Intracellular calcium in hair cells

描述(由申请者提供)：长期目标是了解听觉毛细胞中转导的细胞机制，并描述耳蜗声调组织的潜在因素。实验将集中在转导通道适应和发束机制上，以确定它们受钙离子的调节以及在频率选择性中的作用。一个主要的目标是提高机械刺激器的速度，并利用不同的准备和条件来获得准确的转导和主动束运动的动力学测量。毛细胞的反应将在哺乳动物和鸟类的分离耳蜗中进行测量，并将与细胞内钙成像相结合。其具体目标是：(1)记录沙土鼠和大鼠毛细胞在听力开始前后的机械换能器电流，记录动力学和其他特性随耳蜗位置的变化；(2)表征单个机械换能器通道的特征并研究其随耳蜗位置的变化以及钙离子和环AMP对其的调制，为候选通道蛋白提供基线；(3)测量毛束的力学特性，并寻找哺乳动物毛细胞中自发的和活跃的发束运动。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这两个过程在完整哺乳动物耳蜗的放大和频率调谐中的作用。(4)记录机械换能器电流并测量雏鸡听觉乳头中短(外)毛细胞的毛束力学，以此作为可能的但未经证实的位置，其中主动毛束运动可增加频率选择性。与哺乳动物发束的特性进行比较，将有助于深入了解耳蜗放大的进化；(5)测量和改变发束中的钙离子浓度，并将其与机械转导通道适应和主动毛束运动的控制联系起来。我们将研究细胞内钙缓冲和摄取对限制钙瞬变的作用，特别是线粒体。由于毛细胞承受着巨大的钙负荷，钙稳态的紊乱可能是导致损伤的主要原因。钙离子对机械转导通道的调节可能对所有毛细胞都是常见的，并且可能是噪声暴露、耳毒剂中毒或衰老过程中不可逆转的损伤部位。老化或过度刺激导致的听力损失通常局限于高频，并与耳蜗一端毛细胞的退化有关。这项工作将通过将转导的特性与哺乳动物耳蜗中的位置进行映射来解决这种差异敏感性的原因。推测大多数Ca~(2+)通过MT通道进入毛细胞，高频外毛细胞易损性的增加反映了较大的MT电流导致更大的Ca~(2+)内流。听力障碍是人类最常见的致残性感觉缺陷。严重到严重的听力损失，主要可归因于感觉毛细胞的损伤，每1000名新生儿中就有一人受到影响，70岁以上的人中有60%的听力损失至少为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机械和电学特性以及钙离子对它们的调节来解决这些机制。我们推测，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.