Regulation of hair cell functions

毛细胞功能的调节

• 批准号: 9464520
• 负责人: EBENEZER N YAMOAH
• 金额: $ 42.34万
• 依托单位: UNIVERSITY OF NEVADA RENO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-05 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9464520
• 关键词: Adult; Amplifiers; Anions; Biological Assay; Cell physiology; Characteristics; Chimera organism; Co-Immunoprecipitations; Confocal Microscopy; Data; Dimensions; Electric Capacitance; Family member; Fluorescence Resonance Energy Transfer; Frequencies; Functional Imaging; Functional disorder; Gene Family; Genetic; Hair Cells; Half-Life; Hearing; Immunoelectron Microscopy; Immunofluorescence Immunologic; In Vitro; Labyrinth; Length; Link; Mechanics; Mediating; Membrane Potentials; Microscopy; Molecular; Motor; Motor Neurons; Mus; Mutant Strains Mice; Organ; Outcome Study; Outer Hair Cells; Property; Proteins; Quantitative Reverse Transcriptase PCR; Regulation; Reporting; Resolution; Role; System; Techniques; Testing; Time; Transcript; Visual system structure; cellular imaging; deafness; design; fluorescence imaging; genetic regulatory protein; hearing impairment; human model; in vivo; innovation; interdisciplinary approach; live cell imaging; member; molecular imaging; mouse model; new therapeutic target; otoacoustic emission; protein protein interaction; rat Pres protein; response; sensor; sensory system; solute; sound; voltage

Prestin, a motor protein serves as the cochlear amplifier contributing towards the sensitivity of the mammalian inner ear. Moreover, prestin expressed in heterologous systems yields functional characteristics that are not consistent with the bona-fide OHC properties, raising the possibility that there are missing prestin- interacting proteins that should be identified to understand the mechanisms of this remarkable motor protein. Using innovative genetic, molecular, cellular, and functional imaging strategies, we have invoke this hypothesis. In contrast to the dogma that prestin is uniquely OHC-specific, we hypothesize that the authentic cochlear amplifier consists of prestin and its family member, SLC26A6. We further hypothesize that SLC26A6 is required as an efficient transporter to create intracellular Cl- microdomain for the proper function of prestin. To test the hypothesis, we propose the following three aims: 1) To test the spatial expression and co- localization of prestin and slc26a6 in OHCs. First, we will determine the expression and co-localization of prestin and slc26a6 in OHCs using quantitative real-time PCR (qRT-PCR), immunofluorescence confocal microscopy and immuno-electron microscopy (immuno-EM). Prestin-/- and slc26a6-/- mice will serve as negative controls. 2) To test for the physical interaction or the hetero-oligomerization of prestin and slc26a6. To directly test for possible physical interaction or co-assembly between prestin and SLC26A6, we will use co- immunoprecipitation (co-IP) and fluorescence resonance energy transfer (FRET) techniques. 3) To determine the in vitro and in vivo functional roles of the interaction or hetero-oligomerization of prestin and slc26a6. We will record the anion exchange activities and the nonlinear capacitance of the co-expressed prestin and slc26a6. We will design prestin-slc26a6 chimera constructs to further test for function interaction of the two proteins. Finally, we will test the prediction that in the absence of slc26a6, the motor functions of prestin will be severely altered, compromising the hearing sensitivity of slc26a6 null mutant mice. These studies will reveal a missing functional component of the cochlear amplifier and may divulge a new therapeutic target for the treatment of hearing loss or deafness. The outcomes of these studies will alter our present understanding of the functional mechanisms of prestin in OHCs, and would shift the paradigm from “prestin-centric” towards orchestral proteins that operate to amplify sound in the inner ear.

普雷斯廷是一种运动蛋白，作为耳蜗放大器，对耳蜗的敏感性有贡献。 哺乳动物的内耳此外，在异源系统中表达的普雷斯廷产生功能特性 与真正的OHC性质不一致，增加了缺失普雷斯廷的可能性- 这些相互作用的蛋白质应该被识别出来，以了解这种非凡的马达蛋白的机制。 我们使用创新的遗传、分子、细胞和功能成像策略来调用这一点 假说.与普雷斯廷是唯一的OHC特异性的教条相反，我们假设真正的 耳蜗放大器由普雷斯廷及其家族成员SLC 26 A6组成。我们进一步假设SLC26A6 作为一种有效的转运蛋白，产生细胞内Cl-微区，使普雷斯廷发挥正常功能。 为了验证这一假设，我们提出了以下三个目标：1）检验空间表达和协同效应， 普雷斯廷和slc 26 a6在OHC中的定位。首先，我们将确定的表达和共定位 使用定量实时PCR（qRT-PCR）、免疫荧光共聚焦显微镜和免疫组织化学技术检测OHC中的普雷斯廷和slc26 a6 显微镜和免疫电子显微镜（免疫EM）。普雷斯廷-/-和slc 26 a6-/-小鼠将作为 阴性对照。2)测试普雷斯廷和slc26 a6的物理相互作用或异源寡聚化。到 直接测试普雷斯廷和SLC 26 A6之间可能的物理相互作用或共组装，我们将使用共- 免疫沉淀（co-IP）和荧光共振能量转移（FRET）技术。3)以确定 普雷斯廷和SLC 26 A 6的相互作用或异源寡聚化的体外和体内功能作用。我们 将记录共表达的普雷斯廷的阴离子交换活性和非线性电容， slc26a6.我们将设计prestin-slc26a6嵌合体构建体，以进一步测试两者的功能相互作用。 proteins.最后，我们将测试在slc 26 a6缺失的情况下，普雷斯廷的运动功能将被破坏的预测。 严重改变，损害了SLC 26A 6无效突变小鼠的听觉灵敏度。 这些研究将揭示耳蜗放大器缺失的功能组件，并可能泄露 用于治疗听力损失或耳聋的新的治疗靶点。这些研究的结果将改变 我们目前对普雷斯廷在OHC中的功能机制的理解，并将范式从 “prestin中心”对管弦乐蛋白质的运作，以放大声音在内耳。