Calcium Regulation of Mechanotransduction

描述（由申请人提供）：感觉毛细胞的毛束偏转（机械转导）导致毛细胞产生电反应，这是听觉的主要信号。噪音、耳毒性物质或衰老对这一途径的破坏会导致阈值变化（暂时性和永久性）、听力损失、极端情况下的耳聋等疾病，甚至可能成为某些形式的耳鸣的基础。了解机械转导过程中涉及的机制有助于确定干预和预防听力损失的部位，也可能为毛细胞再生等替代疗法的发展提供指导。虽然在过去的25年里积累了大量关于机械转导的知识，但关于机制和功能的许多基本问题仍有待阐明。这里的实验将使用最先进的光学和电学技术，结合组织培养和药理学工具来探索关于毛细胞机械转导的几个基本问题。特异性目的（SA） 1将通过利用内毛细胞束的形态排列来确定功能性机电转导（MET）通道是位于直立纤毛的顶部还是沿着两侧。它将通过将单个立体纤毛的钙成像与MET电流测量相结合来确定每个立体纤毛的通道数量。最后，它将确定膜张力在通道门化中的作用，这将有助于深入了解MET通道是直接连接到尖端链接或细胞骨架上，还是通过膜拉伸激活。SA2将通过耦合Ca2+成像、Ca2+释放和MET电流的电生理测量来研究适应和激活的Ca2+依赖性。这些实验将尝试从机械刺激中分离钙依赖性反应，并应获得关于钙调节转导和适应的重要新动力学信息。实验还将探讨毛束力学对Ca2+的依赖性。SA3将研究单通道MET特性，以确定Ca2+调节通道电导和打开时间的机制。SA4将测试MET为毛细胞提供机械调节的假设。

DESCRIPTION (provided by applicant): Deflection of the sensory hair cell's hair bundle (mechanotransduction) results in an electrical response from the hair cell that is the primary signal for audition. Disruption of this pathway by noise, ototoxic agents or aging leads to maladies such as threshold shifts (both temporary and permanent), hearing loss, deafness at an extreme and may even underlie some forms of Tinnitus. Understanding the mechanisms involved in the mechanotransduction process should help identify sites for intervention and prevention of hearing loss and may also provide guidelines for development of replacement therapies such as hair cell regeneration. Although a great deal of knowledge regarding mechanotransduction has accrued over the past 25 years, many fundamental questions regarding mechanisms and functions remain to be elucidated. Experiments herein will use state of the art optical and electrical techniques coupled with tissue culture and pharmacological tools to probe several basic questions regarding hair cell mechanotransduction. Specific Aim (SA) 1 will determine whether the functional mechano electric transduction (MET) channels are located at the tops or along the sides of the stereocilia by taking advantage of the morphological arrangement of inner hair cell bundles. It will determine the number of channels per stereocilia by coupling calcium imaging of individual stereocilia with measurements of MET current. Finally, it will determine the role of membrane tension on channel gating which should give insight into whether the MET channel is directly tethered to the tip-link or cytoskeleton or whether it is activated via membrane stretch. SA2 will investigate the Ca2+ dependence of adaptation and activation by coupling Ca2+ imaging, Ca2+ uncaging and electrophysiological measurements of MET currents. These experiments will attempt to separate calcium-dependent responses from mechanical stimulation and should yield important new kinetic information regarding the calcium regulation of transduction and adaptation. Experiments will also explore the Ca2+ dependence of hair bundle mechanics. SA3 will investigate single channel MET properties to determine the mechanism by which Ca2+ regulates channel conductance and open time. SA4 will test the hypothesis that MET provides mechanical tuning to the hair cell.