IN VIVO ROLES OF A DROSOPHILA TRANSMITTER TRANSPORTER

果蝇递质转运蛋白的体内作用

• 批准号: 2684975
• 负责人: Michael J Stern
• 金额: $ 15.08万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2684975
• 关键词: Drosophilidae; Xenopus oocyte; animal genetic material tag; complementary DNA; gamma aminobutyrate; gene mutation; membrane transport proteins; neurogenetics; neuropharmacology; neurotransmitter transport; protein structure function; synapses; tissue /cell culture

One process that is important for nervous system function is synaptic transmission, the process by which neurons communicate with each other and with target muscle cells. Neuronal ion channels play key roles in controlling this process. A more complete understanding of the mechanisms by which synaptic transmission can be regulated requires identification of the ion channel structural and regulatory components. However many of these components have as yet resisted molecular characterization. The long-term objective of this work is to use genetic methodology in Drosophila to identify and characterize these components. With genetic methodology, the genes that regulate synaptic transmission are identified by mutation. Because any gene can be mutated, any protein can be identified by mutation regardless of abundance, homology to previously characterized proteins or even prior knowledge of existence. Thus this approach provides a unique way identifying novel classes of functionally important molecules not accessible by other means. Once identified, the roles of these genes in controlling synaptic transmission are determined with electrophysiological assays, and finally the genes are cloned and sequenced which enables the encoded products to be studied at the molecular level. I previously identified mutations in three new genes that interact behaviorally with Shaker, the structural gene for the A type potassium channel. Electrophysiological analysis of these new mutants has shown that each exhibits aberrant synaptic transmission at the larval neuromuscular junction as a result of aberrant excitability of the motor neuron. In the present application, further functional and molecular characterization of these three genes is proposed. The phenotypes of flies lacking each gene, as well as overexpressing each gene, will be determined. Possible synergistic interactions among the genes will be tested by construction and analysis of double mutants. Effects of each gene on nerve terminal structure and electrophysiological properties will be determined. To facilitate cloning of these genes, mutagenesis with P-elements and X-rays will be performed. Isolation and sequence analysis of cDNAs from these genes will provide clues as to the function of the gene products and provide material for further studies. These genes might encode ion channel subunits or regulatory molecules such as protein kinases, G-proteins, or calcium binding proteins. Because such genes are well conserved in evolution, human homologues of these genes will likely exist and might be involved in hereditable disorders of the nervous or neuromuscular system. In addition, because potassium and calcium channel functions are required for non-neural processes such as the control of blood pressure, insulin release and the activation of T-lymphocytes, these human homologues might be defective in hereditable disorders of these processes as well. Therefore I expect that the study of ion channel structure and regulation in Drosophila will have general medical significance. In the future, this genetic approach will be used further to identify and analyze additional components that control the important process of synaptic transmission.

对神经系统功能很重要的一个过程是突触 传输，神经元相互通信的过程 与目标肌肉细胞。 神经元离子通道在 控制这个过程。 对机制有更全面的了解 通过它可以调节突触传递需要识别 离子通道的结构和调节成分。 然而许多 这些成分尚未被分子表征。 这 这项工作的长期目标是利用遗传方法 果蝇来识别和表征这些成分。 具有遗传性 方法，确定了调节突触传递的基因 通过突变。 因为任何基因都可能发生突变，任何蛋白质都可能发生突变。 通过突变鉴定，无论丰度如何，与先前的同源性 描述了蛋白质的特征，甚至是存在的先验知识。 因此这个 方法提供了一种独特的方法来识别新的功能类别 通过其他方式无法获得的重要分子。 一旦确定， 这些基因在控制突触传递中的作用已确定 通过电生理检测，最后克隆基因并 测序使得编码产物能够在分子水平上进行研究 等级。 我之前发现了三个相互作用的新基因的突变 Shaker（A 型钾的结构基因）的行为 渠道。 这些新突变体的电生理分析表明 每个幼虫神经肌肉都表现出异常的突触传递 运动神经元异常兴奋性导致的交界处。 在 目前的应用，进一步的功能和分子表征 提出了这三个基因。 缺乏每种基因的果蝇的表型， 以及每个基因的过度表达，将被确定。 可能的 基因之间的协同相互作用将通过构建和测试来测试 双突变体分析。 各基因对神经末梢的影响 将确定结构和电生理特性。 到 促进这些基因的克隆、P 元素和 X 射线的诱变 将被执行。 从这些物质中分离 cDNA 并进行序列分析 基因将为基因产物的功能提供线索 为进一步研究提供材料。 这些基因可能编码离子通道 亚基或调节分子，如蛋白激酶、G 蛋白或 钙结合蛋白。 因为这些基因在 随着进化，这些基因的人类同源物很可能存在并且可能存在 涉及神经或神经肌肉系统的遗传性疾病。 另外，由于需要钾和钙通道功能 用于非神经过程，例如控制血压、胰岛素 T 淋巴细胞的释放和激活，这些人类同源物可能 这些过程的遗传性疾病也有缺陷。 因此我期望离子通道结构和调控的研究 在果蝇中具有普遍的医学意义。 未来，这 将进一步使用遗传方法来识别和分析其他 控制突触传递重要过程的成分。