PERMEABILITY MEDIATED BY CONNEXIN CHANNELS

描述（由申请人提供）：连接蛋白通道的基本特性是它们是细胞间细胞质分子直接运动的途径。尽管对连接蛋白通道有相当多的了解，但其最显著的特性-信号分子之间的选择性渗透性-的特征和机制仍然在很大程度上未知。哪些化学信号通过连接蛋白通道，以及它们之间的选择性是如何实现的，是具有深远影响的基础问题。我们试图定义这些过程。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。这种特异性的重要性由以下事实证明：连接蛋白类型的每个功能缺失产生不同的病理，如神经元脱髓鞘、耳聋、心脏缺陷、白内障或不育。由改变的连接蛋白通道功能引起的病理必须由通过连接蛋白孔的异常分子运动引起，无论是在幅度、调节还是分子特异性方面。所提出的研究试图定义连接蛋白通道在细胞质信号分子中选择的机制。我们还寻求进一步开发、表征和利用我们已经鉴定的一类连接蛋白通道的开孔阻断剂。这些实验解决了以下具体问题：连接蛋白通道是否根据特定的分子亲和力在信号分子中进行选择，以及这是如何实现的？环糊精（CD）堵塞孔的机理是什么？CD如何用于研究孔结构？对于这两个问题（选择性渗透率和块），我们试图确定的网站在孔所涉及的。这些位点的鉴定将是至关重要的，并将提供有关蛋白质孔衬部分位置的新信息。阻断剂的工作将导致其作为分子工具，研究连接蛋白的孔结构和功能的利用。该项目利用良好表征的重构系统来研究天然和异源表达的连接蛋白半通道，以获得关于连接蛋白渗透选择性及其结构基础的信息，这是长期以来所期望的，并且通过其他手段不可用。所获得的信息将为疾病和发育中细胞间通讯的细胞和生理学研究提供信息。

DESCRIPTION (provided by applicant): The fundamental property of connexin channels is that they are the pathways for direct movement of cytoplasmic molecules between cells. In spite of considerable knowledge about connexin channels, the character and mechanism of their most salient property - selective permeability among signaling molecules - remains largely unknown. Which chemical signals pass though connexin channel, and how selectivity among them is achieved, are fundamental issues with far-reaching impact. We seek to define these processes. The channels formed by each of the approximately 20 varieties of connexin differ from one another with regard to molecular permeability. The importance of this specificity is demonstrated by the fact that every functional deletion of a connexin type produces a distinct pathology, such as neuronal demyelination, deafness, cardiac defects, cataracts or infertility. The pathologies that arise from altered connexin channel function must arise from abnormal molecular movement through connexin pores, whether in magnitude, regulation or molecular specificity. The proposed studies seek to define mechanism(s) by which connexin channels select among cytoplasmic signaling molecules. We also seek to further develop, characterize and utilize a class of open pore blockers of connexin channels that we have identified. The experiments address the following specific questions: Do connexin channels select among signaling molecules on the basis of specific molecular affinities and how is this achieved? What is the mechanism of pore block by cyclodextrins (CDs) and how can CDs be used to investigate pore structure? For both issues (selective permeability and block) we seek to identify the sites in the pore that are involved. Identification of such sites would be of key importance and will provide new information on the location of pore-lining parts of the protein. The work on the blockers will lead to their utilization as molecular tools to investigate connexin pore structure and function. The projects utilize a well-characterized reconstitution system to study native and heterologously-expressed connexin hemichannels to obtain information about connexin permeation selectivity and its structural basis that has been long desired, and unavailable by other means. The information obtained will inform cellular and physiological studies of intercellular communication in disease and development.