PROJECT SUMMARY/ABSTRACT The main objective of the proposed experimental program is to address questions of mechanotransducer (MET) channel structure and function employing a combination of electrophysiological and genetic techniques. Experiments will focus on the attributes and molecular composition of the hair cell MET channel complex, gathering more evidence that transmembrane channel-like protein isoform TMC1 forms a central component of the channel and dictates its biophysical properties. Hair cell responses will be measured in acutely isolated cochleae of wild-type and mutant mice. Specific aims are: (1) to record and characterize the conductance and ionic properties of MET channels in cochlear hair cells of different mice, each with a distinct Tmc1 missense mutation. The site of the mutations will be selected to define the location of the channel pore, the residues differing between TMC1 and TMC2, and those determining channel block by Ca2+. (2) Using mutations of the accessory protein LHFPL5 to define its contribution to channel conductance, gating and adaptation. One hypothesis is that LHFPL5 provides force transmission between the tip link and the MET channel. (3) To investigate the time course of loss of MET currents and transduction in Tmc1 mutants during the third postnatal week and explore underlying mechanisms. All mutants display MET currents up to postnatal day 12 but lose them over the subsequent five to ten days depending on the mutant. One hypothesis to be tested is that decreased Ca2+ entry into the hair bundle is a major determinant of loss of transduction and that differences between mutants reflect differences in steady Ca2+ influx. The connection between reduced hair bundle Ca2+, lipid scrambling, and hair cell death will also be explored. (4) Determine the origin of a new type of Ca2+ dependent force generation that we previously observed in gecko hair cells. It is hypothesized that this same process exists in mammalian vestibular hair cells. The roles in the process of the kinocilium and non-muscle myosins NMIIB and NMIIC at the endolymphatic surface of the hair cell around the cuticular plate will be studied. It is hoped that the results will supply evidence on the molecular makeup of the hair cell transduction apparatus and yield information about proteins that are mutated in certain forms of human genetic deafness.