Functional Analysis of Inner Ear Gap Junctions

内耳间隙连接处的功能分析

基本信息

批准号：
7418207
负责人：
Hong-Bo Zhao
金额：
$ 24.12万
依托单位：
UNIVERSITY OF KENTUCKY
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-07-15 至 2011-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7418207
关键词：
Back Cell membrane Cells Charge Cochlea Complex Condition Confocal Microscopy Connexins Connexon Coupling Cytoskeleton Diffusion Docking Dyes Endolymph Epithelium Excision Extracellular Space Fluorescence Fluorescence Recovery After Photobleaching Fluorescent Dyes Fluorescent Probes Functional disorder Gap Junctions Gene Family Goals Hair Cells Hearing Hybrids Inherited Intervention Ions Kinetics Labyrinth Location Mammals Measurement Measures Mechanics Membrane Membrane Potentials Metabolic Microscopy Molecular Movement Mutation Nutrient Outer Hair Cells Outer Supporting Cell Patch-Clamp Techniques Performance Perfusion Permeability Physiological Play Potassium Potassium Channel Property Protein Isoforms Recovery Recycling Research Personnel Role Sensory Staining method Stains Structure-Activity Relationship Supporting Cell System Testing Therapeutic Tight Junctions Time Work cell motility cell type deafness extracellular gap junction channel hearing impairment in vivo patch clamp prevent programs research study size small molecule time use uptake voltage

项目摘要

DESCRIPTION (provided by applicant): The long-term goal of this project is to investigate functions of gap junctional coupling in the inner ear. A gap junctional channel is an intercellular cytoplasmic conduit, and is encoded by the connexin gene family in mammals. Six connexin isoforms assemble to form a hexadal connexon (hemichannel), and dock with another connexon in the opposite cell membrane to form an intercellular gap junctional channel through which molecules up to 1 kDa can pass. Cx26 and 30 are the predominant isoforms in cochlear supporting cells. Each connexin mutation can induce hearing loss. It has been found that gap junctions between supporting cells have various asymmetrical voltage gates, indicative of the occurrence of heterotypic and heteromeric hybrid channel configurations in the cochlea. The working hypothesis of this project is that Cx26 and 30 may dock to form heterotypic and heteromeric channels with specific permeability to selectively transfer ions and molecules in the cochlea; asymmetric heterotypic channels may also induce directional intercellular transfer in this multicellular system. Specific aim (SA) 1 is to define connexin-specific functions in the cochlea. We will use immunofluorescent staining to identify connexin expression and distributions, patch clamp recording to measure channel conductance and gating to identify channel types and configurations, and fluorescent probes to assess connexin channel permeability. Charge and size selectivity in gap junctional permeability will be assessed by use of multiple florescent probes with patch clamp recording. Fluorescence recovery after photo bleaching (FRAP) with confocal microscopy will also be employed to quantitatively define gap junctional permeability. The asymmetry of transjunctional diffusion between cochlear supporting cells and directional passage in the cochlear sensory epithelium will be defined by FRAP measurement and time-lapse fluorescent microscopy (SA2). SA3 is to define the activity and permeability of connexin hemi channels in native cochlear supporting cells to further elucidate structure-function relationship of gap junctional coupling in the cochlea. SA 4 is to test the K+-recycling hypothesis that has long been proposed but has yet to be tested. We will use patch clamp technique to directly record K+ passage between cochlear supporting cells. SA5 is to explore the effect of supporting cell's junctional coupling on outer hair cell electro motility. Undoubtedly, completion of these studies will contribute signaificantly towards understanding the mechanisms of gap junctional coupling in the inner ear, and in turn, develop therapeutic and protective interventions for this common hereditary deafness.

描述（由申请人提供）：该项目的长期目标是研究内耳间隙连接耦合的功能。间隙连接通道是细胞间的细胞质导管，由哺乳动物中的连接蛋白基因家族编码。六个连接蛋白亚型组装形成六边形连接子（半通道），并与对面细胞膜中的另一个连接子对接，形成细胞间间隙连接通道，高达 1 kDa 的分子可以通过该通道。 Cx26 和 30 是耳蜗支持细胞中的主要亚型。每个连接蛋白突变都会导致听力损失。已经发现支持细胞之间的间隙连接具有各种不对称电压门，表明耳蜗中存在异型和异聚混合通道配置。该项目的工作假设是Cx26和30可能对接形成具有特定渗透性的异型和异聚通道，以选择性地在耳蜗中转移离子和分子；不对称异型通道也可能诱导该多细胞系统中的定向细胞间转移。具体目标 (SA) 1 是定义耳蜗中连接蛋白的特定功能。我们将使用免疫荧光染色来识别连接蛋白的表达和分布，使用膜片钳记录来测量通道电导和门控来识别通道类型和配置，并使用荧光探针来评估连接蛋白通道的通透性。间隙连接通透性的电荷和尺寸选择性将通过使用具有膜片钳记录的多个荧光探针来评估。共焦显微镜光漂白后荧光恢复 (FRAP) 也将用于定量定义间隙连接渗透性。耳蜗支持细胞之间的跨接点扩散和耳蜗感觉上皮中的定向通道的不对称性将通过 FRAP 测量和延时荧光显微镜 (SA2) 来定义。 SA3旨在定义天然耳蜗支持细胞中连接蛋白半通道的活性和通透性，以进一步阐明耳蜗间隙连接耦合的结构与功能关系。 SA 4是为了检验早已提出但尚未得到检验的K+-回收假设。我们将使用膜片钳技术直接记录耳蜗支持细胞之间的 K+ 通道。 SA5是为了探讨支持细胞的连接耦合对外毛细胞电动性的影响。毫无疑问，这些研究的完成将极大地有助于理解内耳间隙连接耦合的机制，进而为这种常见的遗传性耳聋制定治疗和保护干预措施。

项目成果

期刊论文数量（22）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

The role of an inwardly rectifying K(+) channel (Kir4.1) in the inner ear and hearing loss.

DOI：
10.1016/j.neuroscience.2014.01.036
发表时间：
2014-04-18
期刊：
Neuroscience
影响因子：
3.3
作者：
Chen J;Zhao HB
通讯作者：
Zhao HB

The effects and outcomes of electrolyte disturbances and asphyxia on newborns hearing.

电解质紊乱和窒息对新生儿听力的影响和结果。

DOI：
10.1016/j.ijporl.2013.03.031
发表时间：
2013
期刊：
International journal of pediatric otorhinolaryngology
影响因子：
1.5
作者：
Liang,Chun;Hong,Qi;Jiang,Tao-Tao;Gao,Yan;Yao,Xiao-Fang;Luo,Xiao-Xing;Zhuo,Xiu-Hui;Shinn,JenniferB;Jones,RaleighO;Zhao,Hong-Bo;Lu,Guang-Jin
通讯作者：
Lu,Guang-Jin

Expression and function of pannexins in the inner ear and hearing.