CONNEXIN45 AND NEURAL DEVELOPMENT

GAP功能通道促进细胞间离子交换和 小分子。这种细胞内的通信起着重要的作用 在胚胎和神经发育方面。连接蛋白45(Cx45)是一个亚基间隙 连接蛋白在早期胚胎中表达，在许多 成熟大脑和其他器官的细胞。Cx45通过以下方式形成通道 与其他产品相比，独特的渗透性和监管特性 缝隙连接蛋白。本研究建议进行三组研究。 Cx45及其在神经系统发育中的作用。 目的1.Cx45功能。由于Cx45通常在同一细胞中共表达 在连接蛋白的作用下，它可能通过与连接蛋白混合而发挥“调节”连接蛋白的作用。 其他连接蛋白形成通道改变的异构体半通道 性能(如降低的分子渗透性)。我们将联合快递 带有Cx43或Cx32的Cx45。我们将检查显微注射的转移 染料、细胞间钙波的传播和电耦合。 我们将用生物化学的方法研究混合六聚体的形成。 连接和蔗糖梯度离心法。 目的2.Cx45基因调控。基因组启动子元件与5‘-未翻译 外显子可能与发育和细胞类型特异性有关 Cx45的表达。我们将确定 鸡和小鼠的Cx45基因。RNA印迹、核酸酶保护和RT- 将使用聚合酶链式反应来确定可能的差异转录的mrna。 拼接。带有Cx45基因/荧光素酶报告结构的转基因将 用来定义Cx45表达的最小启动子(S)和 不同于其他连接蛋白的Cx45mRNAs的差异表达。 目的3.Cx45在发育分化中的作用。我们将描述 Cx45在GAP中表达和整合的发育时程 连接斑块及其在早期胚胎中的区域分布 发育中神经细胞的分布及细胞类型的特异性 用RNA和免疫印迹及免疫细胞化学方法进行培养。我们会 产生胚胎干细胞和Cx45缺陷的动物 哪个Cx45被Cx43取代，以检查Cx45的后果- 缺失或用另一种不同的连接蛋白取代 调节和渗透性特性。

Gap functions channels facilitate the intercellular exchange for ions and small molecules. This intracellular communication play a significant role in embryonic and neural development. Connexin45(Cx45) is a subunit gap junction protein which is expressed in early embryos and in a number of cells of the mature brain and other organs. Cx45 forms channels with unique permeability and regulatory properties as compared to other connexins. Three groups of studies are proposed are proposed to examined Cx45 and its role in the developing nervous system. Aim 1. Cx45 function. Since Cx45 is often co-expressed in the same cell with connexins, it may function as a "regulatory" connexin by mixing with other connexins to form heteromeric hemichannels with altered channel properties (such as reduced molecular permeability). We will co-express Cx45 with Cx43 or Cx32. We will examine the transfer of microinjected dyes, propagation of intercellular calcium waves, and electrical coupling. We will examine the formation of mixed hexamers biochemically after cross- linking and sucrose gradient centrifugation. Aim 2. Cx45 gene regulation. Genomic promoter elements and 5'-untranslated exons may contribute to the developmental and cell-type specific expression of Cx45. We will determine the structure and sequence of the chicken and mouse Cx45 genes. RNA blotting, nuclease protection, and RT- PCR will be used to determine possible differential transcription of mRNA splicing. Transfections with Cx45 gene/luciferase reporter constructs will be used to define the minimal promoter(s) for Cx45 expression and the differential expression of Cx45 mRNAs as opposed to other connexins. Aim 3. Cx45 in development and differentiation. We will characterize the developmental time course of Cx45 expression and incorporation into gap junction plaques, its regional distribution in the early embryo, and its cellular distribution in the developing nervous and cell-type specific cultures by RNA- and immuno-blotting and immunocytochemistry. We will generate embryonic stem cells and animals that are Cx45-deficient and in which Cx45 is replaced with Cx43 to examine the consequences of Cx45- deficiency or of its replacement with another connexin with different regulatory and permeability properties.