Second messenger regulation of hair cell mechanotransduction

听觉系统的宽动态范围是一个非常重要的特征，使其能够检测亚原子水平的刺激，同时不会饱和几个数量级的刺激。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性方式将其激活曲线移动数百纳米的能力有关，称为适应。第二信使环腺苷酸（cAMP）也改变了毛束的操作范围，但目前还不知道这个过程的生理相关性或生化机制。本研究的目的是探索cAMP在细胞和系统水平上调节毛束动态范围的作用。本研究的第一个目的是确定毛细胞中腺苷酸环化酶亚型的亚细胞定位，因为这些蛋白质产生cAMP。第二个目的通过五个具体实验探索cAMP作用的机制：1-确定内毛细胞和外毛细胞是否类似地响应cAMP的变化，2-确定cAMP对毛束力学的影响作为鉴定靶位点的手段，3-笼状cAMP的光解释放将确定cAMP作用的动力学，4-PKA在cAMP中的作用将通过基因敲除和使用敲除动物来确定。5-钙是否参与cAMP反应将使用扫描场共聚焦钙成像直接测定。第三个目标将确定，cAMP在耳蜗过程中发挥的作用，通过使用听觉脑干反应，耳蜗微音和失真产品的整个动物听觉测试的突变小鼠的分析，以了解该系统如何调制的属性的毛束。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。

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.