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

为了使神经系统正常工作，不同类型的神经元必须有不同的电活动模式。例如，启动运动的神经元经常“激发”高频的动作电位爆发，而控制缓慢、有节奏的行为的神经元可能只是缓慢地激发，但有非常精确的时间。神经元产生特定活动模式的能力是由它所表达的离子通道的类型和数量决定的。神经细胞通过转录调控选择表达的通道。我们对细胞如何做出这种决定的理解还很不成熟。此外，我们检查基因序列并预测其表达模式的能力是不存在的。目前，DNA序列调控基因表达（控制元件）的目的只能通过经验来确定。Atkinson博士和他的学生将利用基因组计划、进化研究和功能测试来确定控制离子通道基因表达的调控序列的语法。使用果蝇作为模型系统，阿特金森博士在描述缓慢的Ca2+激活的K+通道基因是如何被转录调节方面取得了重大成功。它有一个极其复杂的控制区域。Atkinson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。最值得注意的是鉴定了在四种不同肌肉亚型中不同激活一个启动子的控制元件，以及鉴定了调节发育特异性的内含子区域。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。激振器编码电压门控K+通道。对slowpoke的开发也将继续。对这些基因的研究将使我们能够比较编码两类主要的钾离子通道，电压门控和Ca2+门控钾离子通道的基因是如何调节基因表达的。在所有情况下，重点将放在神经系统中基因的调节上。转录起始位点（~启动子）将被物理定位，并使用转基因动物确定其表达模式。由于整个果蝇是表达系统，因此该小组可以在其自然发育背景下研究所有组织和器官的表达。为了确定控制因素，研究小组将利用重要的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。在序列和位置上都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。这些方法在剖析慢戳基因的调控方面非常成功。此外，还将确定slowpoke和Shaker K+通道基因是否显示坐标调节的证据；也就是说，用一些相同的控制元素组合来调节它们的表达。因为每个控制元件由一个特定的转录因子识别，因此可以推断哪些转录因子参与其中。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最初都是在果蝇中发现或显示存在的。因此，我们期望对果蝇通道基因表达调控的描述也将与对其他生物中相同过程的理解相关。

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.