Regulation of hair cell functions

毛细胞功能的调节

基本信息

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
项目类别：
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

项目摘要

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 是一种运动蛋白，充当耳蜗放大器，有助于提高耳蜗的敏感性此外，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，增加了存在缺失预定义的可能性应该鉴定出相互作用的蛋白质，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，我们援引了这一点与 prestin 是 OHC 独有的教条相反，我们认为真实的。耳蜗放大器由 prestin 及其家族成员 SLC26A6 组成，我们进一步探讨了 SLC26A6。需要作为有效的转运蛋白来创建细胞内 Cl-微结构域，以保证 prestin 的正常功能。为了检验假设，我们提出以下三个目标：1）检验空间表达和协同 prestin 和 slc26a6 在 OHC 中的定位首先，我们将确定 prestin 和 slc26a6 的表达和共定位。使用定量实时 PCR (qRT-PCR)、免疫荧光共聚焦在 OHC 中检测 prestin 和 slc26a6 显微镜和免疫电子显微镜 (immuno-EM) 和 slc26a6-/- 小鼠将作为。 2) 测试 prestin 和 slc26a6 的物理相互作用或异源寡聚化。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 相互作用或异源寡聚化的体外和体内功能作用。将记录共表达的 prestin 和的阴离子交换活动和非线性电容我们将设计 prestin-slc26a6 嵌合体构建体以进一步测试两者的功能相互作用。最后，我们将测试在没有 slc26a6 的情况下 prestin 的运动功能将受到影响的预测。严重改变，损害了 slc26a6 缺失突变小鼠的听力敏感性。这些研究将揭示耳蜗放大器缺失的功能组件，并可能揭示一个治疗听力损失或耳聋的新治疗目标将改变这些研究的结果。我们目前对 prestin 在 OHC 中的功能机制的理解，并将改变范式 “以prestin为中心”的管弦乐蛋白质可以放大内耳的声音。