INTRACELLULAR CALCIUM IN HAIR CELLS

长期的目标是了解听觉毛细胞的细胞转导机制和这些细胞在耳蜗内的tonotopic安排的基础因素。主要重点将是细胞内钙离子在控制传感器适应和毛束力学的作用，通过力学仍然不完全理解。将在完整的海龟耳蜗中测量毛细胞反应，并将与细胞内Ca/2+成像相结合。具体目标是：（1）确定完整毛细胞和已知Ca/2+缓冲液灌注的细胞中内源性Ca/2+缓冲转换器适应性的特性：（2）建立完整毛细胞和分离的静纤毛中单个机械转换器通道的记录方法，并研究Ca/2+对它们的调节作用;（3）Ca/2+和Ca/2+缓冲液对毛束力学性质的影响：（4）通过突触前和突触后细胞的同步记录研究毛细胞与传入神经末梢之间的突触传递。毛细胞Ca/2+的测定将提供有关突触区Ca/2+信号的证据，以及移动的Ca/2+缓冲液和线粒体对Ca/2+稳态的贡献。(5)目的：从海龟毛细胞中克隆Ca/2+激活的K+通道变异体，并通过异源表达来表征其动力学和Ca 2+敏感性。K+通道变体的耳蜗分布的知识可以导致关于如何建立音调组织的见解。这项工作旨在阐明细胞内Ca/2+的控制及其在毛细胞两极的作用机制：对机械转换器通道，对Ca/2+激活的K+通道，和对突触递质的释放。由于毛细胞经历大的Ca 2+负荷，Ca/2+平衡的紊乱-Ca/2+兴奋毒性-可能是毛细胞损伤的主要原因。内部Ca/2+调节机械传感器通道活性的机制可能是所有毛细胞（包括哺乳动物耳蜗中的毛细胞）共有的，并且可能是噪声暴露、耳毒性药物中毒或衰老过程中不可逆损伤的部位。由于机械传感器通道可能是许多疾病的靶点，获得性和遗传性，因此原位探索其机制可以为此类临床问题提供合理的方法。

The long-term objectives are to understand the cellular mechanisms of transduction in auditory hair cells and the factors underlying the tonotopic arrangement of these cells within the cochlea. The main focus will be on the role of intracellular Ca2+ in controlling transducer adaptation and hair bundle mechanics, through mechanics that are still imperfectly understood. Hair cell responses will be measured in the intact turtle cochlea and will be combined with intracellular Ca/2+ imaging. Specific Aims are: (1) to determine the properties of the endogenous Ca/2+ buffering transducer adaptation in intact cells and in cells perfused with known Ca/2+ buffers; (2) to develop methods for recording single mechanotransducer channels both in intact hair cells and in isolated stereocilia, and to investigate their modulation by Ca/2+; (3) to determine the effects of Ca/2+ and Ca/2+ buffers on the mechanical properties of the hair bundles; (4) to study synaptic transmission between the hair cell and afferent nerve terminal by simultaneous recordings from the pre- and post-synaptic cells. Measurements of hair cell Ca/2+ will provide evidence about the Ca/2+ signals at the synaptic zones, and the contributions of mobile Ca/2+ buffers and mitochondria to Ca/2+ homeostasis. (5) to clone Ca/2+- activated K+ channels variants from turtle hair cells and characterize their kinetics and Ca2+ sensitivity by heterologous expression. Knowledge of the cochlear distribution of the K+ channel variants may lead to insights about how the tonotopic organization is established. The work seeks to elucidate the control of intracellular Ca/2+ and its mechanism of action at opposite poles of the hair cell: on the mechanotransducer channels, on the Ca/2+-activated K+ channels, and on the release of synaptic transmitter. Since hair cells experience large Ca2+ loads, disturbances of Ca/2+ balance-Ca/2+ excitotoxicity-may be the leading cause of hair cell damage. The mechanism by which internal Ca/2+ modulates the activity of the mechanotransducer channel is probably common to all hair cells, including those in the mammalian cochlea, and may be the site of irreversible damage during noise exposure, poisoning with ototoxic drugs or aging. Since the mechanotransducer channel may be the target of many diseases, acquired and genetic, exploring its mechanisms in situ could provide a rational approach to such clinical problems.