Connexin mutations in deafness

描述（由申请人提供）：在美国，人类连接蛋白26基因（Cx26，或GJB2）突变是导致非综合征性耳聋的主要原因。其他连接蛋白Cx30 （GJB6）、Cx31 （GJB3）和Cx32 （GJB1）的突变也与人类听力损失有关。虽然这阐明了耳蜗间隙连接的关键功能，但尚不清楚如何从内耳中具有重叠表达模式的不同连接蛋白基因的突变中产生共同的病理，如Cx26， Cx30， Cx31和Cx32。人类感觉毛细胞之间没有间隙连接；但是它们在耳蜗的支持细胞中表达。目前的假设是，这些连接在淋巴末端和淋巴周围之间的钾离子再循环中起作用。很难将这个模型与通过间隙连接通道的钾渗透的现有数据相一致，因为所有的连接蛋白都很容易被这种谨慎渗透，并且单个耳蜗连接蛋白的丢失仍然会留下其他功能连接蛋白来执行这项任务。连接蛋白确实对其他小分子和第二信使表现出不同的渗透性，我们假设这些渗透性差异对耳蜗功能至关重要，并且在几个可用通道亚基之一的功能丧失后更难以补偿。本应用程序的目的是精确定义Cx26的哪些渗透特性是人类正常听觉功能所必需的。为了实现这一目标，我们首先提出在配对非洲爪蟾卵母细胞试验中筛选突变型Cx26等位基因的功能活性。保留通道功能的Cx26突变体将通过双膜片钳方法在横切的哺乳动物细胞系中分析其热选择性特性。最后，我们将产生并表征基因工程小鼠，其中原生Cx26基因已被功能活跃的人类Cx26致病突变所取代。我们将使用基因敲入技术来产生小鼠，这将使我们能够评估动物模型中体外衍生的功能差异。对比野生型和致病型Cx26变体之间的渗透差异，不仅可以提供有关听力损失的机制见解，还可以为连接蛋白基因突变导致人类疾病的其他组织中连接蛋白多样性需求提供一般模型。

DESCRIPTION (provided by applicant): Mutations in the human connexin26 gene (Cx26, or GJB2) are the leading cause of nonsyndromic deafness in the United States. Mutations in other connexins, Cx30 (GJB6), Cx31 (GJB3), and Cx32 (GJB1) have also been linked to hearing loss in humans. While this illuminates a critical function for cochlear gap junctions, it is unclear how a common pathology can arise from mutations within different connexin genes that have an overlapping expression pattern in the inner ear, as is the case for Cx26, Cx30, Cx31 and Cx32. There are no gap junctions between the sensory hair cells in humans; but they are expressed in the supporting cells of the cochlea. The current hypothesis is that these junctions play a role in the re-circulation of potassium ions between the end lymph and perilymph. It is difficult to reconcile this model with the available data on potassium permeation through gap junction channels, as all connexins are readily permeated by this caution and the loss of a single cochlear connexin would still leave other functional connexins available to perform this task. Connexins do show differential permeability to a wide range of other small molecules and second messengers, and we hypothesize that these permeation differences are critical for cochlear function, and more difficult to compensate for following the functional loss of one of the several available channel subunits. The objective of this application is to precisely define which permeation properties of Cx26 are necessary for normal auditory function in humans. To achieve this goal, we first propose to screen mutant Cx26 alleles for functional activity in the paired Xenopus oocyte assay. Cx26 mutants that retain channel function will have their perm selectivity properties analyzed by dual patch clamp methods in transected mammalian cell lines. Finally, We will generate and characterize genetically engineered mice where the native Cx26 gene has been replaced by the functionally active human Cx26 disease causing mutations. We will use genetic knock-in techniques to generate mice that will allow us to evaluate the in vitro derived functional differences in an animal model. Contrasting the differences in permeation between wild type and disease causing variants of Cx26 will not only provide mechanistic insight into hearing loss, but will also provide a general model for the need for connexin diversity in other tissues where human disease results from mutations in connexin genes.