PROPERTIES OF CONNEXIN CHANNELS THAT CAUSE DEAFNESS

描述（由申请人提供）：连接蛋白26 （Cx26）基因（GJB2）突变是人类遗传性综合征感音神经性耳聋的主要原因。虽然大多数突变连接蛋白导致非功能性通道，但有些能够形成功能性间隙连接通道并介导电偶联（例如，可渗透到K+离子），但它们会导致耳聋。这表明病理是由细胞质物质的细胞间运动缺陷引起的，而不是钾离子。现在人们认识到，由大约20种连接蛋白中的每一种形成的通道在分子渗透性方面彼此不同，最近的研究表明，它们在第二信使和其他细胞质分子中具有显著的和高度特异性的选择性。我们的假设是，导致耳聋的功能性Cx26突变体对细胞质分子的渗透性发生了改变，而这种渗透性缺陷不能通过内耳中表达的其他连接蛋白来补偿。我们的实验计划是确定含有WT和引起耳聋的突变体Cx26的通道在第二信使和其他细胞质分子方面的选择性差异。通过这样做，我们希望能够查明或缩小导致耳聋的细胞间分子运动的特定缺陷的可能性。在内耳中，几乎所有含有Cx26的通道也含有Cx30，因此通透性的关键差异可能在于含有WT型Cx26或突变型Cx26的Cx26/Cx30异质通道之间的差异。建议的研究强调对这些异质通道的调查研究。该项目利用表征良好的重组系统来研究原生和异源表达的连接蛋白半通道，以获得有关连接蛋白的选择性及其结构基础的信息，这是人们长期以来所期望的，但通过其他方式无法获得的。我们将使用我们以前应用于研究其他连接蛋白通道的第二信使选择性的技术。我们预计，我们的发现将阐明Cx26突变体导致耳聋的分子机制，并确定正常耳蜗功能所需的细胞间通讯模式。所获得的信息将对其他情况下的连接蛋白通道功能提供信息，并将导致对细胞质渗透剂之间选择性机制的见解。

DESCRIPTION (provided by applicant): Mutations in the connexin26 (Cx26) gene (GJB2) are the predominant cause of inherited syndromic sensorineural deafness in humans. While most mutant connexins result in non-functional channels, some are able to form functional gap junction channels and to mediate electrical coupling (e.g., are permeable to K+ ions), yet they cause deafness. This suggests that the pathology is caused by defect(s) in intercellular movement of cytoplasmic substances other than potassium ions. It is now recognized that channels formed by each of the approximate 20 varieties of connexin differ from one another with regard to molecular permeability, and recent studies show that they can have dramatic and highly specific selectivity's among second messengers and other cytoplasmic molecules. Our hypothesis is that the deafness-causing yet functional mutants of Cx26 have altered permeability to cytoplasmic molecules, and that this defective permeability cannot be compensated for by the other connexins expressed in the inner ear. Our experimental plan is to determine the differences in selectivity of channels that contain WT and deafness-causing mutant Cx26 with regard to second messengers and other cytoplasmic molecules. In doing so, we hope to pinpoint, or narrow down the possibilities for, the specific defect in intercellular molecular movement that causes the deafness. In the inner ear almost all the channels that contain Cx26 also contain Cx30, so it is likely that the key difference in permeability is that between Cx26/Cx30 heteromeric channels that contain WT Cx26 or mutant Cx26. The proposed studies emphasize investigation studies of these heteromeric channels. The projects utilize a well-characterized reconstitution system to study native and heterologously-expressed connexin hemichannels to obtain information about connexin perm selectivity and its structural basis that has been long desired, and unavailable by other means. We will use the techniques that we have previously applied to study second messenger selectivity of other connexin channels. We anticipate that that our findings will elucidate the molecular mechanisms by which Cx26 mutants cause deafness, and identify modes of intercellular communication that are required for normal cochlear function. The information obtained will be informative regarding connexin channel function in other contexts, and will lead to insights about mechanisms of selectivity among cytoplasmic permeants.