Probing how hair bundle mechanical properties shape the mechanotransducer receptor current

摘要：听觉和前庭感觉细胞使用发束，一种阶梯状的肌动蛋白填充阵列 立体纤毛，将机械运动转化为电信号。机械门控(MET)离子通道 位于较短的立体纤毛的顶端，通过拉动延伸的顶端连杆而产生的力来激活 在立体纤毛之间。因为感觉发束是两种遗传性疾病(如引座症)的主要部位 而且也容易受到噪音和老化的破坏，因此了解这些捆绑包的运行方式至关重要 设计预防和恢复功能的治疗方法。哺乳动物的耳蜗毛束有不同寻常的 形态和立体纤毛之间的连接不像其他内耳末端器官那样紧密。的确有 关于影响MET电流和发束的潜在过程的机制有相当大的争论 机械，如快速和慢速适应、门控合规性和电压驱动响应。还有更远的 关于我们是否真的在MET电流反应和机械、分子之间存在因果联系的争论 机械装置。在能够使用新发现的MET分子的基因操作能力之前，我们 需要清楚地了解发束的生物物理特性以及它们如何影响MET受体电流。 我们假设，束运动中缺乏连通性是为了优化发束对自然 立体纤毛的刺激和同步来自覆盖膜(OHC)或液体 刺激(IHC)。我们进一步假设，我们将确定快适应和慢适应的机械关联 以及门禁合规性；然而，我们确实预计与其他头发相比，适应速度不会那么慢 而且，慢适应的机制将与经典理论不符。最后。我们 符合通道属性的假设与发束机制一起工作，以创建受体的调谐 电流。我们将从以下具体目标来研究这些假设。SA1将生成一个 大鼠和大鼠在多个频率位置的MET通道和毛束特性的综合数据集 小鼠P10-12岁。利用三种刺激模式，宽探头、流体喷射和新的 开发了窄探针，我们可以分离出MET通道和发束的特性。SA2将直接 使用新开发的高速成像解决发束机制和已知的发束属性 使用窄探头或流体喷射技术。试验将针对MET通道门控合规性、快速和 缓慢适应和电压依赖的机械性发束反应。SA3将产生频率 使用宽探头和流体喷射来确定过滤特性的生理条件下的响应曲线 通道和发束的颜色。这些目标的完成将提供前所未有的量化水平 关于发束如何运动以及这种运动如何塑造产生的MET受体电流的信息。 它们将成为评估分子操作的标准。

Abstract: Auditory and vestibular sensory cells use the hair bundle, a stair-cased array of actin filled stereocilia, to translate mechanical motion into an electrical signal. Mechanically-gated (MET) ion channels located at the tips of shorter stereocilia are activated by force created by the pulling of a tip link that extends between stereocilia. As sensory hair bundles are a major site for both genetic disorders like Ushers syndrome and are also susceptible to damage from noise and aging, understanding how these bundles operate is critical to designing therapies for prevention and restoration of function. Mammalian cochlear hair bundles have unusual morphologies and interstereocilia connectivity that is not as tight as other inner ear end organs. There is considerable debate as to the mechanisms underlying processes impacting MET currents and hair bundle mechanics, like fast and slow adaptation, gating compliance and voltage driven responses. There is further controversy over whether we truly have causal links between MET current responses and mechanical, molecular mechanisms. Before being able to use the power of genetic manipulation of newly identified MET molecules, we need a clear understanding of hair bundle biophysical properties and how they impact MET receptor currents. We hypothesize that the lack of connectivity in bundle motion is to optimize the hair bundle's response to natural stimulation and that synchronization of stereocilia comes from the tectorial membrane (OHCs) or the fluid stimulation (IHCs). We further hypothesize that we will identify mechanical correlates for fast and slow adaptation as well as gating compliance; however, we do expect there to be less slow adaptation as compared to other hair cell types but also that the mechanism of slow adaptation will not align with classical theories. And finally. we hypothesize that MET channel properties work with hair bundle mechanics to create tuning of the receptor current. We will investigate each of these hypotheses in the following specific aims. SA1 will generate a comprehensive data set of MET channel and hair bundle properties at multiple frequency positions from rats and mice P10-12 of age. By taking advantage of three modes of stimulations, wide probe, fluid jet and the newly developed narrow probe, we can separate between MET channel and hair bundle properties. SA2 will directly address hair bundle mechanics and known hair bundle properties using the newly developed high-speed imaging with either narrow probe or fluid jet technology. Experiments will target MET channel gating compliance, fast and slow adaptation and voltage dependent mechanical hair bundle responses. SA3 will generate frequency response curves under physiological conditions using the wide probe and fluid jet to define the filtering properties of the channel and the hair bundle. Completion of these aims will provide an unprecedented level of quantitative information as to how the hair bundle moves and how this motion shapes the MET receptor current generated. They will be the standard by which molecular manipulations can be assessed.