Nanomechanics of inner-ear hair-cell transduction

项目总结 声音的振动和头部运动的机械刺激转化为电化学 内耳毛细胞机械感受器调节我们的听觉和听觉的大脑处理信号 平衡。毛细胞功能所必需的是构成毛细胞机械转导装置的蛋白质，包括 一种细小的尖端连接细丝，它拉动离子通道复合体来触发感官知觉。提示链接 微丝由钙粘蛋白-23(CDH23)和原钙粘蛋白-15(PCDH15)蛋白形成，而离子通道 复合体被认为是由跨膜通道样蛋白家族TMC1和 TMC2、跨膜内耳蛋白TMIE和毛细胞立体纤毛的四环素膜蛋白 TMHS(也称为LHFPL5)。此外，钙和整合素结合蛋白CIB2与TMC结合 调节机械转导的通道。所有这些蛋白质对听力和平衡都很重要，而且 参与遗传性耳聋，但它们的分子结构和转导的功能结构 人们对它们形成的复杂结构知之甚少。这个项目的总体长期目标是揭示 形成内耳尖端连接和转导离子的蛋白质功能的结构决定因素 航道综合体。在目标1中，我们将使用冷冻电子显微镜、高速原子力显微镜和 CDH23和PCDH15全长胞外结构域的分子动力学模拟 从而建立了内耳机械传导中尖-链功能的结构决定因素。在目标2中，我们 将使用带有偏差的微秒长的分子动力学模拟来生成可测试的预测 膜电位通过实验表征离子和耳毒性氨基糖苷类化合物的渗透 验证了TMC蛋白质孔的结构模型。在目标3中，我们将使用各种计算和生物物理 技术，包括核磁共振和自然质谱学，以探索监管 CIB蛋白的转导机制。从拟议的实验和模拟中获得的结果 将提供内耳蛋白质成分的初始和动态分子视图 随着我们进一步了解它的结构和功能，在正常和 听力和平衡受损。

PROJECT SUMMARY Vibrations from sound and mechanical stimuli from head movements are transformed into electrochemical signals for brain processing by inner-ear hair-cell mechanoreceptors mediating our senses of hearing and balance. Essential to hair-cell function are the proteins that form its mechanotransduction apparatus comprised of a fine tip-link filament that pulls on an ion channel complex to trigger sensory perception. The tip-link filament is formed by cadherin-23 (CDH23) and protocadherin-15 (PCDH15) proteins while the ion channel complex is thought to be formed by members of the transmembrane channel-like protein family TMC1 and TMC2, the transmembrane inner ear protein TMIE, and the tetraspan membrane protein of hair-cell stereocilia TMHS (also known as LHFPL5). In addition, the calcium and integrin binding protein CIB2 binds to TMC channels to regulate mechanotransduction. All these proteins are important for hearing and balance and are involved in inherited deafness, yet their molecular structures and the functional architecture of the transduction complex they form are poorly understood. The overall long-term goal of this project is to reveal the structural determinants of function for the proteins forming the inner-ear tip link and transduction ion channel complex. In Aim 1, we will use cryo-electron microscopy, high-speed atomic force microscopy, and molecular dynamics simulations to study the full-length extracellular domains of CDH23 and PCDH15 and thereby establish the structural determinants of tip-link function in inner ear mechanotransduction. In Aim 2, we will generate testable predictions using microsecond-long molecular dynamics simulations with biasing membrane potentials to characterize permeation of ions and ototoxic aminoglycosides through experimentally validated structural models of TMC protein pores. In Aim 3, we will use various computational and biophysical techniques, including nuclear magnetic resonance and native mass spectrometry, to explore regulatory mechanisms of transduction by CIB proteins. Results obtained from the proposed experiments and simulations will provide an initial and dynamic molecular view of the protein components of the inner ear mechanotransduction apparatus as we advance to understand its architecture and function in normal and impaired hearing and balance.