Molecular Mechanisms of Auditory Transduction

描述(申请人提供)：这项工作旨在了解内耳毛细胞中的强制门控转导通道，该通道执行声音到神经信号的基本转换。这项研究的两个目的是了解这些通道如何定位以对力做出反应，以及它们如何产生反馈力来放大传入的声音。每个毛细胞都有一束以肌动蛋白为基础的立体纤毛，排列高度不断增加；细胞的每个立体纤毛都向下一个更高的立体纤毛延伸一个丝状的“尖端连接”。束的移动会拉紧尖端的连接；它们反过来拉开力门控离子通道，从而打开细胞的去极化。然而，目前尚不清楚转导通道与末端连接的关系，因此我们无法开始详细了解激活它们的机械连接。我们将使用三种新的光学技术来定位转导通道。一种是扫描场共聚焦显微镜，它提供毫秒的时间分辨率。第二种是2P-STED显微镜，这是一种新开发的“超分辨率”显微镜，它提供的空间分辨率是传统光学显微镜的三到五倍。两者都将利用染料来检测通过通道进入的钙离子，从而定位转导通道。第三种是风暴显微镜，这是另一种超分辨率技术，可以观察连接到通道的各个尖端链接，并检测它们的角度和极性。转导通道的打开和关闭涉及几纳米级的蛋白质运动，但这些运动可以将毛细胞的整个发束移动数十纳米。在转导通道打开几毫秒后，通过转导通道进入的钙离子会导致转导通道再次关闭，这一过程被称为快速适应。用膜片钳放大器观察到的通道关闭终止了内向电流，但用玻璃纤维探头观察到它通常会导致发束的快速向后移动。与通道关闭相关的运动虽然很微小，但已被认为是哺乳动物耳蜗里活跃的机械反馈的基础，这种反馈将传入的声音放大100倍或更多，并产生异常尖锐的频率调谐，使人能够对音调进行感觉辨别。然而，目前还不知道基本力生产是如何工作的，即钙是如何关闭通道的。我们将使用柔性玻璃纤维探头来刺激发束并记录它们的运动，并将通过脉冲激光光解释放发束中的钙来直接控制发束中的钙。CA2被释放时，束偏向不同的位置，以绘制钙诱导的运动对位置的依赖关系。结果将与四种不同的钙作用模型的预测结果进行比较。对钙如何产生快速适应的清楚理解可以被纳入到耳蜗放大如何工作的模型中。

DESCRIPTION (provided by applicant): This work is designed to understand the force-gated transduction channels in the hair cells of the inner ear, which perform the fundamental conversion of sound into a neural signal. The two aims of the research are to learn how these channels are positioned to respond to force, and also how they produce a feedback force that amplifies the incoming sound. Each hair cell has a bundle of actin-based stereocilia arranged with increasing heights; each stereocilium of a cell extends a filamentous 'tip link' to the next taller stereocilium. Movement of the bundle tightens tip links; they in turn pull open force-gated ion channels that open to depolarize the cell. Yet it is not clear where the transduction channels are in relation to the tip links, and so we cannot begin to understand in detail the mechanical linkage that activates them. We will use three new optical techniques to locate the transduction channels. One is swept-field confocal microscopy, which offers temporal resolution of milliseconds. A second is 2P-STED microscopy, a newly developed "super-resolution" microscopy that offers spatial resolution three-to-five-fold better than conventional light microscopes. Both will localize transduction channels with the use of dyes that detect Ca2+ entering through the channels. A third is STORM microscopy, another super-resolution technique which can observe the individual tip links that are connected to the channels and detect their angle and polarity. The opening and closing of transduction channels involves protein movements on the scale of a few nanometers, but these movements can move the entire hair bundle of a hair cells by tens of nanometers. Just milliseconds after transduction channels open, the Ca2+ entering through them causes them to close again, a process termed fast adaptation. Channel closure terminates the inward current, observed with a patch-clamp amplifier, but it often produces a fast backwards movement of the hair bundle, observed with a glass fiber probe. Movements associated with channel closure, although minute, have been proposed to underlie an active mechanical feedback in the mammalian cochlea that amplifies the incoming sound by 100-fold or more, and that creates an exceptionally sharp frequency tuning which enables sensory discrimination of tones. Yet it is not known how the basic force production works, i.e., how Ca2+ closes channels. We will use flexible glass fiber probes to stimulate hair bundles and to record their movement, and will control Ca2+ in the hair bundle directly by photolytically releasing it with a pulsed laser. Ca2+ will be released with the bundle biased to different positions, to map out the dependence of Ca2+-induced movement on position. The results will be compared to the predictions of each of four different models for Ca2+ action. A clear understanding of how Ca2+ produces fast adaptation can be incorporated into models for how cochlear amplification works.