Intracellular calcium in hair cells

描述（由申请人提供）：长期目标是了解听觉毛细胞中转导的细胞机制，并描述耳蜗音调组织的潜在因素。实验将集中在换能器通道适应和毛束力学，以确定其调节钙离子和频率选择性的作用。主要目的是提高机械刺激器的速度，并利用不同的制剂和条件，以获得准确的测量动力学的转导和主动束运动。将在哺乳动物和鸟类的分离耳蜗中测量毛细胞反应，并将其与细胞内Ca 2+成像相结合。具体目标是：（1）记录沙土鼠和大鼠毛细胞在听觉发生前后的机械转换器电流，记录动力学和其他特性随耳蜗位置的变化：（2）表征单个机械转换器通道，研究它们随耳蜗位置的变化以及Ca ~（2+）和cAMP的调节，为候选通道蛋白提供基线;（3）测量毛束的力学性质，寻找哺乳动物毛细胞自发主动的毛束运动。主动束运动和外毛细胞收缩之间的相互作用将被用来评估的放大和频率调谐在完整的哺乳动物耳蜗的两个过程中的作用。(4)记录机械传感器电流并测量小鸡听乳头中短（外）毛细胞的毛束力学，听乳头是一个可能但未经证实的部位，其中主动毛束运动用于增强频率选择性。通过与哺乳动物毛束特性的比较，可以深入了解耳蜗放大的演变过程;（5）测量和改变毛束中Ca ~（2+）的浓度，并将其与机械换能器通道适应和主动毛束运动的控制联系起来。将研究Ca 2+缓冲和摄取到细胞内隔室，特别是线粒体，以限制Ca 2+瞬变的贡献。由于毛细胞经历大的Ca 2+负荷，Ca 2+稳态的紊乱可能是损伤的主要原因。机械传感器通道的Ca 2+调节可能是所有毛细胞所共有的，并且可能是噪声暴露、耳毒性剂中毒或衰老过程中不可逆损伤的部位。随着年龄增长或过度刺激而导致的听力损失通常仅限于高频，并且与耳蜗一端毛细胞的退化有关。这项工作将解决的原因，这种差异的敏感性，映射的属性转导与位置在哺乳动物耳蜗。据推测，大部分的Ca 2+通过MT通道进入毛细胞，高频外毛细胞的脆弱性增加反映了更大的Ca 2+内流，因为更大的MT电流。听力障碍是人类最常见的致残性感觉缺陷。严重至极重度听力损失，主要归因于感觉毛细胞的损伤，每1,000名新生儿中就有1人受到影响，60%的70岁以上的人听力缺陷至少为25 dB。它有一系列的原因，包括遗传，噪音或药物诱导，以及与年龄有关，但在大多数情况下，损伤和细胞死亡的基本机制尚不清楚。这项工作将通过记录听觉毛细胞的机械和电学特性以及钙离子对它们的调节来解决机制。我们假设钙超载导致线粒体功能障碍是细胞损伤的主要途径。

DESCRIPTION (provided by applicant): The long-term objectives are to understand the cellular mechanisms of transduction in auditory hair cells and delineate the factors underlying the cochlea's tonotopic organization. Experiments will focus on transducer channel adaptation and hair bundle mechanics to define their regulation by Ca2+ and roles in frequency selectivity. A prime objective is to improve the speed of the mechanical stimulator and utilize different preparations and conditions to obtain accurate measures of the kinetics of transduction and active bundle motion. Hair cell responses will be measured in the isolated cochleae of both mammals and birds and will be combined with intracellular Ca2+ imaging. Specific aims are: (1) to record mechanotransducer currents in gerbil and rat hair cells before and after the onset of hearing, documenting the changes in kinetics and other properties with cochlear location; (2) to characterize single mechanotransducer channels and investigate their variation with cochlear location and modulation by Ca2+ and cyclic AMP, to provide a baseline for candidate channel proteins; (3) to measure the mechanical properties of hair bundles and search for spontaneous and active bundle motion in mammalian hair cells. The interaction between active bundle motion and outer hair cell contractility will be used to assess the roles of the two processes in amplification and frequency tuning in the intact mammalian cochlea. (4) to record mechanotransducer currents and measure hair bundle mechanics of short (outer) hair cells in the chick auditory papilla as a likely but unproven site where active hair bundle motion is used to augment frequency selectivity. Comparison with the properties of the mammalian hair bundles will provide insight into the evolution of cochlear amplification; (5) to measure and alter the concentration of Ca2+ in hair bundles and relate it to control of mechanotransducer channel adaptation and active hair bundle motion. The contributions of Ca2+ buffering and uptake into intracellular compartments, especially the mitochondria, to limit Ca2+ transients will be studied. Since hair cells experience large Ca2+ loads, disturbance of Ca2+ homeostasis may be a leading cause of injury. Ca2+ modulation of mechanotransducer channels is probably common to all hair cells and may be the site of irreversible damage during noise exposure, poisoning with ototoxic agents or aging. Loss of hearing with aging or over-stimulation is often restricted to high frequencies and is linked to degeneration of hair cells at one end of the cochlea. The work will address the reasons for this differential sensitivity by mapping the properties of transduction with location in the mammalian cochlea. It is hypothesized that the majority of Ca2+ enters the hair cells through the MT channels and the increased vulnerability of high frequency outer hair cells reflects a greater Ca2+ influx because of larger MT currents. Hearing impairment is the most common disabling sensory defect in humans. Severe to profound hearing loss, largely attributable to injury to the sensory hair cells, affects 1 in 1,000 newborns, and 60% of people older than 70 years have a hearing deficit of at least 25 dB. It has a range of causes, including genetic, noise or drug induced, as well as being age-related but the basic mechanisms of damage and cell death in most cases are not well understood. The work will address the mechanisms by documenting the mechanical and electrical properties of auditory hair cells and their modulation by calcium ions. We hypothesize that calcium overloading leading to mitochondrial dysfunction is a major route to cell damage.