IN VIVO ROLES OF A DROSOPHILA TRANSMITTER TRANSPORTER

神经系统功能的一个重要过程是突触 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。 更全面地了解这些机制 调节突触传递的方法需要识别 离子通道的结构和调节成分。 然而许多 这些组分至今还不能进行分子表征。 的 这项工作的长期目标是使用遗传方法， 果蝇来识别和描述这些成分。 与遗传 方法，确定了调节突触传递的基因 通过突变。 因为任何基因都可以突变，任何蛋白质都可以 通过突变鉴定，无论丰度如何，与先前 特征蛋白质或甚至存在的先验知识。 因此该 方法提供了一种独特的方式来识别新的功能类别， 重要的分子不能通过其他方式获得。 一旦确定， 确定了这些基因在控制突触传递中的作用 通过电生理检测，最后克隆基因， 测序，这使得编码的产品能够在分子水平上进行研究。 水平 我之前发现了三个新基因的突变， Shaker，A型钾的结构基因， 频道 对这些新突变体的电生理学分析表明， 每种都在幼虫神经肌肉处表现出异常的突触传递 由于运动神经元异常兴奋而导致的连接。 在 本申请的进一步功能和分子表征 这三个基因的基因。 缺乏每种基因的果蝇的表型， 以及每种基因的过表达。 可能 基因间的协同相互作用将通过构建来测试， 双突变体分析 各基因对神经末梢的影响 结构和电生理特性将被确定。 到 促进这些基因的克隆，用P元件和X射线诱变 将被执行。 从这些细胞中分离cDNA并进行序列分析 基因将提供有关基因产物功能的线索， 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 在进化过程中，这些基因的人类同源物可能存在， 与神经或神经肌肉系统的遗传性紊乱有关。 此外，由于钾和钙通道功能是必需的， 对于非神经过程，如控制血压，胰岛素， 释放和激活T淋巴细胞，这些人类同源物可能 在这些过程的遗传性疾病中也有缺陷。 因此我期望离子通道的结构和调控的研究 将具有普遍的医学意义。 未来这个 遗传方法将进一步用于识别和分析其他 控制突触传递的重要过程。

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.