Structural basis of Outer Hair Cell Electromotility at High Resolution

高分辨率外毛细胞电动性的结构基础

• 批准号: 10317974
• 负责人: Eduardo A Perozo
• 金额: $ 50.62万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10317974
• 关键词: Address; Amino Acids; Amplifiers; Anions; Auditory Physiology; Behavior; Binding; Binding Sites; Biochemical; Biological; Charge; Cochlea; Cryoelectron Microscopy; Data; Dependence; Detergents; Development; Devices; Disease; Elbow; Electric Capacitance; Electrophysiology (science); Electrostatics; Elements; Elevator; Environment; Equilibrium; Event; Experimental Models; Family; Fluorescence Microscopy; Frequencies; Glycine; Goals; Health; Hearing; Ion Channel; Light; Lipid Bilayers; Lipids; Mechanics; Membrane; Membrane Proteins; Micelles; Modeling; Molecular; Molecular Conformation; Molecular Motors; Motion; Motor; Motor Neurons; Movement; Mutagenesis; Nature; Noise; Outer Hair Cells; Physiological; Preparation; Process; Proteins; Reproducibility; Resolution; Role; Salicylic Acids; Sensory; Series; Signal Transduction; Site; Site-Directed Mutagenesis; Structure; base; basolateral membrane; crosslink; data modeling; density; design; electric field; experience; experimental study; feasibility research; hearing impairment; inhibitor/antagonist; insight; member; nanodisk; physical model; rat Pres protein; reconstitution; sensor; voltage; voltage clamp

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.

项目摘要 该项目的总体长期目标是了解定义人工耳蜗的分子机制 外毛细胞中的放大器（OHC）。特别是，我们将重点放在电压驱动的电动机prestin上，这是一个独特的 在OHC的基础机理中发现的SCL26转运蛋白家族的成员。虽然普雷斯汀 已经通过功能性的方法进行了广泛的研究，这是对此的基本机理理解 人工耳蜗放大器的基本成分仍有待解决。尽管有现有的丰富性 功能数据，缺乏高分辨率结构是定义其机制在A处的关键缺失元素 分子水平。对于普雷斯汀行动机理的两个基本方面，尤其如此： 电压感应和电动性分子机制的过程。鉴于令人兴奋的新 本提案核心的初步数据我们将能够研究功能行为，高分辨率 Prestin作为生物压电装置的结构和动力学。为此，我们计划通过实验 解决几个基本问​​题：能量传输步骤的物理基础是什么 随着跨膜电压的变化并达到蛋白质（和最终OHC）运动的最终形式？什么是 关键功能状态的结构以其本地双层包裹的形式？分子中的位置 机械转移发生？以及如何？ Prestin-Bilayer相互作用的物理基础是什么？ 功能研究将旨在理解能量转移的物理基础。有关 功能相关构象，构象动力和能量的高分辨率结构 Prestin及其周围脂质双层的关系将从低温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。我们建议新的低温EM冒险 在其天然脂质环境中膜蛋白中结构和动力学分析的方法 应打开一个令人兴奋的新实验大道。这些信息将影响我们对 生理上重要的事件，例如听力，高频扩增和信号转导。