PROJECT SUMMARY The long-term objective is to understand the operation and structure of the mechanotransducer (MT) channel in auditory hair cells. Present evidence about this channel, unlike other ion channels, is very incomplete, and both the molecular identity of the pore-forming subunit and the mechanism of channel gating are controversial. Effects on transduction occur with mutations in TMC1 or LHFPL5 which can interact with the tip link constituent PCDH15. The MT channel is thought to be located at the lower end of the tip link and to be activated by deflections of the hair bundle towards its taller edge, thus tensioning the tip links. However, under some circumstances, including destruction of the tip links, an MT current can also be activated by bundle displacements of opposite polarity. We have preliminary evidence that such `reverse-polarity' MT currents are also prominent during hair bundle development, when they precede the appearance of the normal MT channels. The goal of the project is to investigate these anomalous developmental channels in the hope that it will enlighten how the normal MT channel is transported to its final location, and what trafficking proteins are needed. MT currents will be recorded during mechanical stimulation of hair cells in isolated cochleas of perinatal wild-type and mutant mice. Specific aims are: (1) to document the time of emergence of the reverse- polarity current and its relation to the normal MT current during the days around birth; (2) to compare properties of the reverse-polarity channels with the normal ones and to determine their location in the hair bundle or plasma membrane using fast confocal imaging of Ca2+ influx through these channels; (3) to ascertain whether reverse-polarity channels observed developmentally are susceptible to destruction of inter-ciliary links. Electron microscopy will be used to describe the different types and orientations of links and to describe bundle morphology during development to relate to the reverse-polarity current; (4) to investigate pathways for inserting reverse-polarity MT channels into the plasma membrane, testing the hypothesis that the channels are trafficked to the apical membrane and then translocated to the tips of the stereocilia to become mature MT channels; (5) to investigate the dependence of the reverse-polarity currents on mutations (including those in protocadherin-15, myosin VIIa, LHFPL5 and isoforms of TMC), in which the transduction machinery is perturbed. It is hoped that the results will provide evidence on the molecular composition of the hair cell transduction apparatus and its method of assembly.