CONNEXIN45 AND NEURAL DEVELOPMENT

间隙功能通道促进细胞间离子交换， 小分子。这种细胞内的通讯在 在胚胎和神经发育中。连接蛋白45（Cx45）是一个亚基缺口， 在早期胚胎和许多哺乳动物中表达的连接蛋白， 成熟的大脑和其他器官的细胞。Cx45形成通道， 独特的渗透性和调节特性， 连接蛋白建议审查三组研究 Cx45及其在神经系统发育中的作用 目标1. Cx45功能。由于Cx45通常在同一细胞中共表达， 与连接蛋白，它可以作为一个“调节”连接蛋白混合， 其他连接蛋白形成具有改变的通道的异聚半通道 性能（如降低分子渗透性）。我们将共同表达 Cx45与Cx43或Cx 32。我们将检查显微注射的 染料、细胞间钙波的传播和电耦合。 我们将研究交叉后混合六聚体的生物化学形成- 连接和蔗糖梯度离心。 目标2. Cx45基因调控。基因组启动子元件和5 '-非翻译 外显子可能有助于发育和细胞类型特异性 Cx45的表达。我们将确定的结构和序列的 鸡和小鼠Cx45基因。RNA印迹、核酸酶保护和RT- PCR将用于确定mRNA的可能差异转录 拼接用Cx45基因/荧光素酶报告基因构建体转染将 用于定义Cx45表达的最小启动子， Cx45 mRNA与其他连接蛋白的差异表达。 目标3。Cx45在发育和分化中的作用我们将描述 Cx45表达和掺入间隙的发育时程 连接斑，其在早期胚胎中的区域分布， 发育中神经和细胞类型特异性的细胞分布 通过RNA印迹和免疫印迹以及免疫细胞化学对培养物进行鉴定。我们将 产生胚胎干细胞和Cx45缺陷的动物， 其中Cx45被Cx43取代，以检查Cx45的后果- 缺乏或被另一种具有不同 调节性和渗透性。

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.