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学，其中五分之一的成年人经历 单侧或双边听力损失。在内耳中，听力是在毛细胞水平上介导的： 声音示威者的发束，离子通道在立体中开放，允许机械转移 声音。门控弹簧的身份，控制着这些通道的开口的元素，因此 我们听到的精度和敏感性是未知的。连接相邻的立体丝菌是丝状尖端 链接复合物，其中包括协议蛋白15（PCDH15）的二聚体和Cadherin 23的二聚体。先前的工作 在实验室中，PCDH15的月份在物理力下比预测的 仅在其结构上，表明它具有适当的特性，可以作为门控的组成部分 春天的听力。使用高速光学陷阱，我获得了初步证据表明 PCDH15比月份更硬。在AIM 1中，我将检查PCDH15二聚体的行为 在不同的临界Ca2+浓度下强迫。我将在PCDH15二聚体上执行力斜线实验， 其中的力以恒定的速率增加，以描绘出其对物理力水平的反应。 在细胞外钙粘蛋白（EC）域之间的接头区域中有多个CA2+结合位点 PCDH15和以前的工作显示了PCDH15月份CA2+依赖性结构变化。我 因此假设二聚体将存在类似的Ca2+依赖性，并将在三个 Ca2+级别探究此问题。在AIM 2中，我将研究EC领域的发展如何有助于整体 PCDH15个月的响应。 PCDH15月的先前工作显示了一类 展开事件对应于整个EC领域的展开。因此，我假设EC领域 展开是尖端链路张力的关键调解人。我将通过在A上进行力跨实验来探测此问题 PCDH15构造，其中每个EC域都无法展开。在AIM 3中，我将研究突变 这会导致非综合性死亡性影响PCDH15单体的力学。大约 所有先天性听力损失植物中有50％来自遗传原因。 PCDH15中有许多突变，例如 EC5中的V507D突变，导致非综合性死亡。为了研究 PCDH15受到此突变患者的影响，我将在单体上进行力障碍实验 这个结构。我假设PCDH15 V507D将严重取决于Ca2+浓度，并且会经历 与野生型单体相比，展开的事件更多。综上所述，这些研究将洞悉 PCDH15在正常和异常听力中的作用，并阐明其作为门控弹簧的一部分的能力 听力。这些研究将以A. J. Hudspeth博士的直接心态进行 洛克菲勒大学的实验室，位于三机构的富裕环境中 MD-PHD程序。该建议将极大地支持我成为身体科学家的目标。