Hearing Loss and Calcium Regulation

描述(由申请者提供)：这项有指导的研究培训方案提供了必要的时间和培训，以加强和加强关于有缺陷的钙稳态和信号如何参与听力损失的新研究。指导研究培训将在M.Charles Liberman博士的实验室(目标1)内对耳蜗功能进行活体评估；在David He博士的实验室(目标2)内测量毛细胞的运动性(目标2)；以及在Fabio Mammano博士的实验室内测量毛细胞胞浆钙水平(目标3)。我们的初步数据表明，靶向破坏主要位于外毛细胞的主要钙结合蛋白--肿瘤调节蛋白(OCM)，会导致进行性听力损失。我们假设，OCM调节细胞内钙离子水平，以保护毛细胞免受噪音损伤和老化缺陷的影响。具体地说，这项提案调查了在没有OCM的情况下可能使耳朵更容易受到噪音和老化影响的机制。特异性目标1验证了OCM对钙的缓冲改变传出中介反应的假设。首先，我们将通过测量DPOAE阈值和生长曲线以及测量耳蜗电电位来测试OCM突变体的体内OHC功能，重点是耳蜗微音器(CM)电位。DPOAEs提供了一扇了解耳蜗声放大的窗口。虽然CM反应依赖于频率和SPL，但CM反应可以提供OHC的换能器能力的测量，并且是毛束功能的指示。其次，我们将测试OCM的Lac是否会改变传出中介的反应。传出刺激通常会降低OHC对耳蜗声放大的贡献，从而引起DPOAE波幅的快速抑制。特异性目标2验证了OCM对钙的缓冲改变了细胞内钙依赖的运动反应的假说。细胞内钙水平的增加导致了钙依赖的磷酸化介导的OHC延长。然而，导致OHC缩短或轴向刚度降低的机制却鲜为人知。我们将研究OHC的运动和电生理反应。我们将测量在整个电池，电压钳制条件下，OHC的长度随电压阶跃的变化。为了评估有针对性地删除OCM是否会影响电压门控通道，我们还将测量OHC的电流-电压关系和电容。特定目标3测试了OCM缓冲钙离子改变钙瞬变大小的假设。Ca~(2+)调节缺陷可能导致Ca~(2+)瞬变变宽或Ca~(2+)信号幅度增加。我们将使用基于荧光的测量来研究诱导的[Ca~(2+)]i通量。我们还将比较OCM突变体中细胞外钙离子的内流和内部(储存)钙离子的内流。总而言之，选择用于研究培训的实验室将为PI提供新的策略和工具，供其在自己的实验室内使用，以进一步了解钙离子调节在耳聋中的作用，并增强未来资金的竞争力。

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.