NEUROGENETICS OF SODIUM CHANNEL GENES IN DROSOPHILA

描述：NA通道在生成和 动作电位的传播，这是运动的主要形式 神经系统中的电信号。长期目标 这项拟议的工作是为了阐明 这是神经信号的基础。在这里，调查人员专注于分析 两种钠通道结构、功能和调节的研究进展 果蝇中的基因，称为parA和DSC。这些钠通道基因 组成一个小家庭，其成员似乎是不同的 利用，并可替代生理上不同的功能。这个 目的是了解这两种钠通道各自的功能 果蝇神经系统中的基因，它们区别的基础 表达，以及突变的表型后果。这个 研究人员将结合使用基因、 分子和电生理技术。为了表征DSC， 代表整个开放阅读框架的cDNA将被分离并 测序；零突变将通过不精确的P切除而产生 基因内的元件插入；以及行为和 这些突变体单独和不同类型的电生理表型 将描述双突变组合的特征。刻画 进一步研究了PARA、DSC和TO的空间和发育表达 阐明它们表达的一些调控机制， 调查者将：提纯对位和DSC特异的抗血清 相应通道多肽的免疫定位；检查 利用外显子--对大量类剪接形式的细胞分布的研究 原位杂交的特异探针，剪接依赖的报告 构建或单细胞聚合酶链式反应分析；并识别上游 用于生殖系的PARA转录调控序列 变形实验。探索结构与功能的关系 在PARA和DSC中，调查员将在 大量体内产生的副突变使用敏感的 SSCP技术与序列分析相结合。 培养果蝇钠电流的电生理记录 神经元和异源表达系统将被用来比较 野生型和突变型等位基因编码的通道特性 PARA和DSC以及PARA的不同剪接变体。 最后，调查者建议使用基于PCR的策略来搜索 在果蝇中寻找更多的钠通道基因，这些基因将作为目标 诱变和表型分析。因为Para和DSC是唯一 在任何生物体中发生突变的神经细胞钠通道基因 这些基因为获得新见解提供了独特的机会 探讨钠离子通道表达、功能和分子机制 调节，在体内。一些人类遗传性神经肌肉疾病 已知与结构中的扰动或 离子通道的功能，包括钠通道。因此，结果是 从这些研究中获得的信息可能对 对这些疾病的了解和可能的治疗。

DESCRIPTION: Na channels perform the central role in the generation and propagation of action potentials, which are the primary form of electrical signalling in the nervous system. The long term objective of the proposed work is to elucidate the molecular mechanisms that underlie neural signalling. Here, the investigator focuses on analysis of the structure, function, and regulation of two Na channel structural genes in Drosophila, called para and Dsc. These Na channel genes comprise a small family whose members appear to be differentially utilized and could subserve physiologically distinct functions. The goals are to understand the respective functions of these two Na channel genes in the Drosophila nervous system, the basis of their differential expression, and the phenotypic consequences of mutations. The investigator will approach these goals using a combination of genetic, molecular, and electrophysiological techniques. To characterize Dsc, cDNAs representing the entire open reading frame will be isolated and sequenced; null mutations will be generated by imprecise excision of P element insertions within the gene; and behavioral and electrophysiological phenotypes of these mutants alone and in various double mutant combinations will be characterized. To characterize further the spatial and developmental expression of para and Dsc and to elucidate some of the regulatory mechanisms governing their expression, the investigator will: raise para- and Dsc-specific antisera for immunolocalization of the corresponding channel polypeptides; examine the cellular distribution of the large array of para splice forms using exon- specific probes for in situ hybridization, splice-dependent reporter constructs, or single-cell PCR analysis; and identify the upstream transcriptional regulatory sequences of para for use in germline transformation experiments. To explore structure-function relationships of para and Dsc, the investigator will determine the exact lesion in a large collection of in vivo-generated para mutations using a sensitive SSCP technique in combination with sequence analysis. Electrophysiological recording of Na currents in cultured Drosophila neurons and in heterologous expression systems will be used to compare the properties of channels encoded by wild-type and mutant alleles of para and Dsc as well as by the different splice variants of para. Finally, the investigator proposes to use a PCR-based strategy to search for additional Na channel genes in Drosophila that will be targeted for mutagenesis and phenotypic analysis. Because para and Dsc are the only neuronal Na channel genes in any organism that have been mutated in situ, these genes provide unique opportunities to obtain novel insights into the molecular mechanisms of Na channel expression, function, and regulation, in vivo. A number of human hereditary neuromuscular diseases are known to be associated with perturbation in the structure or function of ion channels, including Na channels. Therefore, the results obtained from these studies could have direct significance for the understanding and possible treatment of these disorders.