Second messenger regulation of hair cell mechanotransduction

描述（由申请人提供）：听觉系统的宽动态范围是一个非常重要的特征，允许它检测亚原子水平的刺激，同时不会对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钙依赖的方式将其激活曲线移动数百纳米的能力有关。第二信使环AMP （cAMP）也改变了毛束的活动范围，但目前还不清楚这一过程的生理相关性或生化机制。本研究的目的是探讨cAMP在细胞和系统水平上调节发束动态范围中的作用。本研究的第一个目的是确定腺苷酸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特定的实验来探索cAMP的作用机制：1-确定内外部毛细胞对cAMP的变化是否有相似的反应，2-确定cAMP对毛束力学的影响，作为识别靶点的手段，3-笼中cAMP的光解释放将决定cAMP的作用动力学，4- PKA在cAMP中的作用将通过药理学和基因敲除动物来确定。5-钙是否参与cAMP反应将通过扫描场共聚焦钙成像直接检测。第三个目标将确定cAMP在耳蜗过程中的作用，通过对突变小鼠的全动物听觉测试，听觉脑干反应，耳蜗微音和失真产品进行分析，以了解系统如何调节毛束的特性。这些数据将为cAMP调节的生化途径以及cAMP在系统水平上调节听力的作用提供新的数据。

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.