INTRACELLULAR CALCIUM IN HAIR CELLS

长期目标是了解听觉毛细胞转导的细胞机制，以及这些细胞在耳蜗内的紧张性排列的潜在因素。主要的焦点将集中在细胞内钙离子在控制换能器适应和毛束机制中的作用，通过仍然不完全了解的机制。毛细胞的反应将在完整的海龟耳蜗中进行测量，并将与细胞内钙/2+成像相结合。具体目标是：(1)确定内源性Ca/2+缓冲转导通路在完整细胞和灌流了已知Ca/2+缓冲液的细胞中的适应性；(2)建立记录完整毛细胞和分离的立体纤毛细胞中单个机械转导通道的方法，并研究Ca/2+对它们的调节；(3)确定Ca/2+和Ca/2+缓冲剂对发束力学特性的影响；(4)通过对突触前和突触后细胞的同步记录，研究毛细胞和传入神经末梢之间的突触传递。毛细胞Ca/2+的测量将为突触区的Ca/2+信号提供证据，以及移动的Ca/2+缓冲器和线粒体对Ca/2+动态平衡的贡献。(5)克隆甲鱼毛细胞Ca/2+激活的K+通道变异体，并通过异源表达研究其动力学和钙敏感性。对K+通道变异的耳蜗区分布的了解可能导致对紧张性组织是如何建立的洞察。本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：对机械转导通道、对Ca/2+激活的K+通道以及对突触递质的释放。由于毛细胞承受较大的钙负荷，钙/2+平衡的紊乱--钙/2+的兴奋性毒性--可能是毛细胞损伤的主要原因。内源性Ca/2+调节机械转导通道活性的机制可能是所有毛细胞，包括哺乳动物耳蜗毛细胞的共同机制，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转的损伤部位。由于机械转导通道可能是许多获得性和遗传性疾病的靶点，在原位探索其机制可以为此类临床问题提供一种合理的途径。

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.