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机械传导的细胞机制和耳蜗音调组织的潜在因素。实验将集中在调节传感器通道适应和毛束力学的细胞内Ca 2+的作用。毛细胞反应将在爬行动物和哺乳动物的离体耳蜗中测量，并将与细胞内Ca 2+成像相结合。具体目标是：（1）记录啮齿动物毛细胞在听力开始之前和之后的换能器电流，记录随着成熟和耳蜗位置的特性变化。(2)研究钙离子对单通道的调制作用及其随频率的变化。比较龟类和啮齿类动物的结果可以说明换能器通道特性的演变;（3）测量哺乳动物外毛细胞静纤毛束的力学特性，寻找自发和主动的束运动。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调谐在哺乳动物耳蜗的两个过程中的作用。(4)从单通道记录和特异性通道亚型的免疫标记两方面测量龟耳蜗中K+通道切片变体的分布。这一结果可能有助于我们了解耳蜗音调组织的起源。(5)测量和改变毛束中的Ca 2+浓度，并将其与传感器通道激活和主动毛束运动的控制相关联。将研究Ca 2+缓冲和摄取到细胞内室，特别是线粒体，以限制Ca 2+瞬变的贡献。由于毛细胞经历大量的Ca 2+负荷，Ca 2+稳态的紊乱可能是细胞死亡的主要原因。Ca 2+对传感器通道的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通道。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，并与耳蜗底部毛细胞的退化有关。这项工作将通过映射数量和耳蜗位置的换能器通道的属性来解决差异灵敏度的原因。

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.