STRUCTURE, FUNCTION, AND REGULATION OF GAP JUNCTION PROTEINS

缝隙连接是允许直接交换的膜特化 许多组织中相邻细胞之间的离子和小分子。在……里面 心肌，通过缝隙连接的电耦合允许传播 动作电位和心肌细胞的同步收缩。 在组织损伤期间，如在缺血中观察到的， 跨越交界处的阻力可能会对受影响的人造成局部损害 地点。因此，缝隙连接在细胞内起着重要的作用。 信号转导的阐明或其结构和功能是 心脏细胞间通讯功能的研究 包括正常生理状态和病理生理状态。编码主要基因的cDNA3 心肌细胞间隙连接蛋白43(Cx43)已被鉴定 在细胞外和细胞外的 不同组织中连接蛋白的跨膜区。然而， 不同来源的连接蛋白胞质结构域不是同源的 并且可能代表独特的监管位置。在本提案中，一个 形态、生化、分子生物学和 将使用电物理方法来研究间隙结结构和 功能。具体地说，我们将：确定细胞质的作用 Cx43结构域在多种秒调节通道功能中的作用 信使和蛋白激酶系统；确定相互作用的性质 通过探测Cx43的胞外结构域来实现相邻细胞之间的连接；以及 确定在功能组装中重要的一些序列 膜上的通道。为了实现这些目标，无论是正常的还是 编码Cx43的一系列突变型cDNAs将在非洲爪蛙中表达 莱维斯卵母细胞。各种电生理、免疫化学和 生化技术将被用来评估它们的功能 属性。

Gap junctions are membrane specializations which permit the direct exchange of ions and small molecules between adjacent cells in many tissues. In myocardium, electrical coupling through gap junctions permits propagation of the action potential and the synchronized contraction of myocyte. During tissue injury, such as the observed in ischemia, an increase in the resistance across junctions may serve to localize damage to the affected site. Thus, gap junctions play a central importance role in intracellular signal transduction an elucidation or their structure and function is of crucial importance to understanding cardiac intercellular communication in both normal and pathophysiologic states. A cDNA encoding the major myocardial gap protein, designated connexin 43 (Cx43), has been identified and there is considerable homology between the extracellular an transmembrane domains of connexins from different tissues. However, the cytoplasmic domains of connexins from different sources are not homologous and may represent unique regulatory sites. In the present proposal, a combination of morphological, biochemical, molecular biological and electrophysical approaches will be used to study gap junction structure and function. Specifically, we will: determine the role of the cytoplasmic domain of Cx43 in regulation of channel function by a variety of second messengers and protein kinase systems; determine the nature of interaction between adjacent cells by probing the extracellular domains of Cx43; and determine some of the sequences important in assembly of functional channels in the membrane. To achieve these goals, both normal, and a series of mutant cDNA's encoding Cx43 will be expressed in the Xenopus laevis oocyte. A variety of electrophysiological, immunochemical and biochemical techniques will be used to evaluate their functional properties.