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.

Prestin，一种运动蛋白作为人工耳蜗放大器，有助于敏感性 哺乳动物内耳。此外，在异源系统中表达的Prestin产生功能特征 与真正的OHC属性不一致的，这增加了缺少Prestin-的可能性 相互作用的蛋白质应被鉴定，以了解这种非凡的运动蛋白的机制。 使用创新的遗传，分子，细胞和功能成像策略，我们已经调用了这一点 假设。与普雷斯汀（Prestin）独特的OHC特异性的教条相反，我们假设是真实的 人工耳蜗放大器由Prestin及其家人SLC26A6组成。我们进一步假设SLC26A6 需要作为有效的转运蛋白来创建细胞内Cl-微区域，以使Prestin的正常功能。 为了检验假设，我们提出以下三个目的：1）测试空间表达和共同 Prestin和Slc26a6在OHC中的定位。首先，我们将确定表达和共定位 使用定量实时PCR（QRT-PCR），免疫荧光共焦 显微镜和免疫电子显微镜（免疫-EM）。 prestin - / - 和slc26a6 - / - 小鼠将作为 负面对照。 2）测试Prestin和SLC26A6的物理相互作用或异差异化。 直接测试Prestin和Slc26a6之间可能的物理相互作用或共同组装，我们将使用共同 免疫沉淀（CO-IP）和荧光共振能量转移（FRET）技术。 3）确定 Prestin和Slc26a6的相互作用或异差异化的体外和体内功能作用。我们 将记录共表达Prestin的阴离子交换活动和非线性电容 SLC26A6。我们将设计prestin-slc26a6嵌合体构造，以进一步测试两者的功能相互作用 蛋白质。最后，我们将测试以下预测：在没有SLC26A6的情况下，Prestin的电机功能将为 严重改变，损害了SLC26A6无效突变小鼠的听力敏感性。 这些研究将揭示人工耳蜗放大器的缺少功能成分，并可能剥离A 治疗听力丧失或死亡的新理论目标。这些研究的结果将改变 我们目前对Prestin在OHC中的功能机制的理解，并将将范式从 “以普雷斯汀为中心”的管弦乐蛋白可以在内耳内放大声音。