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

哺乳动物耳蜗的主动机械放大需要一个称为电运动性的过程，即电压驱动的感觉外毛细胞（OHC）的超快长度变化。产生运动的基本马达是prestin (SLC26A5)，一种与阴离子转运蛋白相关的ohc特异性膜蛋白。人们认为，电运动性是由prestin的构象变化引起的，这种构象变化是由膜电位的变化直接引发的，并涉及阴离子与蛋白质的结合。在之前的资助期内，我们基于同源性建模、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。该结构的性质与prestin的许多功能特征很好地收敛，包括阴离子依赖性和先前确定的结构域的功能影响。基于这些构造信息，我们现在要研究产生运动或力的构象动力学。我们将通过引入的半胱氨酸残基的状态依赖性修饰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，prestin如何促进机械放大也尚不清楚：通过设置主动过程的设定点来缓慢地促进机械放大，或者通过在高声波频率下以循环方式产生力来快速促进机械放大。为了区分这些机制，我们将通过先进的基因组工程方法将动力学改变的prestin突变体引入小鼠，并从耳蜗放大的角度分析周围听力。

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.