Membrane properties of the OHC system

耳蜗由多种细胞类型组成，包括感觉细胞、支持细胞和神经细胞。 综上所述，这些细胞组成了一个功能复杂且凝聚在一起的电子单元，它启动 对我们环境中的声学信息进行分析。我们利用体外培养的方法， 包括分离的耳蜗外植体、单细胞和最近稳定的细胞系，以阐明耳蜗病 单元函数；策略是在集成到内聚性之前表征基本属性 对器官的了解。目前，该项目的总体目标集中在确定 外毛细胞(OHC)的关键膜成分的生物物理性质 听觉功能的参与者，使用电生理学、分子生物学、建模和 高分辨率低温电子显微镜。尽管自上一次会议以来，我们在许多方面都取得了重大进展 在2010年的续订中，我们现在专注于三个特定的研究主题，这三个主题从我们最重要的 成就。特别是，我们调查的主要领域之一一直是，也将是 阴离子对OHC分子马达(Prestin，SLC26A5)机电活性的影响。这 离子是耳蜗放大的根源(Santos-Sacchi等人，2006年)。事实上，NIDCD的2012-2016年 战略计划明确将理解负离子控制听力作为一个关键目标。三位一体 目的是1)评估OHC非线性电容和氯离子依赖的动力学行为 电动学，2)表征细胞内氯离子的运动 和OHC，以及我们创建的新的预熔式YFP氯化物传感器，以及3)确定高 Pretin(SLC26A5)及其最近的哺乳动物家族成员SLC26a6的分辨结构 冷冻-EM，目标是识别由于氯化物和电压引起的构象变化。我们 假设了解这些分子活动将有助于理解OHC如何 使我们能够清楚地听到微环境的影响是如何导致 折磨着数百万人的OHC。

The cochlea is composed of a variety of cell types including sensory, supporting and neural elements. Taken together, these cells comprise a functionally intricate and cohesive electrical unit that initiates the analysis of acoustic information within our environment. We capitalize on the in vitro approach, including isolated cochlea explants, single cell, and more recently stable cell lines to elucidate cochlear cell function; the strategy is to characterize basic properties prior to integration into a cohesive understanding of the organ. Currently, the overarching aim of this project is focused on determining the biophysical properties of key membrane components of the outer hair cell (OHC), one of the major players in auditory function, using a balance of electrophysiology, molecular biology, modelling and high resolution cryo-EM. Though we have made significant progress on many fronts since our last renewal in 2010, we now focus on three specific research topics that evolve from our most significant accomplishments. In particular, one of the main areas of our investigations has been and will be the influence of anions on the OHC molecular motor’s (prestin, SLC26a5) electro-mechanical activity. This ion is at the root of cochlear amplification (Santos-Sacchi et al., 2006). Indeed, the NIDCD’s 2012-2016 Strategic Plan specifically identifies understanding anion control of hearing as a key goal. The three aims are 1) to evaluate the chloride-dependent kinetic behavior of OHC nonlinear capacitance and electromotility, 2) to characterize intracellular chloride movements in the prestin-transfected HEK cell and OHC with a new prestin-fused YFP chloride sensor we created, and 3) to determine the high resolution structure of prestin (SLC26a5) and its closest mammalian family member SLC26a6 using cryo-EM, with the goal of identifying conformational changes due to chloride and voltage. We hypothesize that understanding these molecular activities will aid in understanding how the OHC enables us to hear so well and in turn how micro-environmental influences may lead to pathologies of the OHC that afflict millions.