Functional Analysis of Inner Ear Gap Junctions

项目描述（由申请人提供）：本项目的长期目标是研究内耳间隙连接耦合的功能。缝隙连接通道是哺乳动物细胞间的细胞质通道，由连接蛋白基因家族编码。六个连接蛋白异构体组合形成一个六面连接子（半通道），并与另一个连接子在相反的细胞膜上对接，形成一个细胞间隙连接通道，最高可达1kda的分子可通过该通道。Cx26和30是耳蜗支持细胞的主要亚型。每一种连接蛋白突变都可能导致听力损失。研究发现，支持细胞之间的间隙连接具有各种不对称的电压门，表明耳蜗中存在异型和异型杂交通道构型。本项目的工作假设是：Cx26和30可能对接形成具有特定通透性的异型和异质通道，在耳蜗内选择性传递离子和分子；在这个多细胞系统中，不对称的异型通道也可能诱导定向细胞间转移。特异性目的（SA） 1是确定耳蜗中连接蛋白的特异性功能。我们将使用免疫荧光染色来确定连接蛋白的表达和分布，膜片钳记录来测量通道电导和门控来确定通道的类型和结构，荧光探针来评估连接蛋白通道的渗透率。电荷和尺寸选择性的缝隙连接渗透率将评估使用多荧光探针与膜片钳记录。用共聚焦显微镜进行光漂白后荧光恢复（FRAP）也将用于定量定义间隙连接渗透率。通过FRAP测量和延时荧光显微镜（SA2）来确定耳蜗支持细胞间跨结扩散和耳蜗感觉上皮内定向通道的不对称性。SA3是定义天然耳蜗支持细胞连接蛋白半通道的活性和通透性，进一步阐明耳蜗间隙连接偶联的结构-功能关系。SA 4是为了验证钾离子回收假说，这个假说早就提出了，但尚未得到验证。我们将使用膜片钳技术直接记录K+在耳蜗支持细胞间的传代。SA5是探讨支持细胞的连接耦合对外毛细胞电运动的影响。毫无疑问，这些研究的完成将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性干预措施。

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.