The development of mechanotransduction in cochlear hair cells

项目摘要 长期目标是了解机械换能器（MT）通道的操作和结构 在听觉毛细胞中。与其他离子通道不同，目前关于这种通道的证据非常不完整， 孔形成亚基的分子特性和通道门控的机制都是有争议的。 对转导的影响发生在TMC 1或LHFPL 5突变中，这些突变可与头端连接成分相互作用 PCDH 15。MT通道被认为位于头端连接的下端，并由 发束朝向其较高边缘的偏转，从而张紧尖端链接。然而，在一些 在某些情况下，包括尖端链路的破坏，MT电流也可以被束激活。 相反极性的位移。我们有初步的证据表明，这种“反极性”MT电流是 在毛束发育过程中，当它们先于正常MT的出现时， 渠道该项目的目标是调查这些异常的发展渠道，希望它 将揭示正常的MT通道是如何运输到其最终位置的，以及运输蛋白是什么 needed. MT电流将在机械刺激分离的耳蜗毛细胞期间记录， 围产期野生型和突变型小鼠。具体目标是：（1）记录反转出现的时间- （2）比较出生前后不同时期的极性电流及其与正常MT电流的关系 反向极性通道与正常通道的特性，并确定它们在头发中的位置 通过这些通道的Ca 2+内流的快速共聚焦成像;（3）确定 在发育过程中观察到的反向极性通道是否易受睫状体间连接破坏的影响。 电子显微镜将用于描述不同类型和方向的链接，并描述束 发育过程中的形态与反极性电流有关;（4）研究 将反极性MT通道插入质膜，测试通道是 运输到顶膜，然后移位到静纤毛的尖端，成为成熟的MT （5）研究反向极性电流对突变的依赖性（包括 原钙粘蛋白-15、肌球蛋白VIIa、LHFPL 5和TMC的同种型），其中转导机制是 心烦意乱希望该结果能为毛细胞的分子组成提供证据 换能装置及其组装方法。

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.