外毛细胞功能调控机理的研究

Outer hair cell’s electromotility is key to the cochlear amplifier. By sensing gating current generated by the deflection of stereocilia， motor protein Prestin undergo conformation change which in turn generate the length changes of outer hair cells. Nonlinear capacitance， the membrane charging incurred by the voltage dependent binding of Prestin and chloride anion in the cell，subject to the influence of intracellular chloride concentration and membrane potential. Since the membrane potential and chloride concentration changes are the result of efferent neurotransmitter release by Ach and GABA， we here propose that the efferent nerve regulate cochlear amplifier by modulating outer hair cell intracellular potential and chloride concentration. This study propose to measure changes of outer hair cell nonlinear capacitance and electromotility upon neurotransmitter delivery. Experiment will first be carried out under varies chloride concentrations to observe the effect of changing membrane potential on voltage clamped outer hair cells; then the next set of experiments will use neurotransmitters Ach and GABA deliver to the perforated patched outer hair cell under current clamp mode to observe changes of membrane potential and nonlinear capacitance. Finally we will introduce a new parameter derived from the simultaneous measure of nonlinear capacitance and electromotility to unravel the mechanism for the modulation of cochlear amplifier.

耳蜗放大器的核心是外毛细胞的能动性，能动性源自外毛细胞马达蛋白变构所产生的细胞长度变形; 马达蛋白在感受纤毛摆动所引发的门控电流后与细胞内氯离子结合并产生变构。非线性电容是马达蛋白在特定膜电压范围内与氯离子结合所造成的膜电容变化，并受细胞内氯离子浓度与静息电位影响而改变。因膜电位及氯离子浓度受传出神经递质Ach和GABA所影响，我们提出传出神经调控外毛细胞及耳蜗放大器的机制是通过改变细胞内电位和氯离子浓度来调节马达蛋白的电压响应范围，从而调节耳蜗放大器。本研究通过测量外毛细胞非线性电容和能动性来观察传出神经递质对外毛细胞功能的调控。实验将首先在不同氯离子浓度条件下观察全细胞膜片钳构式下改变膜电压对非线性电容的影响，进而以灌流神经递质Ach和GABA观察非线性电容的变化。最后我们将引入同步测试非线性电容与能动性作为一个全新的观察变量以测试神经递质外毛细胞的调控，以此阐述耳蜗放大器调控的机理。

本研究基于以往对于Prestin外毛细胞耳蜗放大器调控相关关系的系列实验，发现Prestin动力学时间常数可能不足以支持外毛细胞超高频振动及放大的速度，基于此，本研究首先在在体实验确立外毛细胞能动性仅在耳蜗低频段作用这一颠覆性的发现，继而发现外毛细胞Prestin的年龄相关变化导致耳蜗行波时程延长的现象，并提出耳蜗Rescaling of Frequency Place Coding这一全新理论，同时我们找到Prestin promoter关键的1.4kbp片段，为后续基因治疗的实施提供基础。在对V37I动物模型的研究中，阐明Gjb2 V37I突变致病机理，提出环境因素可加速迟发性耳聋进程，为疾病预防提供依据，针对致病机理中离子循环受限，可在后续寻找对症药物过程中有针对性的筛选。