INTRACELLULAR CALCIUM IN HAIR CELLS

长期目标是了解听觉毛细胞的细胞转导机制以及这些细胞在耳蜗内的张力异位排列的潜在因素。主要的焦点将是细胞内Ca2+在控制传感器适应和毛束力学中的作用，通过力学仍然不完全理解。毛细胞反应将在完整的海龟耳蜗中测量，并将与细胞内Ca/2+成像相结合。具体目的是：(1)确定内源性Ca/2+缓冲传感器在完整细胞和灌注了已知Ca/2+缓冲液的细胞中的适应性；(2)开发在完整毛细胞和分离的立体纤毛中记录单个机械传感器通道的方法，并研究Ca/2+对它们的调制；(3)测定Ca/2+和Ca/2+缓冲液对毛束力学性能的影响；(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+的测量将提供突触区Ca/2+信号的证据，以及移动Ca/2+缓冲液和线粒体对Ca/2+稳态的贡献。(5)从龟毛细胞中克隆Ca/2+激活的K+通道变异，并通过异源表达表征其动力学和Ca2+敏感性。对耳蜗K+通道变异分布的了解可能有助于了解异位组织是如何建立的。这项工作旨在阐明细胞内Ca/2+的控制及其在毛细胞相反极的作用机制：在机械换能器通道上，在Ca/2+激活的K+通道上，以及在突触递质的释放上。由于毛细胞承受大量Ca2+负荷，Ca/2+平衡的紊乱可能是毛细胞损伤的主要原因。内部Ca/2+调节机械换能器通道活性的机制可能是所有毛细胞共同的，包括哺乳动物耳蜗中的毛细胞，并且可能是噪音暴露，耳毒性药物中毒或衰老期间不可逆转损伤的部位。由于机械换能器通道可能是许多疾病（获得性和遗传性）的靶点，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.