Structure-based analysis of the molecular mechanisms of prestin-dependent cochlear amplification

基于结构的prestin依赖性耳蜗放大分子机制分析

• 批准号: 218325332
• 负责人: Professor Dr. Dominik Oliver
• 金额: --
• 依托单位: Institut für Physiologie und Pathophysiologie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2012
• 资助国家: 德国
• 起止时间: 2011-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/218325332?language=en
• 关键词: Structure; based; analysis; molecular; mechanisms

Active mechanical amplification in the mammalian cochlea requires a process termed electromotility, i.e. voltage-driven ultrafast length changes of sensory outer hair cells (OHC). The elementary motor that generates motility is prestin (SLC26A5), an anion transporter-related, OHC-specific membrane protein. It is thought that electromotility arises from conformational changes of prestin that are directly triggered by changes in membrane potential and involve the binding of anions to the protein. In the previous funding period, we have established a 3D structural model of prestin and non-mammalian homologs, based on homology modeling, molecular dynamics simulations and - experimentally - extensive mutagenesis. The properties of the structure converge nicely with many of the functional features of prestin including anion dependence and the functional impact of previously identified domains. Based on this structural information we now want to examine the conformational dynamics that give rise to the generation of movement/or force. We will analyze voltage and anion dependent conformational changes by state-dependence of modification of introduced cysteine residues and by environmentally sensitive dyes. Beyond the molecular mode of action, it is also still unclear how prestin contributes to mechanical amplification: in a slow manner by setting the set-point of the active process or ultrafast by producing force in a cycle by cycle manner at high acoustic frequencies. To distinguish between these mechanisms, we will introduce kinetically altered prestin mutants into mice by cutting-edge genome engineering methods and analyze the peripheral hearing in terms of cochlear amplification.

哺乳动物耳蜗中的主动机械放大需要一个称为电动性的过程，即电压驱动的感觉外毛细胞（OHC）的超快长度变化。产生运动的基本运动是 prestin (SLC26A5)，一种阴离子转运蛋白相关的 OHC 特异性膜蛋白。人们认为电动性是由 prestin 的构象变化引起的，这种变化是由膜电位的变化直接触发的，并且涉及阴离子与蛋白质的结合。在上一个资助期间，我们基于同源建模、分子动力学模拟和实验上广泛的诱变，建立了 prestin 和非哺乳动物同源物的 3D 结构模型。该结构的特性与 prestin 的许多功能特征很好地融合，包括阴离子依赖性和先前识别的结构域的功能影响。基于这些结构信息，我们现在想要检查引起运动/或力产生的构象动力学。我们将通过引入的半胱氨酸残基修饰的状态依赖性和环境敏感染料来分析电压和阴离子依赖性构象变化。除了分子作用模式之外，目前还不清楚 prestin 如何促进机械放大：通过设置活性过程的设定点以缓慢的方式，或者通过在高声频下以循环方式产生力来超快地实现。为了区分这些机制，我们将通过尖端的基因组工程方法将动力学改变的 prestin 突变体引入小鼠体内，并根据耳蜗放大来分析外周听力。