Prestin动力传递对外毛细胞电运动调控的研究

Siting above the basllar membrane, outer hair cells（OHCs）appear able to perceive its vibration through their mechanosensitive hair bundles and to feed back mechanical forces that enhance both auditory system's sensitivity and its frsquency selectivity．They are mechanical components of the cochlea and have been the subject of many experiments designed to discover just exactly how they generate mechanical forces and how these forces contribute to the micromechanics of the cochlea. .Prestin is a protein that in humans is encoded by the SLC26A5 (solute carrier anion transporter family 26, member 5) gene. Prestin is the motor protein of the outer hair cells of the inner ear of the mammalian cochlea. It is highly expressed in the outer hair cells, and is not expressed in the nonmotile inner hair cells. Immunolocalization shows prestin is expressed in the lateral plasma membrane of the outer hair cells, the region where electromotility occurs. The expression pattern correlates with the appearance of outer hair cell electromotility..Prestin is a transmembrane protein also that mechanically contracts and elongates leading to electromotility of outer hair cells (OHC). Eletromotility is the driving force behind the somatic motor of the cochlear amplifier which is a mammalian evolution that increases sensitivity to incoming sound wave frequencies and thus amplifies the signal. Previous research has suggested that this modulation takes place via an extrinsic voltage-sensor (Partial Anion Transporter model) whereby chloride binds to the intracellular side of prestin and enters a defunct transporter causing prestin elongation..Prestin is required for OHC motility and plays a central role in OHC electromotility. Today, we still have no knowledge of Prestin's most nature, such as the three dimensional organization. Without definitive information, it is difficult to understand how is alters electromechanical coupleing the OHC PM, how is involved in the mammalian cochlear amplifier. In this study, we design experiment to verify the force model or correlationship between Prestin and other membrane proteins.

耳聋是重要的感觉系统疾病，严重影响人们的工作生活。1978年Kemp发现耳声发射，即人耳在听到声音的同时也向外发出特定的声音。随后的研究发现，耳声发射由耳蜗外毛细胞的电运动产生，Presin是电运动的分子基础，但目前Prestin在相互作用、动力传递及功能调控等方面仍缺乏直接实验依据。本研究拟以Prestin动力传递为切入点，通过综合运用免疫电镜、原子力显微镜、基因转染、膜片钳及高速影像系统等病理生理学的技术手段，动态观察正常外毛细胞及Prestin转染细胞的动力蛋白表达水平、细胞结构、电运动的强度和频率特性的异同，揭示Prestin与外毛细胞侧膜中的肌动蛋白、肌球蛋白等细胞结构的相互作用，阐明Prestin动力传递的机制和调控方式。以Prestin动力传递及其调控机制的研究入手，可以为耳聋的发生机制及临床治疗提供新理论依据和思路，对提高国民健康水平、改善耳聋人群的生活质量具有重要意议。

本项目应用能够直接反映Prestin蛋白、外毛细胞与支持细胞相互作用的电生理方法，并结合原子力显微镜、细胞膜受体蛋白免疫荧光染色等的研究手段，在蛋白及细胞水平对外毛细胞及Prestin动力传递的机制进行探索性研究。耳蜗是哺乳动物的听觉终器。具有极高频率响应的外毛细胞电运动（Outer hair cell electromotility）是耳蜗机-电转换过程中独特的功能形式。听觉器官因耳蜗主动放大机制的存在才使听觉灵敏度及频率选择性得以大大提高，而外毛细胞电运动正是耳蜗主动放大机制能够发挥作用的前提条件。以往的研究发现耳蜗支持细胞上有大量缝隙连接蛋白（Connexin）的表达，支持细胞间依靠缝隙连接而相互连接藕合。虽然外毛细胞上没有缝隙连接蛋白的表达，外毛细胞间也不存在缝隙连接耦合，但编码组成缝隙连接蛋白的Connexin基因突变致缝隙连接失功能则会引起严重的听力损失。Deiters细胞（Deiters cell，DC）是耳蜗主要的支持细胞之一，外毛细胞处于支持细胞特别是Deiters细胞的支撑和环绕之中。Deiters细胞细长的指突与外毛细胞顶部的表皮板相连，宽大的胞体与外毛细胞底部相连接构成稳定的弓形结构以支撑Cortis器的外部形态。Deiters细胞上有Connexin基因表达，细胞间借助广泛的缝隙连接通道相互耦合。Deiters细胞与外毛细胞之间虽有组织连接，但以往并未观察到发现两者之间存在直接电导联的证据。临床病例及实验室数据均已证实，Connexin 26（Cx26）基因突变后畸变产物耳声发射（Distortion Product of Otoacoustic Emissions, DPOAEs）会明显降低甚至完全不能被引出。这就提示支持细胞及细胞间的缝隙连接耦合状态与源自Prestin蛋白的外毛细胞电运动之间，可能存在极大相关性。为证实这一推测，我们利用双膜片钳技术对Deiters细胞及细胞间的缝隙连接通道耦合对外毛细胞电运动影响进行了观察，实验发现Deiters细胞能够通过细胞骨架或改变细胞膜的张力来直接影响外毛细胞的电运动。这将为我们更深入地理解听器动力传递的机制及精细调控在内耳中的功能提供新的思路。