Structural basis of Outer Hair Cell Electromotility at High Resolution

项目摘要 该项目的总体长期目标是了解耳蜗的分子机制， 外毛细胞（OHC）。具体来说，我们将重点介绍电压驱动电机普雷斯廷，一个独特的 在OHC的基底外侧膜中发现的转运蛋白SCL 26家族的成员。虽然普雷斯廷 已经通过功能性方法进行了广泛的研究， 耳蜗放大器的基本元件仍有待解决。尽管现有的财富 功能数据，缺乏高分辨率的结构是一个关键的缺失元素，在定义其机制， 分子水平。这对于普雷斯廷作用机制的两个基本方面尤其如此： 电压感知的基础过程和电运动的分子机制。鉴于令人兴奋的新 初步数据的核心，这一建议，我们将能够研究的功能行为，高分辨率 生物压电器件普雷斯廷结构和动力学为此，我们计划通过实验 解决几个基本问题：能量转换步骤的物理基础是什么， 与跨膜电压的变化，并在蛋白质（最终OHC）运动的高潮？有哪些 结构的关键功能国家在其本机，双层嵌入的形式？在分子中 机械传导发生了吗怎么做？Prestin-双层相互作用的物理基础是什么？ 功能研究将旨在了解能量转导的物理基础。信息 功能相关构象、构象动力学和能量的高分辨率结构 普雷斯廷与其周围脂质双层的关系将从冷冻电镜、电生理学和 荧光显微镜实验。数据将被解释以产生高分辨率的结构， 机电转换的不同阶段。我们认为新的冷冻电镜的出现 分析膜蛋白在其自然环境中的结构和动力学的方法 将开辟一条激动人心的新实验大道这些信息将影响我们对 生理上重要的事件，如听觉，高频放大和信号转导。

Project Summary The overall, long-term goal of this project is to understand the molecular mechanism of that define the cochlear amplifier in outer hair Cells (OHC). Specifically, we will focus on the voltage-driven motor Prestin, a unique member of the SCL26 family of transporters found in the basolateral membranes of OHCs. Although Prestin has been studied extensively though functional approaches, the basic mechanistic understanding of this fundamental component of the cochlear amplifier remain to be solved. In spite of the richness of the existing functional data, the lack of a high resolution structure is a key missing element in defining its mechanism at a molecular level. This is particularly so for the two fundamental aspects of Prestin’s mechanism of action: the process underlying voltage sensing and the molecular mechanism of electromotility. In light of exciting new preliminary data at the core of this proposal we will be able to study the functional behavior, high resolution structure and dynamics of Prestin as a biological piezoelectric device. To do so, we plan to experimentally address several fundamental questions: What is the physical basis of the energy transduction steps, starting with transmembrane voltage changes and culminating in protein (and ultimately OHC) motion? What are the structures of the key functional states in its native, bilayer-embedded form? Where in the molecule does mechanical transduction occur? And how? What are the physical basis of the Prestin-bilayer interaction? Functional studies will be designed to understand the physical basis of energy transduction. Information on the high resolution structure of functionally relevant conformations, conformational dynamics and energetic relationship of Prestin with its surrounding lipid bilayer will be obtained from cryo-EM, electrophysiology and Fluorescence microscopy experiments. The data will be interpreted to generate high resolution structures of the different stages of the electromechanical transduction. We suggest that the advent of new cryo-EM approaches to the analysis of structure and dynamics in membrane proteins in their native lipidic environment shall open an exciting new experimental avenue. This information will impact our understanding of physiologically important events such as hearing, high frequency amplification and signal transduction.