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

内耳异聚 cx26 和 cx30 间隙连接通道的功能意义。

• 批准号: BB/D009669/1
• 负责人: Andrew Forge
• 金额: $ 39.08万
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2006
• 资助国家: 英国
• 起止时间: 2006 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FD009669%2F1
• 关键词: functional; significance; heteromeric; cx26; cx30

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.

间隙连接是相邻细胞之间直接通信的部位。一个细胞膜上的通道与邻近细胞膜上的通道精确地对齐，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“连接蛋白”家族的成员组成。人类有21种连接蛋白类型。不同的连接形成特定的通道，可以选择允许通过的内容。缝隙连接几乎存在于所有的身体组织中，但每个组织只产生少数连接蛋白，可能是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会导致蛋白质异常。这会影响间隙连接允许细胞间转移的能力。两个特殊的家族成员——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。通过“基因工程”从耳蜗中去除cx26或cx30的老鼠也会失聪，但没有表现出其他症状。因此，cx26和cx30对听力都很重要。我们之前的工作表明，在内耳中，cx26和cx30可以结合在一起，形成一种独特的间隙连接通道（“异质”cx26/cx30通道）。Cx26和cx30不会同时存在于任何其他组织的同一细胞中。鸟的耳朵既不含cx26也不含cx30。相反，它拥有另一种连接蛋白，叫做chicken-(c-)cx31，这种连接蛋白只存在于内耳中。因此，Cx26/cx30和c-cx31通道可能具有对听力至关重要的特殊特性。该项目将确定由耳蜗中存在的连接蛋白形成的间隙连接的一些特征。我们将首先使用通常不会形成间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，将被注射到单个细胞中，以发现每一种染料是否以及如何有效地转移到相邻的细胞中。这将告诉我们通道通常允许通过的分子的性质。某些自然发生的信号离子和分子的转移也将被测试。我们预测具有cx26/cx30通道的间隙连接将具有与含有c-cx31的间隙连接相似的特性，但与仅含有cx26或仅含有cx30的间隙连接不同。我们还将使用这种细胞培养系统来测试导致耳聋的cx26突变是否会影响cx26/cx30异质通道。这将进一步测试cx26/cx30通道在内耳中是否可能很重要。然后，我们将使用小鼠耳蜗的薄片来检查间隙连接在真实环境中的特性。这些切片提供了对处于活状态的耳蜗细胞的访问，细胞的排列不受干扰。在细胞培养中测试的染料转移和信号分子的传递将显示耳蜗中是否存在具有培养中定义的特征的间隙连接以及在何处存在。耳蜗细胞间通讯的途径将使用一种可以通过几乎所有类型连接蛋白通道的染料进行追踪。这些正常的特性将与间隙连接的特性进行对比，在一只被改造成遗传性连接蛋白相关耳聋的老鼠身上进行对比。这将发现突变是如何以及在哪里影响细胞间通讯的。结果将有助于解释特定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信如何支持听力。