The functional significance of heteromeric cx26 and cx30 gap junction channels in the inner ear.

缝隙连接是相邻细胞之间直接通讯的场所。通过一个细胞膜的通道与通过其相邻细胞膜的通道精确对齐，允许离子，一些营养物质和小信使分子从一个细胞传递到另一个细胞。这些通道由蛋白质的"连接蛋白"家族的成员形成。人类有21种连接蛋白。不同的连接蛋白形成特定的通道，可以选择允许什么通过。缝隙连接存在于几乎所有的身体组织中，但每个组织仅产生少数连接蛋白，推测那些具有适合该组织功能的通道特性的连接蛋白。编码特定连接蛋白的基因突变可能会导致蛋白质异常。这影响了差距连接允许细胞间转移的能力。两个特殊家族成员连接蛋白（cx）26和cx30的基因突变会导致耳聋。其中一些突变只会导致耳聋，尽管这两种连接蛋白都是在其他组织中产生的。经过“基因工程”去除耳蜗中cx26或cx30的老鼠也会失聪，但没有其他症状。因此，cx26和cx30对听力都很重要。我们先前的工作表明，在内耳中，cx26和cx30可以联合收割机一起形成一种独特的缝隙连接通道（"异聚体" cx26/cx30通道）。Cx26和Cx30并不同时存在于任何其他组织的同一细胞中。鸟类的耳朵既不含cx26也不含cx30。相反，它拥有另一种连接蛋白，叫做chicken-（c-）cx31，只在内耳中发现。因此，cx26/cx30和c-cx31通道可能具有对听力至关重要的特定特性。本计画将探讨耳蜗内连接蛋白所形成的差距连接的特性。我们将首先使用通常不形成缝隙连接的细胞培养物，并迫使它们产生我们感兴趣的连接蛋白。不同的荧光染料，其分子大小和电荷不同，将被注入单个细胞，以发现是否，以及如何有效地，每一个可以转移到相邻的细胞。这将告诉我们通道通常允许通过的分子的特性。还将测试某些天然存在的信号传导离子和分子的转移。我们预测，缝隙连接与cx26/cx30通道将有类似的特性，含有c-cx31，但不同于那些只含有cx26或cx30。我们还将使用这种细胞培养系统来测试cx26的致突变突变是否会影响异聚体cx26/cx30通道。这将进一步测试cx26/cx30通道是否可能在内耳中很重要。然后，我们将使用小鼠耳蜗的薄片来检查缝隙连接在其真实的环境中的特性。这些切片提供了在细胞排列不受干扰的情况下进入活态耳蜗中细胞的途径。在细胞培养物中测试的染料转移和信号分子的通过将显示具有培养物中定义的特征的间隙连接是否存在于耳蜗中以及在耳蜗中的何处存在。将使用可以通过几乎所有类型的连接蛋白通道的染料来追踪耳蜗中细胞间通信的途径。这些正常的特性将与那些设计成显示遗传性连接蛋白相关耳聋的小鼠中的间隙连接的特性进行对比。这将发现突变如何以及在何处影响细胞间的通讯。这些结果将有助于解释特定的连接蛋白如何支持特定的细胞功能，以及间隙连接细胞间通讯如何支持听力。

Gap junctions are sites of direct communication between adjacent cells. Channels through the membrane of one cell are aligned precisely with channels through the membrane of its neighbour allowing the passage of ions, some nutrients and small messenger molecules from one cell to another. These channels are formed by members of the 'connexin' family of proteins. There are 21connexin types in humans. The different connexins form specific channels that can select what is allowed through. Gap junctions are present in almost all body tissues, but each tissue makes only a few connexins, presumably those with channel properties suited for the functioning of that tissue. Mutations in genes that code for a particular connexin can result in an abnormal protein. That affects the ability of the gap junction to allow intercellular transfer. Mutations in the genes for two particular family members, connexin(cx)26 and cx30, cause deafness. Some of these mutations only cause deafness, even though both connexins are produced in other tissues. Mice which have been 'genetically engineered' to remove either cx26 or cx30 from the cochlea are also deaf, but show no other symptoms. Thus, both cx26 and cx30 must be important for hearing. Our previous work has suggested that in the inner ear cx26 and cx30 can combine together to make a unique kind of gap junction channel ('heteromeric' cx26/cx30 channels). Cx26 and cx30 are not present together in the same cell in any other tissue. The ear of birds contains neither cx26 nor cx30. Instead it possesses another connexin called chicken-(c-)cx31 that is found only in the inner ear. Cx26/cx30 and c-cx31 channels may therefore have particular properties that are essential to hearing. This project will determine some of the characteristics of the gap junctions formed by the connexins present in the cochlea. We will first use cultures of cells that do not normally form gap junctions and force them to produce the connexins in which we are interested. Different fluorescent dyes, whose molecules differ in size and charge, will be injected into a single cell to discover whether, and how efficiently, each one can transfer to adjacent cells. This will tell us about the properties of molecules that the channels normally allow to pass. The transfer of certain naturally occurring signalling ions and molecules will also be tested. We predict that gap junctions with cx26/cx30 channels will have similar characteristics to those that contain c-cx31 but different from those which contain only cx26 or only cx30. We will also use this cell culture system to test whether deafness-causing mutations of cx26 affect heteromeric cx26/cx30 channels. This will further test whether cx26/cx30 channels are likely to be important in the inner ear. We will then examine the properties of gap junctions in their real environment using thin slices of the cochlea of mice. These slices provide access to the cells in the cochlea in a living state with the arrangement of cells undisturbed. Dye transfer and passage of signalling molecules as tested in the cell cultures, will show whether and where gap junctions with the characteristics defined in the cultures exist in the cochlea. The pathways of intercellular communication in the cochlea will be traced using a dye that can pass through almost all types of connexin channel. These normal properties will be contrasted with those of gap junctions in a mouse engineered to display an inherited connexin-related deafness. This will find out how and where the mutation affects intercellular communication. The results will help explain how specific connexins support particular cellular functions, and how gap junctional intercellular communication supports hearing.