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扩增需要一种称为电动性的过程，即电压驱动的超快长度变化的感觉外毛细胞（OHC）。产生运动性的基本电动机是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，电动性是由普雷斯汀的构象变化引起的，这些变化是由膜电位变化直接触发的，并涉及阴离子与蛋白质的结合。在上一个资金期间，我们基于同源性建模，分子动力学模拟以及 - 实验性的广泛诱变，建立了Prestin和非哺乳动物同源物的3D结构模型。结构的性质与普雷斯汀的许多功能特征（包括阴离子依赖性和先前鉴定的域的功能影响）很好地收敛。基于这些结构信息，我们现在想检查产生运动/或力的产生的构象动力学。我们将通过对引入半胱氨酸残基的修饰和环境敏感染料的状态依赖性分析电压和阴离子依赖性构象变化。除了分子的作用模式之外，还不清楚普雷斯汀如何促进机械放大：通过在高声频率下通过周期以周期产生循环的方式来设置活性过程的设定点或超快，以缓慢的方式进行机械扩增。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，并通过人工耳蜗扩增分析外围听力。