Hearing Loss and Calcium Regulation

听力损失和钙调节

• 批准号: 9335140
• 负责人: DWAYNE D SIMMONS
• 金额: $ 0.01万
• 依托单位: BAYLOR UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9335140
• 关键词: Hearing; Loss; Calcium; Regulation

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.

描述（由申请人提供）：该指导的研究培训建议提供了必要的时间和培训，以增加和增强有缺陷的CA2+稳态和信号传导可能涉及听力损失的新研究。在M. Charles Liberman博士的实验室内对人工耳蜗功能的体内评估将进行指导的研究培训（AIM 1）；在测量戴维HE博士实验室内的毛细胞运动中（AIM 2）；在测量Fabio Mammano博士实验室内的毛细胞胞质Ca2+水平（AIM 3）中。我们的初步数据表明，靶向启用性的CA2+结合蛋白（OCM）主要是外毛细胞（OHC）（OHC）的靶向破坏会导致渐进的听力损失。我们假设OCM调节保护OHC免受噪声损伤和衰老缺陷所需的ca2+水平。具体而言，该提案调查了机制，即在没有OCM的情况下，可能会使耳朵更容易受到噪音和衰老的影响。特定的目标1检验了以下假设：OCM通过OCM缓冲会改变传出介导的反应。首先，我们将通过测量DPOAE阈值和生长曲线和测量耳蜗电位来测试OCM突变体中的体内OHC函数，重点是耳蜗显微关节（CM）电位。 DPOAE为人工耳蜗提供了一个窗口。尽管取决于频率和SPL，但CM响应可以提供OHCS传感器能力的度量，并且是发束功能的指示。其次，我们将测试OCM的紫胶是否改变了传出介导的响应。传出刺激通常会降低OHC对耳蜗扩增的贡献，从而引起对DPOAE幅度的快速抑制。具体目标2检验了以下假设：OCM的Ca2+缓冲改变了OHC的Ca2+依赖性运动响应。0increases在胞质Ca2+水平中导致由Ca2+依赖性磷酸磷酸化介导的OHC延伸。但是，几乎没有理解诱导OHC缩短或轴向刚度降低的机制。我们将研究OHC运动和电生理反应。我们将测量OHC在整个电池（电压夹条件）中向电压步骤的长度变化。为了评估OCM的靶向缺失是否影响电压门控通道，我们还将测量OHC电流 - 电压关系和电容。特定的目标3检验了以下假设：OCM的Ca2+缓冲改变了Ca2+瞬变的大小。 Ca2+调节中的缺陷可能导致Ca2+瞬变的扩大或CA2+信号振幅增加。我们将使用基于荧光的测量结果研究诱导的[Ca2+] I通量。我们还将将细胞外Ca2+的涌入与OCM突变体中的内部（商店）Ca2+进行比较。总之，选择用于研究培训的实验室将为PI提供新的策略和工具，以进一步了解CA2+调节在耳聋中的作用，并增强对未来资金的竞争力。