Connexin mutations in deafness

描述（由申请人提供）：在美国，人类连接蛋白26基因（Cx 26或GJB 2）突变是非综合征性耳聋的主要原因。其他连接蛋白，Cx 30（GJB 6），Cx 31（GJB 3）和Cx 32（GJB 1）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，但尚不清楚如何从在内耳中具有重叠表达模式的不同连接蛋白基因内的突变引起常见病理学，如Cx 26，Cx 30，Cx 31和Cx 32的情况。在人类的感觉毛细胞之间没有缝隙连接;但它们在耳蜗的支持细胞中表达。目前的假设是，这些连接在终末淋巴和外淋巴之间的钾离子再循环中发挥作用。很难将该模型与关于钾通过间隙连接通道渗透的可用数据相协调，因为所有连接蛋白都容易被这种谨慎渗透，并且单个耳蜗连接蛋白的丢失仍将留下其他功能性连接蛋白可用于执行该任务。连接蛋白确实显示出对广泛的其他小分子和第二信使的不同渗透性，我们假设这些渗透差异对耳蜗功能至关重要，并且更难以补偿几个可用通道亚基之一的功能丧失。本申请的目的是精确定义Cx 26的哪些渗透性质是人类正常听觉功能所必需的。为了实现这一目标，我们首先提出了筛选突变Cx 26等位基因的功能活性配对非洲爪蟾卵母细胞测定。保留通道功能的Cx 26突变体将在横切的哺乳动物细胞系中通过双膜片钳方法分析其烫发选择性特性。最后，我们将产生和表征基因工程小鼠，其中天然Cx 26基因已被功能活性的人Cx 26致病突变所取代。我们将使用基因敲入技术来产生小鼠，这将使我们能够在动物模型中评估体外衍生的功能差异。对比Cx 26的野生型和致病变体之间的渗透差异不仅将提供对听力损失的机制性洞察，而且还将提供在其他组织中需要连接蛋白多样性的一般模型，其中人类疾病由连接蛋白基因突变引起。

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.