GROWTH FACTOR REGULATION OF THE PN1 SODIUM CHANNEL IN PC12 CELLS

导致神经元分化的事件已经被证明 显然要依赖于生长因子与 神经细胞表面的受体。在外围设备和 中枢神经系统神经生长因子(NGF)是 体内不同神经元群体的发育。那只老鼠 嗜铬细胞瘤细胞系PC12是主要的细胞培养物 神经营养因子的分化作用模型。一个 NGF对PC12细胞的主要作用是诱导膜 兴奋性。通过本计划中描述的协作 我们已经确定了膜的兴奋性是 诱导CNS和PNS型钠通道表达的结果 基因通过不同的信号转导途径。我们还有 揭示了NGF利用的一种新的信号转导途径，AS 以及通过成纤维细胞生长因子、表皮生长因子和细胞因子干扰素-γ，以 选择性诱导外周神经型钠的表达 通道基因，PN1。我们将这条新路径命名为“触发的” 因为只有一分钟的接触就会刺激它 这些因素。项目2的一个主要目标是定义分子 负责触发PN1基因诱导的中间体。我们 将决定神经生长因子和成纤维细胞生长因子受体中的哪些区域 负责PN1的监管(目标1)，使用现场技术- 定向诱变及转基因PC12克隆的产生 表达突变受体蛋白。我们将使用以下技术 显微注射、免疫化学和遗传学检查可能的 STAT蛋白家族在PN1基因诱导中的作用 2)。通过定义NGF和成纤维细胞生长因子受体中的结构域 对于II型钠通道诱导(目标3)，我们将开始 定义调节中枢神经系统(类型)的独立信号通路 Ii)和PNS(PN1)通道类型。我们的研究将为我们提供见解 进入调节神经元的独特信号通路 三叉神经节和中枢神经系统的兴奋性，以及这些信号如何 通路汇聚到转录因子上，最终 调节钠离子通道基因。这些研究的结果将 纳入项目3和项目4，其目标是 了解生长因子对小学生兴奋性的调节 神经细胞培养和体内实验。

The events leading to neuronal differentiation have been shown clearly to depend upon interactions of growth factors with receptors on the neuronal cell surface. In both the peripheral and central nervous systems Nerve Growth Factor (NGF) is required for the development of distinct neuronal populations in vivo. The rat pheochromocytoma cell line, PC 12, is the premier cell culture model for the differentiating actions of the neurotrophin, NGF. A major action of NGF on PC12 cells is the induction of membrane excitability. Through collaborations described in this program project we have determined that the membrane excitability is the result of induced expression of CNS- and PNS-type sodium channel genes through distinct signal transduction pathways. We have also revealed a new signal transduction pathway utilized by NGF, as well as by FGF, EGF and the cytokine interferon-gamma, to selectively induce expression of the peripheral nerve type sodium channel gene, PN1. We have termed the new pathway the "triggered" pathway because it is stimulated by only a one-minute exposure to the factors. A major goal of project 2 is to define the molecular intermediates responsible for triggered PN1 gene induction. We will determine which domains in the NGF and FGF receptors are responsible for PN1 regulation (aim 1), using techniques of site- directed mutagenesis and generation of transfected PC12 clones expressing the mutant receptor proteins. We will use techniques of microinjection, immunochemistry and genetics to examine a possible role for the STAT family of proteins in PN1 gene induction (aim 2). By defining the domains in the NGF and FGF receptors that lead to type II sodium channel induction (aim 3), we will begin to define the independent signaling pathways regulating the CNS (type II) and PNS (PN1) channel types. Our studies will provide insights into the unique signaling pathways regulating neuronal excitability in both the PNS and CNS, and into how these signaling pathways converge onto the transcriptional factors that ultimately regulate the sodium channel genes. Results from these studies will be integrated into projects 3 and 4, which are aimed at understanding growth factor regulation of excitability in primary neuronal cell cultures and in vivo.