel adaptation in mammalian hair cells, testing the hypothesis that it differs from that established in non-mammals. Ca2+ will be manipulated by internal perfusion and photolysis of caged Ca2+. Mice with mutations in myosin VIIa will also be characterized to determine the contribution of this myosin to adaptation; (3) to define the function of the hair cell Ca2+-binding protein oncomodulin in Ca2+ buffering and development of hair cells by generating conditional mutants and exploring their properties; the hypothesis is that embryonic expression of oncomodulin is essential for hair cell development but that post-natal loss is without effect; (4) to record MT currents and measure hair bundle mechanics of short (outer) hair cells in the chick auditory papilla as a likely but unproven site where active hair bundle motion is used to augment frequency selectivity. The interaction between active bundle motion derived from channel gating and a possible prestin-induced motility will be used to assess the contributions of the two processes to amplification and frequency tuning. Comparison with the properties of the mammalian outer hair cells will provide insight into the evolution of cochlear amplification in amniotes; (5) to examine the embryonic maturation of frequency tuning in the chicken auditory papilla, testing a previously reported shift in best frequency; also charting the embryonic development of the MT current, the role of Tmc proteins and of parvalbumin-3, the avian equivalent of oncomodulin. It is hoped that the results will supply evidence on the molecular composition of the hair cell transduction apparatus and will yield information about proteins that are mutated in certain forms of human genetic deafness.
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