Hearing Loss and Calcium Regulation

听力损失和钙调节

• 批准号: 8700820
• 负责人: DWAYNE D SIMMONS
• 金额: $ 15.8万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 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 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8700820
• 关键词: Affect; Aging; Alleles; Apoptosis; Auditory; Backcrossings; Binding Proteins; Buffers; Calcium; Calcium Binding; Calmodulin; Cell physiology; Cells; Cochlear Microphonic Potentials; Cytoskeleton; Data; Defect; Development; Ear; Electric Capacitance; Environment; Feedback; Foundations; Frequencies; Funding; Future; Genes; Genetic Transcription; Growth; Hair; Hair Cells; Hearing; Homeostasis; Image; In Vitro; Injury; Investigation; Knock-out; Laboratories; Labyrinth; Lead; Length; Link; Measurement; Measures; Mediating; Mentors; Mus; Muscle; Muscle Contraction; Noise; Noise-Induced Hearing Loss; Organ of Corti; Outer Hair Cells; Parvalbumins; Phosphorylation; Physiologic pulse; Play; Presbycusis; Regulation; Relaxation; Reporting; Research; Research Proposals; Research Training; Role; Signal Pathway; Signal Transduction; Synapses; System; Techniques; Testing; Time; Training; Transducers; base; cell motility; deafness; extracellular; hearing impairment; immunoreactivity; in vivo; interest; mutant; oncomodulin; overexpression; protective effect; public health relevance; rat Pres protein; response; tool; voltage; voltage clamp; voltage gated channel

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）：该指导研究培训计划提供了必要的时间和培训，以增加和加强关于缺陷Ca 2+稳态和信号传导如何参与听力损失的新研究。指导研究培训将在M博士实验室内进行耳蜗功能的体内评估。Charles Liberman（目标1）;大卫贺博士实验室内毛细胞运动性的测量（目标2）;以及Fabio Mammano博士实验室内毛细胞胞质Ca 2+水平的测量（目标3）。我们的初步数据表明，有针对性地破坏癌调节蛋白（Ocm），主要在外毛细胞（OHC）中的主要Ca 2+结合蛋白，导致进行性听力损失。我们假设Ocm调节细胞质Ca 2+水平，这是保护OHC免受噪声损伤和老化缺陷所必需的。具体来说，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衰老的影响的机制。具体目标1测试的假设，Ca 2+缓冲OCM改变传出介导的反应。首先，我们将通过测量DPOAE阈值和生长曲线以及测量耳蜗电位来测试OCM突变体中的体内OHC功能，重点是耳蜗颤噪（CM）电位。DPOAEs提供了一个进入耳蜗放大的窗口。尽管取决于频率和SPL，CM响应可以提供OHC的换能器能力的测量，并且是发束功能的指示。其次，我们将测试缺乏OCM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检验了OCM对Ca 2+的缓冲作用改变OHC的Ca 2+依赖性运动反应的假设。胞浆Ca 2+水平的增加导致OHC由Ca 2+依赖性磷酸化介导的延长。然而，引起OHC缩短或轴向刚度降低的机制知之甚少。我们将研究OHC运动和电生理反应。我们将在整个电池、电压钳位条件下测量OHC长度随电压阶跃的变化。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。具体目标3检验了Ocm对Ca 2+缓冲作用改变Ca 2+瞬变大小的假设。Ca 2+调节的缺陷可能导致Ca 2+瞬变的加宽或Ca 2+信号的幅度增加。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Ca 2+与内部（储存）Ca 2+的流入。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，以在自己的实验室内使用，以进一步了解Ca 2+调节在耳聋中的作用，并提高未来资金的竞争力。