Connexin mutations in deafness

耳聋中的连接蛋白突变

• 批准号: 6857085
• 负责人: THOMAS W WHITE
• 金额: $ 27.39万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6857085
• 关键词: Xenopus oocyte; cell line; cochlea; deafness; gap junctions; gene deletion mutation; gene mutation; genetically modified animals; laboratory mouse; membrane channels; membrane permeability; transfection; voltage /patch clamp

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 Finally, We will generate and characterize genetically engineered mice where the native Cx26 gene has been replaced by the functionally active human Cx26 disease causing mutations.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.

描述（由申请人提供）：人类连接蛋白 26 基因（Cx26 或 GJB2）的突变是美国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，但尚不清楚内耳中具有重叠表达模式的不同连接蛋白基因的突变如何产生常见的病理学，如 Cx26、Cx30、Cx31 和 Cx32 的情况。人类的感觉毛细胞之间没有间隙连接；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末端淋巴和外淋巴之间的钾离子再循环中发挥作用。很难使该模型与间隙连接通道钾渗透的现有数据相一致，因为所有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损失仍然会留下其他功能性连接蛋白可用于执行此任务。连接蛋白确实对多种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于耳蜗功能至关重要，并且在几个可用通道亚基之一功能丧失后更难以补偿。本申请的目的是精确定义 Cx26 的哪些渗透特性对于人类正常听觉功能是必需的。为了实现这一目标，我们首先建议在配对非洲爪蟾卵母细胞测定中筛选突变型 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烫发选择性特性。最后，我们将生成并表征基因工程小鼠，其中天然 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。对比野生型和引起疾病的 Cx26 变体之间的渗透差异不仅可以提供对听力损失的机制见解，而且还可以为其他组织中连接蛋白多样性的需求提供通用模型，在这些组织中，人类疾病是由连接蛋白基因突变引起的。