Role of myosin 1c in adaptation in the inner ear

描述（由申请人提供）：本研究的广泛、长期目标是确定听力和平衡的分子机制。内耳感觉毛细胞上充满肌动蛋白的突起，或称静纤毛，将声音和机械振动产生的力转化为神经冲动。机械转导的流行模型，被认为是耳蜗和前庭毛细胞都很保守，是其中相邻的静纤毛连接的细胞外丝称为尖端链接，偏转响应于刺激，从而导致打开或关闭的转导通道。这些通道连接到一个适应-运动复合体，该复合体控制尖端-链接张力。在兴奋性刺激期间，张力最初很高，打开通道;然后运动复合体沿着肌动蛋白细胞骨架滑动，降低尖端连接张力并允许通道关闭。相反，在负刺激期间，张力最初较低，通道关闭;然后运动复合体上升到细胞骨架，恢复静息张力并重新打开通道。分子马达肌球蛋白1c（Myo 1c）在静纤毛中的关键位置的定位;并且使用表达可被选择性抑制的突变Myo 1c的小鼠的研究已经显示Myo 1c参与该称为适应的过程（Holt等人，2002; Stauffer等人，2005年）。适应使毛细胞在长时间的刺激下对新的刺激保持敏感。该提案的目标是通过测量表达具有确定分子特性的Myo 1c突变体的小鼠的适应性来确定Myo 1c如何支持适应的特定方面。Myo 1c突变体将包括（i）影响Myo 1c适应机械负荷的能力的突变体，这是本实验室先前研究中预测的Myo 1c的特性（Batters et al.，2004 a; 2004 b）;（ii）对Ca 2+异常敏感的人，Ca 2+调节Myo 1c和适应。具体目标是：体外表达Myo 1c突变体，并使用ATP酶测定、运动测定、动力学分析和单分子力学研究来表征Myo 1c突变体的生物化学和力学性质。接下来，将产生表达这些突变Myo 1c分子的敲入小鼠，并在来自敲入小鼠的毛细胞中测量适应性。体外和动物研究的结合有望为Myo 1c的分子机制及其在适应中的作用提供关键的新见解。这些知识最终可能导致设计合理的诊断和治疗方法来治疗听力和/或平衡疾病。这项研究的重点是分子马达蛋白Myo 1c，它被认为是内耳毛细胞中的适应-马达复合物。确定毛细胞中转导装置的关键成分（如Myo 1c）的生物化学和生物物理特性及其在适应中的作用对于揭示听力和平衡的分子机制至关重要，因此可以开发新的和适当的诊断和治疗方法来预防或治疗听力损失和眩晕。

DESCRIPTION (provided by applicant): The broad, long-range goal of this study is to determine the molecular mechanism of hearing and balance. Actin-filled projections, or stereocilia, on the sensory hair cells of the inner ear convert force produced by sounds and mechanical vibrations into nerve impulses. The prevailing model for mechanotransduction, thought to be well-conserved for both cochlear and vestibular hair cells, is one in which neighboring stereocilia connected by extracellular filaments called tip links, deflect in response to stimuli, thereby causing the opening or closing of transduction channels. The channels are attached to an adaptation-motor complex, which controls tip-link tension. During an excitatory stimulus, tension is initially high, opening channels; the motor complex then slips down the actin cytoskeleton, reducing tip-link tension and allowing the channels to close. By contrast, during a negative stimulus, tension is initially low and channels close; the motor complex then ascends the cytoskeleton, restoring the resting tension and reopening the channels. Localization of the molecular motor, myosin 1c (Myo1c) at strategic places in the stereocilia; and studies using mice expressing a mutant Myo1c that can be selectively inhibited have shown Myo1c's involvement in this process known as adaptation (Holt et al., 2002; Stauffer et al., 2005). Adaptation allows hair cells under prolonged stimuli to remain sensitive to new stimuli. The goal of this proposal is to determine how Myo1c supports specific aspects of adaptation by measuring adaptation in mice expressing Myo1c mutants with defined molecular properties. Myo1c mutants will include (i) those that affect the ability of Myo1c to adapt to mechanical load, a property predicted for Myo1c from previous studies in this laboratory (Batters et al., 2004a; 2004b); and (ii) those with aberrant sensitivity to Ca2+, which regulates Myo1c and adaptation. The specific aims are: to express in vitro and characterize the biochemical and mechanical properties of Myo1c mutants using ATPase assays, motility assays, kinetic analyses and single-molecule mechanical studies. Next, knock-in mice expressing these mutant Myo1c molecules will be generated and adaptation will be measured in hair cells from the knock-in mice. The combined in vitro and animal studies are expected to provide critical, new insight into the molecular mechanism of Myo1c and its role in adaptation. This knowledge could ultimately lead to the design of rational diagnostics and therapies to treat diseases of hearing and/or balance. The proposed study focuses on the molecular motor protein, Myo1c, which is implicated as the adaptation-motor complex in the hair cells of the inner ear. Determining the biochemical and biophysical properties of key components of the transduction apparatus in hair cells, like Myo1c, and its role in adaptation are of fundamental importance to revealing the molecular mechanisms of hearing and balance, so that new and appropriate diagnostics and therapies to prevent or treat hearing loss and vertigo can be developed.