Homeostatic Regulation of Neuronal Ion Channel Expression

神经元离子通道表达的稳态调节

• 批准号: 0641370
• 负责人: Nigel Atkinson
• 金额: --
• 依托单位: University of Texas at Austin
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-15 至 2011-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0641370&HistoricalAwards=false
• 关键词: Homeostatic; Regulation; Neuronal; Ion; Channel

Ion channel proteins produce the electrical impulses used to transmit information in the nervous system. Each neuron encodes a large complement of channel genes and the electrical character of the cell is dependent on the subset and quantity of channels that it expresses. In the mature nervous system, the cell must coordinately regulate the activity of ion channel genes to ensure that the properties of the cell do not drift. The Atkinson lab focuses on how channel gene expression is regulated in adult animals. They use the Drosophila model system because of the unusual molecular tools that it provides. The slo gene that they study encodes a BK type Ca2+-activated K+ channel which is highly conserved in structure and function from insects to mammals. The Drosophila slo gene has one of the best described transcriptional control regions of any channel gene. The Atkinson lab has shown that the slo gene responds to aberrant changes in neural excitability in a fashion predicted to restore normal activity. They use organic solvents as anesthetics to reduce neural excitability and observe that the animals up regulate slo gene expression. This change makes the animals resistant to further attempts at sedation (a behavior called tolerance). It is the transcriptional mechanics that link neural excitability to slo gene expression that the lab seeks to determine. The Atkinson lab has long studied the regulation of ion channel gene expression and has the tools and experience to address this question. The first objective is to use mutant analysis to identify transcription factors that up regulate slo after sedation. Flies carrying mutant transcription factor genes are tested to see if the mutation interferes with sedation-induced up regulation of slo. To date, two mutations have been identified and additional reasonable candidates exist. The second objective will be to determine if these factors act by directly by binding the slo promoter region or through an intermediary transcription factor. The chromatin immunoprecipitation assay will be used to see if the protein is physically present on the slo promoter region. Furthermore, by quantifying the amount of bound transcription factor in sedated and mocksedated animals, the lab will determine if sedation increases the binding of the transcription factor to its binding site. The third objective will be to determine the order of action of each transcription factor in the process. Previously, the lab used the chromatin immunoprecipitation assay to describe various epigenetic changes that occur across the slo promoter region following sedation. These changes are thought to be produced by transcription factor activity. Mutations in the responsible transcription factor gene are expected to block specific steps and interrupt the progression of these changes. By determining where each mutation truncates the process, the order of transcription factor activity can be determined. IThis project is designed from the ground up to have aspects that are readily accessible to undergraduate and high school students. This will enable the students the chance to join the lab and to perform important experimental work while they acquire the skills required for the technically difficult molecular portion of the project. Importantly, the undergraduates selected for the project will originate from both U.T. and from the minority institution, Texas A&M Kingsville. This project will be an important tool for encouraging minority participation in the life sciences.

离子通道蛋白产生用于在神经系统中传递信息的电脉冲。每个神经元编码大量的通道基因，细胞的电学特性取决于它所表达的通道的子集和数量。在成熟的神经系统中，细胞必须协调调节离子通道基因的活性，以确保细胞的特性不会漂移。阿特金森实验室专注于成年动物中通道基因表达的调控。他们使用果蝇模型系统，因为它提供了不同寻常的分子工具。他们研究的slo基因编码一种BK型钙激活的K+通道，该通道在结构和功能上从昆虫到哺乳动物都是高度保守的。果蝇slo基因是所有通道基因中描述得最好的转录控制区之一。阿特金森实验室已经证明，slo基因对神经兴奋性的异常变化做出反应，这种方式被预测为恢复正常活动。他们使用有机溶剂作为麻醉剂来降低神经兴奋性，并观察到动物上调了slo基因的表达。这种变化使动物对进一步尝试镇静(一种称为耐受性的行为)具有抵抗力。实验室试图确定的是将神经兴奋性与slo基因表达联系起来的转录机制。阿特金森实验室长期以来一直研究离子通道基因表达的调控，并拥有解决这个问题的工具和经验。第一个目标是使用突变分析来确定在镇静后上调sLO的转录因子。携带突变转录因子基因的果蝇被测试，以确定该突变是否干扰镇静诱导的SLO上调。到目前为止，已经确定了两个突变，并存在其他合理的候选基因。第二个目标是确定这些因子是通过直接结合SLO启动子区域还是通过中介转录因子发挥作用。染色质免疫沉淀试验将被用来确定蛋白质是否实际存在于SLO启动子区域。此外，通过量化镇静和模拟镇静动物中结合的转录因子的数量，实验室将确定镇静是否增加了转录因子与其结合部位的结合。第三个目标是确定每个转录因子在这一过程中的作用顺序。此前，该实验室使用染色质免疫沉淀分析来描述镇静后SLO启动子区域发生的各种表观遗传变化。这些变化被认为是由转录因子活性引起的。负责的转录因子基因的突变预计会阻止特定的步骤，并中断这些变化的进展。通过确定每个突变在哪里截断该过程，就可以确定转录因子活性的顺序。这个项目从一开始就被设计成有本科生和高中生容易接触到的方面。这将使学生有机会加入实验室并执行重要的实验工作，同时获得项目中技术困难的分子部分所需的技能。重要的是，被选入该项目的本科生将来自德克萨斯大学和少数族裔院校德克萨斯A&M金斯维尔。该项目将成为鼓励少数群体参与生命科学的重要工具。