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Biophysics and Development of Cochlear Hair Cells

耳蜗毛细胞的生物物理学和发育

基本信息

批准号：
8969599
负责人：
Zhi-Zhou He
金额：
$ 36.94万
依托单位：
CREIGHTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-12-12 至 2016-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8969599
关键词：
Amino Acid Motifs Amino Acid Sequence Amino Acids Anions Biological Assay Biophysics Carrier Proteins Cells Charge Chickens Chimeric Proteins Chloride Ion Chlorides Constitution Data Development Diagnosis Disease Electric Capacitance Electrophysiology (science)Embryo Epithelium Family Family member Formates Future Genes Gerbils Goals Goiter Hair Cells Human Individual Investigation Iodides Kidney Label Length Measurement Measures Mechanics Modeling Molecular Molecular Motors Motor Motor Activity Movement Mutate Mutation Orthologous Gene Outer Hair Cells Pendred Syndrome Prevention Property Proteins Radioactive Radioisotopes Research Sensorineural Hearing Loss Side Site-Directed Mutagenesis Sodium System Techniques Testing Time Translating Transmembrane Domain Vertebrates Voltage-Clamp Technics Work Zebrafish base cell motility deafness flexibility gain of function gene therapy hearing impairment kidney cell member mutant novel paralogous gene rat Pres protein research study solute uptake voltage

项目摘要

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.

描述（申请人提供）：Prestin是耳蜗外毛细胞的运动蛋白，是耳蜗放大的基础。Prestin属于一个独特的阴离子转运蛋白家族，称为溶质载体蛋白26A，或SLC26A。这个由11个成员组成的家族的每个成员都有两个根本不同的功能。虽然所有prestin类似物（即该家族的所有其他成员）在各种上皮中运输不同的阴离子底物，但prestin （SLC26A5）是唯一作为分子马达的成员。提出的研究目标是研究prestin如何起作用的分子机制。我们已经确定了两个区域（残基158-168和残基256-276），它们在哺乳动物物种中保存良好，但在现有的同源物和SLC26A相似物中变化很大。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prestin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构成是否对电压传感/运动功能至关重要。Aim 3将确定位于prestin分子跨膜结构域6的残基256-276是否对增强运动功能很重要。位点导向突变将用于在沙鼠prestin和人类pendrin之间交换这些区域。Pendrin （SLC26A4）是一种不依赖钠的氯/碘离子转运体。该基因的突变与彭德雷德综合征有关，彭德雷德综合征是第二常见的综合征性耳聋。非线性电容和体细胞运动，prestin的运动功能的两个标志，将测量嵌合pendin转染HEK细胞，使用电压钳技术和光电二极管为基础的位移测量系统。在嵌合pendin中，运动功能有望获得。放射性同位素吸收技术将用于确定嵌合pendrin是否保留其运输功能。提出的实验对于基本理解prestin和pendrin如何工作具有重要意义。