Second messenger regulation of hair cell mechanotransduction

描述(申请人提供)：听觉系统的宽广动态范围是一个极其重要的特征，使其能够检测亚原子水平的刺激，同时不会对大几个数量级的刺激饱和。在某种程度上，该系统的动态范围与毛细胞机械转导过程以一种称为适应的钙依赖方式将其激活曲线移动数百纳米的能力有关。第二信使环磷酸腺苷(CAMP)也改变了毛束的工作范围，但目前还不知道这一过程的生理相关性或生化机制。这项建议的目的是探索cAMP在细胞和系统水平上调节发束动态范围中的作用。这项研究的第一个目的是确定腺苷环化酶异构体在毛细胞中的亚细胞定位，因为这些蛋白质产生cAMP。第二个目的是通过五个具体的实验来探索cAMP的作用机制：1-确定内外毛细胞是否对cAMP的变化做出类似的反应，2-确定cAMP对毛束力学的影响作为识别靶点的手段，3-cAMP的光解释放将决定cAMP作用的动力学，4-将通过药理学和使用敲除动物来确定PKA在cAMP中的作用。5-钙是否参与cAMP反应将直接使用扫描场共聚焦钙成像进行检测。第三个目标将通过使用听觉脑干反应、耳蜗微音学和失真产物的全动物听觉测试来分析突变小鼠，以确定cAMP在耳蜗过程中所扮演的角色，以了解该系统如何调节毛束的特性。总之，这些数据将提供有关cAMP调节的生化途径以及cAMP在系统水平上调节听力的作用的新数据。 公共卫生相关性：听力障碍影响2800万美国人。此外，听力损失的发生率随着年龄的增长而增加；65岁以上的美国人中约有30%，75岁及以上的美国人中约有40%至50%患有听力损失。听力损失的流行，以及不断增长的患有这种潜在残疾的人口，清楚地表明，我们需要更好地了解听觉系统是如何运作的，以便更好地治疗听力障碍患者。

DESCRIPTION (provided by applicant): The broad dynamic range of the auditory system is a critically important feature, allowing it to detect stimuli at the subatomic level while at the same time not saturating for stimuli several orders of magnitude larger. In part, the dynamic range of the system is related to the ability of the hair cell mechanotransduction process to shift its activation curve by hundreds of nanometers in a calcium dependent manner known as adaptation. The second messenger cyclic AMP (cAMP) also shifts the operating range of the hair bundle, but no physiological relevance or biochemical mechanism is currently known for this process. The goal of this proposal is to explore the role of cAMP in modulating the hair bundle dynamic range at both the cellular and system level. The first aim of this research is to determine the subcellular localization of adenylate cyclase isoforms in the hair cell, since these proteins generate cAMP. The second aim explores the mechanism of cAMP action with five specific experiments: 1- determine whether inner and outer hair cells respond similarly to changes in cAMP, 2- determine the effects of cAMP on hair bundle mechanics as a means to identify the target site, 3- Photolytic release of caged cAMP will determine the kinetics of cAMP action, 4- The role of PKA in the cAMP will be determined pharmacologically and also with the use of knockout animals. 5- Whether calcium is involved in the cAMP response will be directly assayed using swept field confocal calcium imaging. The third aim will determine, the role cAMP plays in cochlear processes via the analysis of mutant mice using whole animal auditory testing of auditory brainstem responses, cochlear microphonics, and distortion products in order to understand how the system modulates the properties of the hair bundle. Together these data will provide new data regarding the biochemical pathway of cAMP regulation and also the role of cAMP in modulating hearing at the systems level. PUBLIC HEALTH RELEVANCE: Hearing impairment affects 28 million Americans. Furthermore, the incidence of hearing loss increases with age; about 30% of Americans over the age of 65 and 40-50% of Americans age 75 and older suffer from hearing loss. The prevalence of hearing loss along with the ever growing population of people afflicted with this potential disability is a clear indication that we need to better understand how the auditory system functions in order to better treat patients with hearing impairments.