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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要该项目的总体长期目标是了解定义耳蜗的分子机制具体来说，我们将重点关注电压驱动电机 Prestin，这是一种独特的技术。 Prestin 是 OHC 基底外侧膜中发现的 SCL26 转运蛋白家族的成员。主要通过功能方法进行研究，对此的基本机制理解尽管现有的技术很丰富，但人工耳蜗放大器的基本组成部分仍有待解决。功能数据，缺乏高分辨率结构是定义其机制的关键缺失元素对于 Prestin 作用机制的两个基本方面尤其如此：电压传感的过程和电动性的分子机制。作为该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 作为生物压电器件的结构和动力学为此，我们计划进行实验。解决几个基本问题：能量转换步骤的物理基础是什么？跨膜电压变化并最终导致蛋白质（最终是 OHC）运动是什么？其天然双层嵌入形式的关键功能状态的结构在分子的什么位置？ Prestin-双层相互作用的物理基础是什么？功能研究旨在了解能量转换信息的物理基础。功能相关构象、构象动力学和能量的高分辨率结构 Prestin与其周围脂质双层的关系将通过冷冻电镜、电生理学和荧光显微镜实验将解释数据以生成高分辨率结构。我们建议新冷冻电镜的出现。膜蛋白在其天然脂质环境中的结构和动力学分析方法将开辟一条令人兴奋的新实验途径，这些信息将影响我们的理解。重要的生理事件，例如听力、高频放大和信号传导。