Nanomechanics of Inner Ear Hair Cell Transduction

 描述(申请人提供)：内耳毛细胞通过将声音和头部运动中的机械刺激转化为可由大脑处理的电化学信号来调节我们的听觉和平衡。毛细胞功能的核心是“尖端连接”，这是正常听力所必需的细小蛋白质丝，与遗传有关。 耳聋。两个巨大的钙粘蛋白蛋白被证明形成了成熟的末端链接(钙粘蛋白-23和原钙粘蛋白-15)，这被认为是一个直丝，具有紧密结合的钙离子，使其具有刚性和强度。瞬时的、未成熟的Protocadherin-15末端链变体被认为在噪声诱导损伤后的发育和末端链再生过程中起重要作用。可能还有其他与生理相关的变异体，由多种异构体的钙粘蛋白-23和原钙粘蛋白-15形成。该项目的总体目标是确定尖端链接在暴露于不同钙浓度时、由不同蛋白质变体产生时以及当它们携带与耳聋相关的突变时的生物物理和生化行为。在目标1中，力光谱和各种生物物理和生化方法将被用来确定改变钙浓度如何改变末端连接属性，以及原钙粘附素-15的非正则钙结合位点如何改变其弹性。在……里面 目的2，X射线结晶学和计算模拟将被用来获得和表征涉及所有细胞外钙粘蛋白-23和原钙粘蛋白-15亚型的末端连接变体的模型。在目标3中，将使用各种方法来确定与遗传性耳聋相关的错义突变和缺失的生化和生物物理后果，以解释观察到的表型的严重性，并预测TIP连接蛋白中尚未发现的突变和缺失的影响。这个项目的结果将提供一个清晰的分子观点，在各种生理相关的情况下，末端连接的性质和功能，并可能提供遗传性耳聋的治疗策略和分子治疗。

 DESCRIPTION (provided by applicant): Inner ear hair cells mediate our senses of hearing and balance by transforming mechanical stimuli from sound and head movements into electrochemical signals that can be processed by the brain. At the core of hair cell function are "tip links", fine protein filaments essential for normal hearing and that are involved in inherited deafness. Two enormous cadherin proteins have been shown to form the mature tip link (cadherin-23 and protocadherin-15), which is thought to be a straight filament with tightly, bound calcium ions that give it rigidity and strength. Transient, immature protocadherin-15 tip link variants have been suggested to be important during development and tip link regeneration after noise-induced damage. There might be additional physiologically relevant variants formed by multiple isoforms of cadherin-23 and protocadherin-15. The overall goal of this project is to determine the biophysical and biochemical behavior of tip links when exposed to different calcium concentrations, when made by different protein variants, and when they carry deafness-related mutations. In aim 1, force spectroscopy and various biophysical and biochemical methods will be used to determine how changing calcium concentration modifies tip link properties and how non-canonical calcium binding sites of protocadherin-15 alter its elasticity. In aim 2, x-ray crystallography and computational modeling will be used to obtain and characterize models of tip link variants involving all extracellular isoforms of cadherin-23 and protocadherin-15. In aim 3, various methods will be used to determine the biochemical and biophysical consequences of missense mutations and deletions associated with inherited deafness, both to explain the severity of observed phenotypes and to predict the effects of yet to be found mutations and deletions in tip link proteins. Results of this project will provide a clear molecula view of tip link properties and function under various physiologically relevant circumstances, and may inform treatment strategies and molecular therapies for inherited deafness.