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中机械转导的机制，并描述耳蜗声调组织的潜在因素。实验将集中在机械换能器(MT)通道的属性和分子组成以及钙离子对其调节。毛细胞的反应将在分离的小鼠和鸡的耳蜗中进行测量。这项工作的一个特点是使用了小鼠突变体，并将其与培养的听觉上皮细胞相结合。具体目标是：(1)记录MT通道候选分子跨膜通道样(TMC)蛋白TMC1和TMC2突变小鼠耳蜗毛细胞的MT电流。将研究现有的小鼠突变以及那些将被导入耳蜗培养的点突变；(2)研究哺乳动物毛细胞中MT通道适应的钙依赖和机制，验证其不同于非哺乳动物中建立的假设。Ca~(2+)将通过笼中钙离子的内部灌流和光解进行调控。具有肌球蛋白VIIa突变的小鼠也将被表征以确定这种肌球蛋白对适应的贡献；(3)通过产生条件突变并探索它们的性质来确定毛细胞钙结合蛋白oncomodrin在钙缓冲和毛细胞发育中的功能； (4)记录MT电流和测量短(外)毛细胞的毛束力学，作为一个可能但未经证实的位置，在那里主动的毛束运动来增加频率的选择性。通道门控产生的主动束运动和可能的prestin诱导的运动之间的相互作用将被用来评估这两个过程对放大和频率调谐的贡献。通过与哺乳动物外毛细胞的特性进行比较，可以深入了解羊膜动物中耳蜗放大的进化过程；(5)研究胚胎发育过程中鸡听觉乳头中频率调谐的成熟过程，测试先前报道的最佳频率的变化；(3)绘制MT电流的变化图，TMC蛋白质和禽类中的oncomodrin的类似物--小白蛋白-3的作用。希望这些结果将提供毛细胞转导装置的分子组成的证据，并提供有关某些形式的人类遗传性耳聋突变的蛋白质的信息。

DESCRIPTION (provided by applicant): The long-term objectives are to understand the mechanisms of mechanotransduction in auditory hair cells and delineate the factors underlying the cochlea's tonotopic organization. Experiments will focus on the attributes and molecular composition of the mechanotransducer (MT) channel and its modulation by Ca2+. Hair cell responses will be measured in the isolated cochleas of both mouse and chicken. A feature of the work is the use of mouse mutants combined with transfection of cultured auditory epithelia. Specific aims are: (1) to record MT currents in cochlear hair cells from mice with mutations in the transmembrane channel-like (Tmc) proteins Tmc1 and Tmc2, which are molecular candidates for the MT channel. Available mouse mutants will be studied as well as those with engineered point mutations which will be transfected into cochlear cultures; (2) to examine the Ca2+ dependence and mechanism of MT channel adaptation in mammalian hair cells, testing the hypothesis that it differs from that established in non-mammals. Ca2+ will be manipulated by internal perfusion and photolysis of caged Ca2+. Mice with mutations in myosin VIIa will also be characterized to determine the contribution of this myosin to adaptation; (3) to define the function of the hair cell Ca2+-binding protein oncomodulin in Ca2+ buffering and development of hair cells by generating conditional mutants and exploring their properties; the hypothesis is that embryonic expression of oncomodulin is essential for hair cell development but that post-natal loss is without effect; (4) to record MT currents and measure hair bundle mechanics of short (outer) hair cells in the chick auditory papilla as a likely but unproven site where active hair bundle motion is used to augment frequency selectivity. The interaction between active bundle motion derived from channel gating and a possible prestin-induced motility will be used to assess the contributions of the two processes to amplification and frequency tuning. Comparison with the properties of the mammalian outer hair cells will provide insight into the evolution of cochlear amplification in amniotes; (5) to examine the embryonic maturation of frequency tuning in the chicken auditory papilla, testing a previously reported shift in best frequency; also charting the embryonic development of the MT current, the role of Tmc proteins and of parvalbumin-3, the avian equivalent of oncomodulin. It is hoped that the results will supply evidence on the molecular composition of the hair cell transduction apparatus and will yield information about proteins that are mutated in certain forms of human genetic deafness.