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（由申请人提供）：Prestin是人工耳蜗外毛细胞的运动蛋白，是人工耳蜗扩增的基础。 Prestin属于一个不同的阴离子转运蛋白家族，称为溶质载体蛋白26a或SLC26A。这个11人家庭的个体成员提供了两个根本不同的功能。虽然所有普雷斯汀旁系同源物（即，该家族中的所有其他成员）在各种上皮上运输不同的阴离子底物，而普雷斯汀（SLC26A5）是唯一用作分子运动的成员。拟议的研究的目的是研究普雷斯汀如何工作的分子机制。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守，但在Prestin直系同源物和SLC26A旁系同源物中有很大变化。我们的总体假设是这些氨基酸序列代表了运动功能的必要基序。该项目具有三个特定的目标：AIM 1将确定位于普雷斯汀分子外部环2中的残基158-168是否代表运动功能的基本基序。 AIM 2将确定11-氨基酸序列的长度和构成对于电压传感/运动功能至关重要。 AIM 3将确定位于Prestin分子的跨膜结构域6中的残基256-276是否对于增强运动功能很重要。定点诱变将用于在沙鼠普雷斯汀和人钟蛋白之间交换这些区域。 Pendrin（SLC26A4）是旁系同源物之一，是独立于钠的氯化物/碘化物转运蛋白。该基因中的突变与Pendred综合征有关，这是综合综合性耳聋的第二大最常见形式。非线性电容和躯体运动，Prestin的运动功能的两个标志，将使用嵌合内形蛋白转染的HEK细胞测量，使用电压钳技术和基于光电二极管的位移测量系统。预计在嵌合束中会增长运动功能。放射性同位素摄取技术将用于确定嵌合束是否保留其运输功能。提出的实验对于对Prestin和Pendrin的工作方式的基本了解至关重要。