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的细胞转导机制，并描述耳蜗张力异位组织的潜在因素。实验将集中在换能器通道适应和毛束力学来定义Ca2+对它们的调节和频率选择性的作用。主要目标是提高机械刺激器的速度，并利用不同的制备和条件来获得转导动力学和主动束运动的准确测量。将在哺乳动物和鸟类的分离耳蜗中测量毛细胞反应，并将与细胞内Ca2+成像相结合。具体目标是：(1)记录沙鼠和大鼠毛细胞在听力发生前后的机械传感器电流，记录其动力学和其他特性随耳蜗位置的变化；(2)表征单个机械换能器通道，并研究其随耳蜗定位和Ca2+和环AMP调节的变化，为候选通道蛋白提供基线；(3)测量毛束的力学特性，寻找哺乳动物毛细胞中自发和主动的毛束运动。活动束运动和外毛细胞收缩之间的相互作用将用于评估这两个过程在完整哺乳动物耳蜗放大和频率调谐中的作用。(4)记录机械传感器电流并测量小鸡听觉乳头短毛细胞（外部）的毛束力学，作为一个可能但未经证实的部位，活跃的毛束运动被用来增加频率选择性。与哺乳动物毛束的特性进行比较将有助于深入了解耳蜗放大的进化过程；(5)测量和改变毛束中Ca2+的浓度，并将其与机械传感器通道适应和活跃的毛束运动的控制联系起来。将研究Ca2+缓冲和摄取到细胞内室，特别是线粒体，以限制Ca2+瞬态的贡献。由于毛细胞经历较大的Ca2+负荷，Ca2+稳态的紊乱可能是损伤的主要原因。Ca2+对机械换能器通道的调节可能是所有毛细胞共同的，并且可能是在噪声暴露，耳毒性物质中毒或衰老期间不可逆损伤的部位。老化或过度刺激导致的听力丧失通常局限于高频，并与耳蜗一端毛细胞的退化有关。这项工作将通过在哺乳动物耳蜗中定位转导的特性来解决这种差异敏感性的原因。据推测，大多数Ca2+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由于较大的MT电流而增加的Ca2+内流。听力障碍是人类最常见的致残性感觉缺陷。严重到深度的听力损失，主要是由于感觉毛细胞的损伤，影响1千分之一的新生儿，60%的70岁以上的人听力损失至少为25分贝。它有一系列的原因，包括遗传、噪音或药物引起的，以及与年龄有关的，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力学和电学特性以及钙离子对它们的调节来解决这些机制。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.