Molecular Mechanisms of Auditory Transduction

描述（由申请人提供）：本研究旨在了解内耳毛细胞中的强制门控转导通道，该通道将声音基本转换为神经信号。研究的两个目的是了解这些通道如何定位以响应力，以及它们如何产生放大传入声音的反馈力。每个毛细胞都有一束以肌动蛋白为基础的立体纤毛，排列高度增加；细胞的每根立体纤毛延伸一根丝状的“尖端连接”到下一根更高的立体纤毛。束的运动使尖端环节收紧；它们反过来打开强制门控离子通道，打开离子通道使细胞去极化。然而，目前尚不清楚转导通道与尖端连接的关系，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，它提供毫秒级的时间分辨率。第二种是2P-STED显微镜，这是一种新开发的“超分辨率”显微镜，其空间分辨率比传统光学显微镜高三到五倍。两者都将使用检测Ca2+进入通道的染料来定位转导通道。第三种是STORM显微镜，这是另一种超分辨率技术，可以观察连接到通道的单个尖端链接，并检测它们的角度和极性。转导通道的打开和关闭涉及到蛋白质在几纳米尺度上的运动，但这些运动可以使毛细胞的整个毛束移动几十纳米。在转导通道打开几毫秒后，Ca2+通过通道进入，导致通道再次关闭，这一过程被称为快速适应。通道闭合终止向内的电流，用膜片钳放大器观察到，但用玻璃纤维探针观察到，它经常产生发束的快速向后运动。与通道关闭相关的运动，虽然是微小的，但被认为是哺乳动物耳蜗中主动机械反馈的基础，它将传入的声音放大100倍或更多，并产生异常尖锐的频率调谐，使音调的感官辨别成为可能。然而，我们还不知道基本的力是如何产生的，即Ca2+是如何关闭通道的。我们将使用柔性玻璃纤维探针来刺激头发束并记录它们的运动，并将通过脉冲激光光解释放头发束中的Ca2+直接控制头发束中的Ca2+。Ca2+将随着束偏向于不同的位置而释放，以绘制Ca2+诱导的运动对位置的依赖性。这些结果将与四种不同的Ca2+作用模型的预测结果进行比较。对Ca2+如何产生快速适应的清晰理解可以纳入耳蜗放大如何工作的模型。

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. PUBLIC HEALTH RELEVANCE: These experiments are aimed at resolving two very fundamental issues in auditory transduction. First, we need to understand the location of mechanotransduction channels for further studies of the transduction apparatus, in particular to identify new proteins that-like the tip-link proteins-may be mutated in inherited deafness. Second, we need to understand the active mechanical feedback by hair cells that is essential for cochlear tuning, so as to understand the perceptual deficits produced by age-related loss of hair cells.