Functional Analysis of Inner Ear Gap Junctions

描述(申请人提供)：本项目的长期目标是研究内耳缝隙连接耦合的功能。缝隙连接通道是一种细胞间的胞质通道，由哺乳动物的连接蛋白基因家族编码。六种连接蛋白异构体组装成一个十六进制的连接蛋白(半通道)，并与另一个连接蛋白对接在相对的细胞膜上，形成一个细胞间缝隙连接通道，分子可以通过该通道。Cx26和Cx30是耳蜗肌支持细胞的主要亚型。每一种连接蛋白突变都会导致听力损失。研究发现，支持细胞之间的缝隙连接具有各种不对称的电压门，这表明耳蜗中出现了异型和异质混合通道配置。该项目的工作假设是，Cx26和Cx30可能对接形成具有特定通透性的异型和异构体通道，以选择性地转移耳蜗内的离子和分子；不对称的异型通道也可能在这个多细胞系统中诱导定向的细胞间转移。特异性目的(SA)1是为了确定连接蛋白在耳蜗中的特定功能。我们将使用免疫荧光染色来确定连接蛋白的表达和分布，使用膜片钳记录来测量通道电导，使用门控来确定通道的类型和配置，并使用荧光探针来评估连接蛋白通道的通透性。缝隙连接渗透性的电荷和尺寸选择性将通过使用多个带膜片钳记录的荧光探头来评估。还将使用共聚焦显微镜的光漂白后荧光恢复(FRAP)技术来定量定义缝隙连接的渗透性。通过FRAP测量和时间推移荧光显微镜(SA2)可以确定耳蜗区支持细胞之间的跨连接扩散的不对称性和耳蜗感觉上皮中的定向通道。SA3是为了进一步阐明耳蜗缝隙连接偶联的结构与功能关系，确定天然耳蜗区支持细胞中连接蛋白半通道的活性和通透性。SA 4将测试K+循环假说，这一假说早已被提出，但尚未得到检验。我们将使用膜片钳技术直接记录耳蜗肌支持细胞之间的K+通道。SA5是为了探讨支持细胞的连接偶联对外毛细胞电活动的影响。毫无疑问，这些研究的完成将有助于了解内耳缝隙连接耦合的机制，进而开发针对这种常见遗传性耳聋的治疗和保护措施。

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.