Regulation of hair cell functions

Prestin，一种运动蛋白作为耳蜗放大器，有助于提高 哺乳动物的内耳。此外，在异源系统中表达的prestin产生了功能特征 与真正的OHC性质不一致，增加了丢失预置的可能性- 相互作用的蛋白质，应该确定，以了解这种显着的马达蛋白的机制。 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 假设。与预设是唯一特定于OHC的教条相反，我们假设真实的 耳蜗放大器由Prestin及其家族成员SLC26A6组成。我们进一步假设SLC26A6 作为一种有效的转运蛋白，为Prestin的正常功能创造了细胞内的氯微域。 为了检验这一假设，我们提出了以下三个目标：1)检验空间表达和协同效应。 Prestin和slc26a6在毛细胞中的定位。首先，我们将确定的表达和共本地化 用实时定量聚合酶链式反应和免疫荧光共聚焦技术检测毛细胞中Prestin和sLc26a6的表达 显微镜和免疫电子显微镜(Ig-EM)。Prestin-/-和slc26a6-/-小鼠将作为 阴性对照。2)检测prestin和slc26a6的物理相互作用或异位齐聚作用。至 直接测试Prestin和SLC26A6之间可能的物理相互作用或联合组装，我们将使用联合 免疫共沉淀(co-IP)和荧光共振能量转移(FRET)技术。3)确定 Prestin与slc26a6相互作用或异位齐聚的体内外功能作用。我们 将记录共表达的Prestin的阴离子交换活性和非线性电容 Slc26a6.我们将设计prestin-slc26a6嵌合体结构，以进一步测试两者的功能相互作用。 蛋白质。最后，我们将检验预测，在没有slc26a6的情况下，prestin的运动功能将是 严重改变，损害了slc26a6基因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，并可能泄露一种 治疗听力损失或耳聋的新治疗靶点。这些研究的结果将会改变 我们目前对Prestin在毛细胞中的作用机制的理解，并将把范式从 以“Prestin为中心”的管弦乐蛋白，其作用是放大内耳的声音。

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.