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

哺乳动物耳蜗中的主动机械放大需要称为电运动的过程，即感觉外毛细胞（OHC）的电压驱动的超快长度变化。产生运动性的基本马达是普雷斯廷（SLC 26 A5），一种阴离子转运蛋白相关的OHC特异性膜蛋白。据认为，电运动性源于普雷斯廷的构象变化，其直接由膜电位的变化触发，并涉及阴离子与蛋白质的结合。在上一个资助期，我们已经建立了一个三维结构模型的普雷斯廷和非哺乳动物同源物，同源性建模，分子动力学模拟和实验的基础上广泛的诱变。该结构的性质与普雷斯廷的许多功能特征，包括阴离子依赖性和先前确定的结构域的功能影响很好地收敛。基于这些结构信息，我们现在想要研究引起运动/或力产生的构象动力学。我们将分析电压和阴离子依赖的构象变化的状态依赖的半胱氨酸残基和环境敏感的染料的修改。除了分子作用模式之外，还不清楚普雷斯廷如何有助于机械放大：以缓慢的方式通过设置主动过程的设定点或通过在高声学频率下以循环接循环的方式产生力而超快。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普雷斯廷突变体引入小鼠，并从耳蜗放大的角度分析外周听力。

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.