Structural basis of Outer Hair Cell Electromotility at High Resolution

项目摘要 这个项目的总体的、长期的目标是了解定义耳蜗的分子机制。 外毛细胞(OHC)中的放大器。具体地说，我们将重点介绍电压驱动电机Prestin，这是一种独特的 SCL26转运蛋白家族的成员，存在于毛细胞的基侧膜上。尽管演示 已经通过函数方法进行了广泛的研究，对此的基本机理理解 耳蜗放大器的基波元件还有待解决。尽管现有的丰富的 功能数据，缺乏高分辨率结构是在定义其机制时缺少的一个关键因素 分子水平。普雷斯汀作用机制的两个基本方面尤其如此： 电压传感的基本过程和电动的分子机制。鉴于令人振奋的新消息 在这项建议的核心初步数据我们将能够研究的功能行为，高分辨率 Prestin作为生物压电器件的结构和动力学。为了做到这一点，我们计划试验 解决几个基本问题：能量传递步骤的物理基础是什么，开始 随着跨膜电压的变化并最终导致蛋白质(最终是OHC)运动？什么是 天然双层嵌入形式的关键功能状态的结构？在分子中的什么地方 机械转导发生了吗？那又是怎么做的呢？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。有关的资料 功能相关构象、构象动力学和能量学的高分辨结构 Prestin与其周围脂双层的关系将通过冷冻-EM、电生理学和 荧光显微镜实验。数据将被解释为生成高分辨率结构 机电转导的不同阶段。我们认为，新的低温EM的出现 膜蛋白在天然脂环境中的结构和动力学分析方法 将开辟一条激动人心的新实验大道。这一信息将影响我们对 生理上重要的事件，如听力、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.