Biophysics and Development of Cochlear Hair Cells

耳蜗毛细胞的生物物理学和发育

• 批准号: 8585048
• 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
• 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/8585048
• 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是耳蜗外毛细胞的运动蛋白，是耳蜗放大的基础。 Prestin 属于一种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 名成员组成的大家庭中的各个成员承担着两种截然不同的职能。虽然所有 prestin 旁系同源物（即该家族中的所有其他成员）在各种上皮细胞上转运不同的阴离子底物，但 prestin (SLC26A5) 是唯一充当分子马达的成员。拟议研究的目标是研究 prestin 工作的分子机制。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高度保守，但在prestin直系同源物和SLC26A旁系同源物中高度可变。我们的总体假设是这些氨基酸序列代表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 中的残基 158-168 是否代表运动功能的基本基序。目标 2 将确定 11 个氨基酸序列的长度和构成对于电压传感/运动功能是否至关重要。目标 3 将确定位于 prestin 分子跨膜结构域 6 中的残基 256-276 对于增强运动功能是否重要。定点诱变将用于在沙鼠 prestin 和人 pendrin 之间交换这些区域。 Pendrin (SLC26A4) 是旁系同源物之一，是一种不依赖于钠的氯/碘转运蛋白。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种最常见的综合征性耳聋。非线性电容和躯体运动是 prestin 运动功能的两个标志，将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 细胞中测量。嵌合 pendrin 有望增强运动功能。将使用放射性同位素摄取技术来确定嵌合pendrin是否保留其转运功能。所提出的实验对于从根本上理解 prestin 和 pendrin 的工作原理具有重要意义。