Hearing Loss and Calcium Regulation

描述（由申请人提供）：这一指导研究培训提案提供了必要的时间和培训，以增加和加强钙离子平衡缺陷和信号传导如何参与听力损失的新研究。指导研究训练将在M. Charles Liberman博士的实验室进行人工耳蜗功能的体内评估（Aim 1）；在何大卫博士的实验室测量毛细胞运动（Aim 2）；以及在Fabio Mammano博士的实验室中测量毛细胞胞浆Ca2+水平（Aim 3）。我们的初步数据表明，肿瘤调节素（Ocm）是一种主要存在于外毛细胞（ohc）中的Ca2+结合蛋白，其靶向破坏可导致进行性听力损失。我们假设Ocm调节细胞-固体Ca2+水平，这是保护OHCs免受噪声损伤和老化缺陷所必需的。具体来说，这项提议调查了在没有Ocm的情况下，耳朵更容易受到噪音和衰老的影响的机制。特异性目的1检验了钙离子缓冲由Ocm改变传入介导的反应的假设。首先，我们将通过测量DPOAE阈值和生长曲线以及测量耳蜗电位来测试Ocm突变体的体内OHC功能，重点是耳蜗麦克风（CM）电位。dpoae为耳蜗放大提供了一个窗口。尽管依赖于频率和声压级，CM响应可以提供OHCs换能器能力的测量，并且是毛束功能的指示。其次，我们将测试Ocm缺失是否会改变传出介导的反应。传出刺激通常会降低OHC对耳蜗放大的贡献，从而引起DPOAE振幅的快速抑制。特异性目的2验证了Ocm对Ca2+的缓冲作用改变OHCs的Ca2+依赖性运动反应的假设。细胞质内Ca2+水平的增加导致Ca2+依赖性磷酸化介导的OHC延伸。然而，引起OHC缩短或轴向刚度降低的机制尚不清楚。我们将研究OHC运动和电生理反应。我们将测量在整个电池、电压箝位条件下ohc对电压阶跃的长度变化。为了评估定向删除Ocm是否会影响电压门控通道，我们还将测量OHC电流-电压关系和电容。特异性目标3测试了钙离子缓冲由Ocm改变Ca2+瞬态的大小的假设。Ca2+调控缺陷可能导致Ca2+瞬态变宽或Ca2+信号幅度增加。我们将使用基于荧光的测量来研究诱导的[Ca2+]i通量。我们还将比较Ocm突变体中细胞外Ca2+和内部（储存）Ca2+的流入。总之，选择进行研究培训的实验室将为PI提供新的策略和工具，以便在他自己的实验室中使用，以进一步了解Ca2+调节在耳聋中的作用，并提高未来资助的竞争力。

DESCRIPTION (provided by applicant): This mentored research training proposal provides the necessary time and training to augment and enhance new research into how defective Ca2+ homeostasis and signaling may be involved in hearing loss. Mentored research training will occur in the in vivo assessment of cochlear function within the laboratory of Dr. M. Charles Liberman (Aim 1); in the measurement of hair cell motility within the laboratory of Dr. David He (Aim 2); and in the measurement of hair cell cytosolic Ca2+ levels within the laboratory of Dr. Fabio Mammano (Aim 3). Our preliminary data suggest that targeted disruption of oncomodulin (Ocm), a major Ca2+ binding protein mostly in outer hair cells (OHCs), leads to progressive hearing loss. We hypothesize that Ocm regulates cyto- solic Ca2+ levels necessary to protect OHCs from noise damage and aging defects. Specifically, this proposal investigates mechanisms that in the absence of Ocm might make the ear more vulnerable to noise and aging. Specific Aim 1 tests the hypothesis that Ca2+ buffering by Ocm alters efferent-mediated responses. First, we will test in vivo OHC function in Ocm mutants by measuring DPOAE thresholds and growth curves and measuring cochlear potentials, with a focus on the cochlear microphonic (CM) potential. DPOAEs provide a window into cochlear amplification. Although dependent on frequency and SPL, the CM response can provide a measure of the transducer capability of OHCs and is an indication of hair bundle functionality. Second, we will test if a lac of Ocm alters efferent-mediated responses. Efferent stimulation normally decreases the OHC contribution to cochlear amplification, thereby eliciting fast suppression of DPOAE amplitudes. Specific Aim 2 tests the hypothesis that Ca2+ buffering by Ocm alters Ca2+-dependent motile responses of OHCs.0Increases in cytosolic Ca2+ levels lead to OHC elongation mediated by Ca2+-dependent phosphoryla- tion. However, the mechanisms that induce OHC shortening or a decrease in axial stiffness are little understood. We will investigate OHC motility and electrophysiological responses. We will measure length changes of OHCs to voltage steps in whole cell, voltage clamp conditions. To assess whether targeted deletion of Ocm affects voltage-gated channels, we will also measure OHC current-voltage relations and capacitance. Specific Aim 3 tests the hypothesis that Ca2+ buffering by Ocm alters the size of Ca2+ transients. Defects in Ca2+ regulation could lead either to a broadening of Ca2+ transients or increases in the amplitude of Ca2+ signals. We will investigate induced [Ca2+]i flux using fluorescent-based measurements. We will also compare influx of extracellular Ca2+ to internal (store) Ca2+ in Ocm mutants. In summary, the laboratories chosen for research training will provide the PI with new strategies and tools to employ within his own laboratory to further understand the role of Ca2+ regulation in deafness and enhance competitiveness for future funding.