REGULATION OF SODIUM CHANNEL EXPRESSION

钠通道表达的调节

• 批准号: 3478102
• 负责人: Robert A. Maue
• 金额: $ 9.15万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-04-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3478102
• 关键词: DNA binding protein; action potentials; electrophysiology; epidermal growth factor; fibroblast growth factor; gene expression; genetic regulatory element; messenger RNA; molecular biology; neural information processing; neurogenesis; neurons; neurotrophic factors; northern blottings; nucleic acid sequence; sodium channel; voltage /patch clamp

The expression of voltage-gated sodium (Na) channels is a crucial aspect of neuronal differentiation. As the molecular basis of electrical excitability in most neurons, Na channels play a central role in the production and propagation of action potentials and ultimately in the coding and transfer of information in the nervous system. Despite the importance of Na channel expression, the mechanisms regulating the Na channel gene family are unknown. Therefore, as part of a long term goal to understand the regulation of the Na channel gene family, the specific aims of this proposal are designed to elucidate cellular and molecular mechanisms controlling the expression of the type II Na channel gene. First, regulatory elements in the 5' and 3' flanking regions of the type II gene and the proteins that interact with them will be identified and characterized. Genomic fragments of the 5' and 3' flanking regions that have already been isolated will be dissected by deletional analysis and fused to a reliably assayed reporter gene. The biological activity of the fusion genes in neuronal and non-neuronal cell lines will be determined in transient expression assays. Upon identification of the precise sequences necessary for the neuronal specific expression of the gene, the interaction of specific DNA-binding proteins with these sequences will be analyzed using mobility shift assays and nuclear extracts from neuronal and non- neuronal cells. Results of this analysis will then be used in initial attempts to clone the DNA-binding proteins by screening lambdagt11 expression libraries with DNA recognition-site probes. Second, the mechanism(s) underlying the induction of the type II gene by nerve growth factor (NGF) will be clarified using molecular biological approaches. The influence of NGF on the stability of type II Na channel mRNA will be determined using in vivo pulse chase labeling and filter binding assays, as well as RNA synthesis inhibitors and Northern Blot analysis. Biochemical and molecular biological reagents will be used to alter the activity of protein kinase A and determine its role in the NGF induction reagents will be used to alter the activity of protein kinase A and determine its role in the NGF induction of type II Na channel mRNA. The ability of biochemical treatments that activate protein kinase A to mimic the induction of type II Na channel mRNA by NGF, as well as the ability of NGF to induce type II Na channel mRNA in mutant cells deficient in A kinase activity and in cells stably transfected with an A kinase inhibitor, will be determined using Northern Blot analysis. Third, other growth factors influencing the expression of the type II Na channel will be identified using both electrophysiological and molecular biological approaches. In particular, the effects of fibroblast growth factor and epidermal growth factor will be determined using whole-cell patch clamp recordings and previously developed cell lines stably transfected with Na channel fusion genes. The results will provide a basis for understanding the changes in Na channel expression that occur in response to injury and disease, and will clarify the processes underlying neuronal differentiation and development of the nervous system.

电压门控钠（Na）通道的表达是 神经元分化。 作为电气的分子基础 NA通道在大多数神经元中的兴奋性在 行动潜力的生产和传播，最终 神经系统中信息的编码和传输。 尽管有 NA通道表达的重要性，调节Na的机制 渠道基因家族是未知的。 因此，作为长期目标的一部分 了解NA通道基因家族的调节，具体目的 该建议的设计旨在阐明细胞和分子 控制II型Na通道基因表达的机制。 首先，II型的5'和3'侧面区域中的调节元素 基因及其相互作用的蛋白质将被识别，并且 特征。 5'和3'侧面区域的基因组碎片 已经被隔离了，将通过缺失分析和 与可靠的测定记者基因融合在一起。 的生物活性 神经元和非神经元细胞系中的融合基因将在 瞬态表达测定。 确定精确序列 对于基因的神经元特异性表达所必需的，相互作用 将分析具有这些序列的特定DNA结合蛋白的 使用来自神经元和非神经元和非神经元的迁移性转移测定和核提取物 神经元细胞。 然后将在初始中使用此分析的结果 尝试通过筛选lambdagt11克隆DNA结合蛋白 具有DNA识别位点探针的表达文库。 第二， 通过神经生长诱导II型基因的机制 因子（NGF）将使用分子生物学方法来阐明。 这 NGF对II型NA通道mRNA稳定性的影响将是 使用体内脉冲追逐标记和过滤结合测定确定，为 以及RNA合成抑制剂和北印迹分析。 生化 和分子生物试剂将用于改变 蛋白激酶A并确定其在NGF诱导试剂中的作用 用于改变蛋白激酶A的活性并确定其在 II型NA通道mRNA的NGF诱导。 生化的能力 激活蛋白激酶A以模仿II型诱导的处理 NGF以及NGF诱导II型NA的能力 在激酶活性和细胞中缺乏突变细胞中的mRNA 用A激酶抑制剂稳定转染的稳定转染将使用 北印迹分析。 第三，其他影响的增长因素 使用两者都会识别II型Na通道的表达 电生理和分子生物学方法。 尤其， 成纤维细胞生长因子和表皮生长因子的影响将是 使用全细胞贴片夹记录确定并先前开发 细胞系稳定地用Na通道融合基因转染。 结果 将提供理解NA频道表达的变化的基础 这是对伤害和疾病的响应，并将阐明 神经元分化和发展的过程 神经系统。