Intracochlear Electrochemical Gradients

描述（由申请人提供）：本研究的长期目标是研究外毛细胞电活动的物理决定因素。外毛细胞通过将耳蜗内电化学梯度的能量转化为机械能来增强哺乳动物听觉的灵敏度和频率选择性。负责的运动机制位于外毛细胞的侧壁，这是一个100纳米厚的三层结构，由两层膜组成，细胞骨架网络夹在它们之间。侧壁的能量转换是双向的（电到机械和机械到电）。电极化和机械位移之间的紧密耦合是压电性的特征。本项目期间的具体目标是：1）确定有助于驱动电运动的高频膜电位的机制，2）确定侧壁对细胞功能所需的电化学梯度的调节和维持的贡献。协调的理论和实验方法确定的压电和离子的贡献细胞的高频响应。他们还解决了侧壁的独特分子组织如何影响膜成分的扩散，并有助于离子，水和其他分子通过其膜之间的狭窄空间的运输。实验将通过记录分离自正常和普雷斯廷无效突变小鼠的外毛细胞来评估膜蛋白普雷斯廷的作用。荧光脂质类似物和膜蛋白的横向流动性将被确定。方法包括开发新的转基因小鼠，微电阻抗谱，电压和电流钳，包括双吸管记录;荧光漂白后的恢复，共聚焦显微镜，全内反射光解，视频显微镜和计算建模。这些研究将进一步阐明外毛细胞作为耳蜗放大器的作用。阐明电运动的物理原理也将有助于新兴的生物纳米技术领域。

DESCRIPTION (provided by applicant): The long-term goal of this research is to study the physical determinants of outer hair cell electromotility. The outer hair cell enhances the sensitivity and frequency selectivity of mammalian hearing by converting the energy of intracochlear electrochemical gradients into mechanical energy. The motor mechanism responsible resides in the outer hair cell's lateral wall, a 100 nanometer thick, three-layer structure composed of two membranes with a cytoskeletal network sandwiched between them. Energy conversion by the lateral wall is bidirectional (electrical to mechanical and mechanical to electrical). The tight coupling between electrical polarization and mechanical displacement is characteristic of piezoelectricity. The specific objectives of this project period are to 1) identify the mechanisms that contribute to the high frequency membrane potentials that drive electromotility and 2) to identify the contributions of the lateral wall to the modulation and maintenance of the electrochemical gradients necessary for cell function. Coordinated theoretical and experimental approaches identify the piezoelectric and ionic contributions to the cell's high frequency response. They also address how the unique molecular organization of the lateral wall influence the diffusion of membrane constituents and contribute to the transport of ions, water and other molecules through the narrow space between its membranes. Experiments will assess the role of the membrane protein prestin by recording from outer hair cells isolated from normal and prestin null mutant mice. The lateral mobility of fluorescent lipid analogues and membrane proteins will be determined. Methods include the development of new transgenic mice, micro electro impedance spectroscopy, voltage and current-clamp, including two-pipette recording; fluorescence recovery after photobleaching, confocal microscopy, total internal reflection photolysis, video microscopy and computational modeling. The studies will further clarify the role of the outer hair cell as the cochlear amplifier. Clarification of the physical principles underlying electromotility will also contribute to the emerging field of biological nanotechnology.