Intracochlear Electrochemical Gradients

描述(申请人提供)：这项研究的长期目标是研究外毛细胞电动的物理决定因素。外毛细胞通过将耳壳内电化学梯度的能量转化为机械能，提高了哺乳动物听力的敏感性和频率选择性。负责的马达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，中间夹着一个细胞骨架网络。侧壁的能量转换是双向的(电气到机械和机械到电气)。电极化和机械位移之间的紧密耦合是压电性的特征。本项目的具体目标是：1)确定驱动电动的高频膜电位的机制；2)确定侧壁对调节和维持细胞功能所需的电化学梯度的作用。协调的理论和实验方法确定了压电和离子对电池高频响应的贡献。它们还阐述了侧壁独特的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进行运输。实验将通过记录从正常和prestin缺失突变小鼠分离的外毛细胞来评估膜蛋白prestin的作用。荧光类脂类似物和膜蛋白的侧向迁移率将被确定。方法包括新转基因小鼠的建立、微阻抗谱、电压和电流钳，包括双针记录；光漂白后的荧光恢复、共聚焦显微镜、全内反射光解、视频显微镜和计算模拟。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.