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机械转导的细胞机制和耳蜗声强直组织的潜在因素。实验将集中在细胞内钙离子在调节转导通道适应和毛束力学中的作用。毛细胞的反应将在爬行动物和哺乳动物的分离耳蜗中进行测量，并将与细胞内钙成像相结合。具体的目的是：(1)记录啮齿动物毛细胞在听力开始前后的换能器电流，记录随着成熟和耳蜗位置的特性的变化。(2)研究单个转导通道的特性以及钙离子对其的调制作用和频率变化。比较海龟和啮齿类动物的结果可能说明转导通道特性的进化；(3)测量毛细胞立体纤毛束的机械特性，并寻找哺乳动物外毛细胞中自发和主动的毛细胞束运动。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哺乳动物耳蜗的放大和调节中的作用。(4)通过单通道记录和特定通道亚型的免疫标记，测量K+通道切片变体在甲鱼耳蜗组织中的分布。这一结果可能为深入了解耳蜗声调组织的起源提供依据。(5)测定和改变发束内的钙离子浓度，并将其与传导通道激活和主动发束运动的控制联系起来。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。由于毛细胞承受较大的钙负荷，钙稳态失调可能是导致细胞死亡的主要原因。钙离子对转导通道的调节可能是所有毛细胞共有的，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的通道。老化或过度刺激导致的听力损失通常局限于高频，并与耳蜗基底部毛细胞的退化有关。这项工作将通过将换能器通道的数量和属性与耳蜗位置进行映射来解决差动敏感性的原因。

DESCRIPTION (provided by applicant): The long-term objectives are to understand the cellular mechanisms of mechanotransduction in auditory hair cells and the factors underlying the cochlea's tonotopic organization. Experiments will focus on the role of intracellular Ca 2+ in regulating transducer channel adaptation and hair bundle mechanics. Hair cell responses will be measured in the isolated cochleas of both reptiles and mammals and will be combined with intracellular Ca 2+ imaging. Specific aims are: (1) to record transducer currents in rodent hair cells before and after the onset of hearing, documenting the changes in properties with maturation and with cochlear location. (2) to characterize single transducer channels and investigate their modulation by Ca 2+ and variation with frequency. Comparison of turtle and rodent results may illustrate the evolution of transducer channel properties; (3) to measure the mechanical properties of hair cell stereociliary bundles and search for spontaneous and active bundle motion in mammalian outer 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 tuning in the mammalian cochlea. (4) to measure the distribution of K+ channel slice variants in the turtle cochlea from both single channel recording and immunolabeling for specific channel isoforms. The results may provide insight into the origin of the cochlea's tonotopic organization. (5) to measure and alter Ca 2+ concentration in hair bundles and relate it to control of transducer channel activation and active hair bundle motion. The contributions of Ca 2+ buffering and uptake into intracellular compartments, especially the mitochondria, to limit Ca 2+ transients will be studied. Since hair cells experience large Ca 2+ loads, disturbance of Ca 2+ homeostasis may be a leading cause of cell death. Ca 2+ modulation of transducer channels is probably common to all hair cells and may be the conduit 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 linked to degeneration of hair cells at the base of the cochlea. The work will address reasons for the differential sensitivity by mapping number and properties of transducer channels with cochlear location.