Homeostatic Regulation of Neuronal Ion Channel Expression

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漂移。阿特金森实验室专注于通道基因表达如何在成年动物中被调节。他们使用果蝇模型系统是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BK型Ca2+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。果蝇的slo基因是所有通道基因中被描述得最好的转录控制区之一。阿特金森实验室已经证明，慢速基因对神经兴奋性的异常变化作出反应，以一种可以恢复正常活动的方式。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慢速基因表达。这种变化使动物抵抗进一步的镇静尝试（一种称为耐受性的行为）。实验室试图确定的是将神经兴奋性与缓慢基因表达联系起来的转录机制。Atkinson实验室长期以来一直在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体分析来鉴定镇静后上调缓慢的转录因子。携带突变转录因子基因的果蝇进行了测试，看看突变是否干扰镇静诱导的慢速调节。迄今为止，已经确定了两种突变，并且存在其他合理的候选突变。第二个目标将是确定这些因子是否直接通过结合慢启动子区域或通过中间转录因子起作用。染色质免疫沉淀试验将用于观察蛋白质是否物理存在于慢启动子区域。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位点的结合。第三个目标将是确定过程中每个转录因子的作用顺序。此前，该实验室使用染色质免疫沉淀法来描述镇静后慢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产生的。预期相关转录因子基因的突变会阻断特定步骤并中断这些变化的进展。通过确定每个突变截断该过程的位置，可以确定转录因子活性的顺序。这个项目的设计从一开始就很容易让本科生和高中生接触到。这将使学生有机会加入实验室并进行重要的实验工作，同时他们将获得项目中技术困难的分子部分所需的技能。重要的是，入选该项目的本科生将同时来自德州大学和少数族裔院校德州A&amp；M Kingsville。这个项目将成为鼓励少数民族参与生命科学的一个重要工具。

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.