Membrane properties of the OHC system

OHC 系统的膜特性

• 批准号: 9364111
• 负责人: JOSEPH R SANTOS-SACCHI
• 金额: $ 64.77万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-12 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9364111
• 关键词: Achievement; Acoustics; Anions; Area; Auditory; Automobile Driving; Basilar Membrane; Behavior; Binding; Biophysics; CRISPR/Cas technology; Calibration; Cavia; Cell membrane; Cell physiology; Cells; Characteristics; Charge; Chloride Ion; Chlorides; Cochlea; Consult; Coupling; Cryoelectron Microscopy; Data; Development; Distant; Drops; Electric Capacitance; Electrophysiology (science); Elements; Ensure; Environment; Equilibrium; Event; Family member; Frequencies; Functional disorder; Goals; Hair Cells; Hearing; Homologous Gene; Human; In Vitro; Investigation; Ion Channel; Ions; Kinetics; Knock-in Mouse; Lateral; Lead; Mammals; Measurement; Measures; Mechanics; Membrane; Membrane Potentials; Modeling; Modification; Molecular; Molecular Biology; Molecular Conformation; Molecular Motors; Monitor; Motor; Motor Neurons; Movement; Mus; National Institute on Deafness and Other Communication Disorders; Organ; Outer Hair Cells; Pathology; Phase; Physiological; Plant Roots; Predisposition; Process; Property; Proteins; Publishing; Reporting; Research; Resolution; Role; Saint Jude Children' s Research Hospital; Sensory; Speech; Strategic Planning; Stretching; Structure; Study models; System; Techniques; Testing; Time; Viral; base; biophysical analysis; biophysical properties; cell type; cohesion; data modeling; experimental study; extracellular; interest; non-invasive monitor; rat Pres protein; relating to nervous system; sensor; single molecule; stable cell line; vibration; voltage

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.

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。 总而言之，这些细胞组成了一个功能复杂且具有凝聚力的电气单元，可以启动 对我们环境中的声学信息进行分析。我们利用体外方法， 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 细胞功能；该策略是在整合为有凝聚力的产品之前先表征基本属性 对器官的了解。目前，该项目的总体目标是确定 外毛细胞 (OHC) 关键膜成分的生物物理特性，是主要的细胞膜成分之一 听觉功能的参与者，利用电生理学、分子生物学、建模和 高分辨率冷冻电镜。尽管自上次以来我们在许多方面取得了重大进展 2010 年更新后，我们现在专注于三个具体的研究主题，这些主题是从我们最重要的 成就。特别是，我们调查的主要领域之一已经并将是 阴离子对 OHC 分子马达（prestin，SLC26a5）机电活动的影响。这 离子是耳蜗放大的根源（Santos-Sacchi 等，2006）。事实上，NIDCD 2012-2016 年 战略计划明确将了解阴离子控制听力作为关键目标。三个 目标是 1) 评估 OHC 非线性电容的氯化物依赖性动力学行为，以及 电动性，2) 表征 prestin 转染的 HEK 细胞中的细胞内氯离子运动 和 OHC 与我们创建的新型预熔 YFP 氯化物传感器，以及 3) 确定高 使用 prestin (SLC26a5) 及其最接近的哺乳动物家族成员 SLC26a6 的分辨率结构 冷冻电镜，目的是识别氯离子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了解 OHC 如何 使我们能够很好地听到声音，进而了解微环境的影响如何导致疾病 困扰数百万人的 OHC。