Biophysics and Development of Cochlear Hair Cells

描述（由申请人提供）：普雷斯廷是耳蜗外毛细胞的运动蛋白，是耳蜗放大的基础。普雷斯廷属于一个独特的阴离子转运蛋白家族，称为溶质载体蛋白26 A，或SLC 26 A。这11个成员家庭的每个成员都有两个基本不同的功能。虽然所有的普雷斯廷旁系同源物（即，该家族中的所有其他成员）将不同的阴离子底物转运通过多种上皮细胞，普雷斯廷（SLC 26 A5）是充当分子马达的唯一成员。这项研究的目标是研究普雷斯廷如何起作用的分子机制。我们已经鉴定了两个区域（残基158-168和残基256-276），它们在哺乳动物物种中是很保守的，但在普雷斯廷直系同源物和SLC 26 A旁系同源物中是高度可变的。我们的总体假设是，这些氨基酸序列代表了运动功能的基本基序。该项目有三个具体目标：目标1将确定位于普雷斯廷分子外环2的残基158-168是否代表运动功能的基本基序。目的2将确定11个氨基酸序列的长度和组成是否对电压感知/运动功能至关重要。目的3将确定位于普雷斯廷分子的跨膜结构域6中的残基256-276对于增强运动功能是否重要。将使用定点诱变在沙鼠普雷斯廷和人pendrin之间交换这些区域。Pendrin（SLC 26 A4）是一种非钠依赖性氯/碘转运蛋白。该基因的突变与Pendred综合征有关，Pendred综合征是综合征性耳聋的第二种最常见形式。使用电压钳技术和基于光电二极管的位移测量系统，从嵌合pendrin转染的HEK细胞测量非线性电容和体细胞运动性，这是普雷斯廷运动功能的两个标志。在嵌合pendrin中预期获得运动功能。放射性同位素摄取技术将用于确定嵌合pendrin是否保留其转运功能。这些实验对于理解普雷斯廷和pendrin的作用机制具有重要意义。

DESCRIPTION (provided by applicant): Prestin, a motor protein of cochlear outer hair cells, is the basis for cochlear amplification. Prestin belongs to a distinct anion transporter family called solute carrier protein 26A, or SLC26A. Individual members of this 11-member family serve two fundamentally distinct functions. While all prestin paralogs (i.e., all other members in this family) transport different anion substrates across a variety of epithelia, prestin (SLC26A5) is the only member serving as a molecular motor. The goal of the proposed research is to investigate the molecular mechanisms of how prestin works. We have identified two regions (residues 158-168 and residues 256-276) that are well conserved among mammalian species but highly variable among prestin orthologs and SLC26A paralogs. Our overall hypothesis is that these amino acid sequences represent the essential motif for motor function. This project has three specific aims: Aim 1 will determine whether residues 158-168, located in the external loop 2 of the prestin molecule, represent the essential motif for motor function. Aim 2 will determine whether the length and constitution of the 11-amino acid sequence are critical for voltage-sensing/motor function. Aim 3 will determine whether residues 256-276, located in the transmembrane domain 6 of the prestin molecule, are important for enhancing motor function. Site-directed mutagenesis will be used to swap these regions between gerbil prestin and human pendrin. Pendrin (SLC26A4), one of the paralogs, is a sodium-independent chloride/iodide transporter. Mutations in this gene are associated with Pendred syndrome, the second most common form of syndromic deafness. Nonlinear capacitance and somatic motility, two hallmarks of the motor function of prestin, will be measured from chimeric pendrin-transfected HEK cells, using the voltage-clamp technique and a photodiode-based displacement measurement system. A gain of motor function is expected in the chimeric pendrin. A radioisotope uptake technique will be used to determine whether the chimeric pendrin retains its transport function. The proposed experiments are significant for the fundamental understanding of how prestin and pendrin work.