Protocadherin 15 as a critical component of the gating spring of human hearing

项目摘要 听力损失是美国最常见的感觉病理学，五分之一的成年人患有听力损失。 单侧或双侧听力损失。在内耳中，听觉是在毛细胞的水平上介导的： 声音使发束偏转，静纤毛顶部的离子通道打开，允许机械转导， 声音门控弹簧的身份，控制这些通道的打开的元件，以及因此 精确度和灵敏度是未知的。连接相邻静纤毛的是丝状顶端 连接复合物，其包含原钙粘蛋白15（PCDH 15）的二聚体和钙粘蛋白23的二聚体。以前的工作 在实验室中的研究表明，PCDH 15的单体在生理力下比基于 仅就其结构而言，这表明它具有适当的性质，可以作为门控的组成部分 听觉的春天使用高速光阱，我已经获得了初步的证据， PCDH 15比单体更硬。在目标1中，我将检查PCDH 15二聚体响应 在不同的临界Ca 2+浓度下。我会对PCDH 15二聚体进行力梯度实验 其中力以恒定速率增加，以描绘其对力的生理水平的响应。 在细胞外钙粘蛋白（EC）结构域之间的连接区中存在多个Ca 2+结合位点， PCDH 15，和以前的工作已经表明，在PCDH 15的单体钙依赖性的结构变化。我 因此，假设二聚体将表现出类似的Ca 2+依赖性，并将在三个 Ca 2+水平来探测这一点。在目标2中，我将研究EC结构域展开如何有助于整体 PCDH 15单体对力的响应。先前对PCDH 15单体的研究揭示了一类 解折叠事件对应于整个EC结构域的解折叠。因此，我假设EC域 展开是尖端-连杆张力的关键媒介。我将通过在一个 PCDH 15构建体，其中每个EC结构域被阻止解折叠。在目标3中，我将研究突变如何 导致非综合征性耳聋的因素会影响PCDH 15单体的结构。约 所有先天性听力损失的50%源于遗传原因。PCDH 15中存在许多突变，例如 EC 5中V507 D突变导致非综合征性耳聋。为了研究 PCDH 15在具有这种突变的患者中受到影响，我将对PCDH 15的单体进行力斜坡实验。 这个建筑。我假设PCDH 15 V507 D将严重依赖于Ca 2+浓度，并将经历 比野生型单体更多的解折叠事件。综合起来，这些研究将深入了解 PCDH 15在正常和异常听力中的作用，并阐明其作为门控弹簧一部分的能力 听觉的。这些研究将在A博士的直接指导下进行。J. Hudspeth在该组织的 洛克菲勒大学的实验室，位于三机构的丰富支持环境中， MD-PhD项目。这个建议将极大地支持我成为一名医生科学家的目标。

Project Summary Hearing loss is the most common sensory pathology in the United States, with one in five adults experiencing unilateral or bilateral hearing loss. In the inner ear, hearing is mediated at the level of the hair cells: when a sound deflects the hair bundle, ion channels atop the stereocilia open, allowing for the mechanotransduction of sound. The identity of the gating spring, the element that controls the opening of these channels, and thus the precision and sensitivity with which we hear, is unknown. Connecting adjacent stereocilia is the filamentous tip link complex, which comprises a dimer of protocadherin 15 (PCDH15) and a dimer of cadherin 23. Previous work in the laboratory showed that the monomer of PCDH15 is softer under physiological forces than predicted based on its structure alone, suggesting that it has the appropriate properties to serve as a component of the gating spring of hearing. Using a high-speed optical trap, I have obtained preliminary evidence that the dimer of PCDH15 is stiffer than the monomer. In Aim 1, I will examine the behavior of the PCDH15 dimer in response to force at different critical Ca2+ concentrations. I will perform force-ramp experiments on the PCDH15 dimer, in which force is increased at a constant rate, in order to delineate its response to physiological levels of force. There are multiple Ca2+ binding sites in the linker regions between extracellular cadherin (EC) domains in PCDH15, and previous work has shown Ca2+-dependent structural changes in the monomer of PCDH15. I therefore hypothesize that the dimer will exhibit a similar Ca2+ dependence and will perform experiments at three Ca2+ levels to probe this. In Aim 2, I will investigate how EC domain unfolding contributes to the overall response of the PCDH15 monomer to force. Previous work on the monomer of PCDH15 revealed a class of unfolding events corresponding to the unfolding of an entire EC domain. I therefore hypothesize that EC domain unfolding is a critical mediator of tip-link tension. I will probe this by performing force-ramp experiments on a PCDH15 construct in which each EC domain is prevented from unfolding. In Aim 3, I will study how a mutation that results in non-syndromic deafness affects the mechanics of the PCDH15 monomer. Approximately 50 % of all congenital hearing loss stems from genetic causes. There are many mutations in PCDH15, such as the V507D mutation in EC5, that result in non-syndromic deafness. In order to study how the mechanics of PCDH15 are affected in patients with this mutation, I will perform force-ramp experiments on the monomer of this construct. I hypothesize that PCDH15 V507D will depend critically on Ca2+ concentration and will undergo more unfolding events than does the wildtype monomer. Taken together, these studies will yield insight into the role of PCDH15 in normal and aberrant hearing and elucidate its ability to serve as a portion of the gating spring of hearing. These studies will be carried out with the direct mentorship of Dr. A. J. Hudspeth in the group’s laboratory at The Rockefeller University, situated within the richly supportive environment of the Tri-Institutional MD-PhD Program. This proposal will greatly support my goal of becoming a physician-scientist.