GROWTH FACTOR REGULATION OF THE PN1 SODIUM CHANNEL IN PC12 CELLS

导致神经元分化的事件已经显示 显然依赖于生长因子与 神经元细胞表面的受体。在外围和 中枢神经系统需要神经生长因子（NGF） 体内不同神经元群体的发育。大鼠 嗜铬细胞瘤细胞系PC 12是主要的细胞培养物， 神经营养因子（NGF）分化作用的模型。一 NGF对PC 12细胞的主要作用是诱导细胞膜形成， 兴奋性通过本计划中描述的合作 我们已经确定，膜兴奋性是 CNS和PNS型钠通道诱导表达的结果 基因通过不同的信号转导途径。我们还 揭示了一个新的信号转导途径所利用的神经生长因子， 以及FGF、EGF和细胞因子干扰素-γ， 选择性诱导周围神经型钠的表达 通道基因PN 1。我们把这条新的途径称为“触发的” 因为它只受到一分钟的刺激， 的因素。项目2的一个主要目标是定义分子 负责触发PN 1基因诱导的中间体。我们 将决定NGF和FGF受体中的哪些结构域 负责PN 1调节（目标1），使用现场技术- 定向诱变和转染的PC 12克隆的产生 表达突变受体蛋白。我们将使用的技术 显微注射，免疫化学和遗传学来检查一种可能的 STAT家族蛋白在PN 1基因诱导中的作用（目的 2）。通过定义导致神经生长因子和成纤维细胞生长因子受体的结构域， 到II型钠通道诱导（目标3），我们将开始 定义调节CNS的独立信号通路（类型 II）和PNS（PN 1）通道类型。我们的研究将提供 进入调节神经元的独特信号通路 PNS和CNS的兴奋性，以及这些信号如何传递 这些通路汇聚到转录因子上， 调节钠通道基因。这些研究的结果将 纳入项目3和项目4， 了解生长因子对原发性肝癌兴奋性的调节 神经元细胞培养物和体内。

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.