Biophysics and Development of Cochlear Hair Cells

耳蜗毛细胞的生物物理学和发育

• 批准号: 8769947
• 负责人: Zhi-Zhou He
• 金额: $ 36.57万
• 依托单位: CREIGHTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-12-12 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8769947
• 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; 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

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是耳蜗外毛细胞的一种运动蛋白，是耳蜗放大的基础。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。这个由11名成员组成的家庭的个别成员承担着两项从根本上不同的职能。虽然所有的prestin paralog(即，这个家族中的所有其他成员)都可以通过各种上皮细胞运输不同的阴离子底物，但prestin(SLC26A5)是唯一充当分子马达的成员。这项拟议的研究的目标是研究prestin如何发挥作用的分子机制。我们已经确定了两个区域(残基158-168和残基256-276)，这两个区域在哺乳动物物种中非常保守，但在prestin同源基因和SLC26a同源基因中差异很大。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158-168残基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敏感/运动功能至关重要。目的3将确定位于prestin分子跨膜区6的残基256-276是否对增强运动功能重要。定点突变将被用来在沙土鼠prestin和人类垂垂蛋白之间交换这些区域。Pendrin(SLC26A4)是一种钠离子非依赖性的氯/碘转运蛋白。该基因的突变与Pendred综合征有关，Pendred综合征是第二种最常见的综合征性耳聋。利用电压钳技术和基于光电二极管的位移测量系统，将从嵌合的摆动蛋白基因转染的HEK细胞中测量Prestin运动功能的两个标志--非线性电容和躯体运动。嵌合式吊环有望获得运动功能的增强。将使用放射性同位素摄取技术来确定嵌合吊环是否保留其运输功能。所提出的实验对于从根本上理解Prestin和Pendrin是如何工作的具有重要意义。