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

原钙粘蛋白 15 作为人类听力门控弹簧的重要组成部分

• 批准号: 10684942
• 负责人: Camila Marie Villasante
• 金额: $ 5.27万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10684942
• 关键词: Adult; Affect; Amino Acids; Behavior; Bilateral Hearing Loss; Binding; Binding Sites; CDH23 gene; Cadherin Domain; Complex; Data; Dependence; Doctor of Philosophy; Elasticity; Electrons; Elements; Endolymph; Event; Exhibits; Functional disorder; Future; Genetic; Goals; Hair; Hair Cells; Hearing; Human; Individual; Institution; Ion Channel; Ions; Laboratories; Labyrinth; Lead; Link; Measurement; Mechanics; Mediating; Mediator; Mentorship; Molecular Conformation; Mutation; PCDH15 gene; Pathologic; Pathology; Patients; Physicians; Physiological; Property; Proteins; Ramp; Rod; Role; Scientist; Sensory; Series; Speed; Stereocilium; Structure; Testing; United States; Universities; Variant; Work; biophysical properties; congenital hearing loss; deafness; dimer; disulfide bond; experience; experimental study; extracellular; hearing impairment; hereditary hearing loss; in vivo; insight; mechanical properties; mechanotransduction; monomer; novel; optic trap; optical traps; physical property; prevent; programs; response; single molecule; sound; stem; supportive environment; treatment strategy

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.

项目摘要 听力损失是美国最常见的感官病理，五分之一的成年人经历 单侧或双侧听力损失。在内耳，听力是在毛细胞水平上调节的：当一个 声音使发束偏转，体毛纤毛顶部的离子通道打开，允许机械转导 声音。闸门弹簧的身份，控制这些通道打开的元件，从而 我们听到的精确度和敏感度是未知的。连接相邻的立体纤毛的是丝状尖端 连接复合体，由原钙粘附素15(PCDH15)的二聚体和钙粘附素23的二聚体组成。以前的工作 在实验室中显示PCDH15的单体在生理作用力下比预测的基础上更软 仅就其结构而言，这表明它具有用作浇注组件的适当属性 听觉的春天。使用高速光学陷阱，我已经获得了初步证据 PCDH15的硬度比单体高。在目标1中，我将研究PCDH15二聚体的行为作为响应 在不同的临界钙离子浓度下施加压力。我将在PCDH15二聚体上进行力变实验， 其中，力以恒定的速度增加，以描绘其对力的生理水平的反应。 细胞外钙粘蛋白(EC)结构域之间的连接区存在多个钙离子结合位点 PCDH15，以前的工作表明PCDH15单体的结构变化依赖于钙离子。我 因此，假设二聚体将表现出类似的钙依赖关系，并将在三个月的 Ca2+水平来探测这一点。在目标2中，我将调查EC领域的展开如何有助于整体 PCDH15单体对力的响应。先前对PCDH15单体的研究揭示了一类 展开与整个EC域展开相对应的事件。因此，我假设EC域 展开是尖端-连杆张力的关键调节因素。我将通过在一个 PCDH15结构，其中每个EC结构域被阻止展开。在目标3中，我将研究突变是如何 这会导致非综合征性耳聋，影响PCDH15单体的机械性能。大致 50%的先天听力损失是由遗传因素引起的。PCDH15有许多突变，例如 EC5中的V507D突变，导致非综合征性耳聋。为了研究它的力学原理 PCDH15都是受这种突变影响的患者，我将对其单体进行力变实验 这个构造。我推测PCDH15 V507D将严重依赖于钙离子浓度，并将经历 比野生型单体更多的事件在展开。综上所述，这些研究将深入了解 PCDH15在正常和异常听力中的作用及其作为门控弹簧一部分的能力 倾听的声音。这些研究将在A·J·哈德斯佩斯博士的直接指导下进行 洛克菲勒大学的实验室，位于三所大学非常支持的环境中 医学博士学位课程。这项提议将极大地支持我成为一名内科科学家的目标。