The Regulation of Ca2+-Gated and Voltage-Gated K+ Channel Genes in the Nervous System

神经系统中 Ca2 门控和电压门控 K 通道基因的调节

• 批准号: 0114716
• 负责人: Nigel Atkinson
• 金额: $ 72.53万
• 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: 2001
• 资助国家: 美国
• 起止时间: 2001-09-01 至 2006-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0114716&HistoricalAwards=false
• 关键词: Regulation; Ca2+; Gated; Voltage; K+

PI: Nigel AtkinsonAbstractDifferent types of neurons must have different patterns of electrical activity for the nervous system to function properly. For example, neurons that initiate movement often "fire" high frequency bursts of action potentials, whereas neurons that control slow, rhythmic behaviors may fire only slowly, but with very precise timing. A neuron's ability to generate specific activity patterns is determined by the types and numbers of ion channels it expresses. Nerve cells pick and choose which channels to express by transcriptional regulation. Our understanding of how cells make this decision is very immature. Furthermore, our ability to inspect the sequence of a gene and to then predict its expression pattern is nonexistent. Presently, the purpose of DNA sequences that regulate gene expression (control elements) can only be empirically ascertained. Dr. Atkinson and his students will utilize the genome project, evolutionary studies and functional testing to determine the grammar of regulatory sequences that control ion channel gene expression. Using Drosophila melanogaster as a model system, Dr. Atkinson has had substantial success describing how the slowpoke Ca2+-activated K+ channel gene is transcriptionally regulated. It has an extremely complex control region. The Atkinson lab has described control elements that specify the developmental- and tissue-specific expression pattern. Most notable was the identification of control elements that differentially activate one promoter in four different muscle subtypes and the identification of an intronic region that modulates developmental specificity. In the this project Dr. Atkinson will add the Shaker gene to their list of channel genes to be studied. Shaker encodes a voltage-gated K+ channel. Work on slowpoke will also continue. Work on these to genes will enable a comparison of how genes encoding two major classes of K+ channels, the voltage-gated and Ca2+-gated K+ channels, regulate gene expression. In all cases, the focus will be on the regulation of the genes in the nervous system. Transcriptional start sites (~promoters) will be physically mapped and their expression pattern determined using transgenic animals. Because the entire fly is the expression system, the group can study expression in all tissues and organs in their natural developmental context. To identify the control elements the group will make use of the fact that important DNA sequences tend to be conserved over evolutionary time. For each gene, the transcriptional control regions from four different insect species will be sequenced and compared. Small sequences conserved in both sequence and position will be assumed to be control elements. The candidate elements will be tested for function by deleting them from a transgene and then asking how expression had been altered. These methods have been very successful in dissecting the regulation of the slowpoke gene. Furthermore, it will be determined whether the slowpoke and Shaker K+ channels genes show evidence of coordinate regulation; that is, do some of the same combinations of control elements regulate their expression. Because each control element is recognized by a specific transcription factor(s), it will be possible to infer which transcription factors are involved. Drosophila have the same families of ion channel genes found in vertebrates. In addition, most of the important regulatory cascades affecting developmental gene expression were originally discovered in or shown to exist in Drosophila. Therefore, it is expected that the description of the regulation of channel gene expression in Drosophila will also be relevant to the understanding of the same process in other organisms.

PI：奈杰尔阿特金森摘要不同类型的神经元必须有不同的电活动模式，神经系统才能正常工作。例如，启动运动的神经元通常会“激发”高频率的动作电位爆发，而控制缓慢、有节奏行为的神经元可能只会缓慢地激发，但时间非常精确。神经元产生特定活动模式的能力由其表达的离子通道的类型和数量决定。神经细胞通过转录调节挑选和选择表达哪些通道。我们对细胞如何做出这个决定的理解还非常不成熟。此外，我们没有能力检查基因序列，然后预测其表达模式。目前，调节基因表达的DNA序列（控制元件）的目的只能凭经验确定。阿特金森博士和他的学生将利用基因组计划，进化研究和功能测试，以确定控制离子通道基因表达的调控序列的语法。 Atkinson博士利用果蝇作为模型系统，成功描述了慢波钙激活钾通道基因是如何转录调控的。它有一个非常复杂的控制区域。阿特金森实验室已经描述了指定发育和组织特异性表达模式的控制元件。最值得注意的是控制元件，差异激活一个启动子在四个不同的肌肉亚型和一个内含子区域，调节发育特异性的鉴定。 在这个项目中，Atkinson博士将把Shaker基因添加到他们要研究的通道基因列表中。Shaker编码电压门控K+通道。 关于slowpoke的工作也将继续。对这些基因的研究将能够比较编码两种主要类型的K+通道（电压门控和Ca 2+门控K+通道）的基因如何调节基因表达。在所有情况下，重点将放在神经系统中基因的调节上。将对转录起始位点（~启动子）进行物理作图，并使用转基因动物确定其表达模式。因为整个果蝇都是表达系统，所以该小组可以研究所有组织和器官在其自然发育环境中的表达。为了确定控制元件，该小组将利用重要的DNA序列在进化过程中趋于保守的事实。对于每个基因，将对来自四种不同昆虫物种的转录控制区进行测序和比较。在序列和位置上保守的小序列将被假定为控制元件。候选元件将通过从转基因中删除它们来测试功能，然后询问表达是如何改变的。这些方法在剖析慢波基因的调控方面非常成功。此外，还将确定慢波钾离子通道和震荡钾离子通道基因是否显示出协同调节的证据;也就是说，是否有一些相同的控制元件组合调节它们的表达。 因为每个控制元件都被特定的转录因子识别，所以可以推断出涉及哪些转录因子。 果蝇具有与脊椎动物相同的离子通道基因家族。此外，大多数影响发育基因表达的重要调控级联反应最初在果蝇中发现或显示存在于果蝇中。因此，可以预期的是，果蝇中通道基因表达调控的描述也将与其他生物体中相同过程的理解相关。