NEUROTRANSMITTER RELEASE IN SYNAPTOTAGMIN MUTANTS

突触结合蛋白突变体中神经递质的释放

• 批准号: 2270089
• 负责人: MARK S PERIN
• 金额: $ 14.93万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2270089
• 关键词: Drosophilidae; calcium flux; electrophysiology; exocytosis; gene mutation; immunocytochemistry; intermolecular interaction; laboratory rabbit; laboratory rat; larva; membrane proteins; molecular cloning; muscle contraction; mutant; neural transmission; neuromuscular junction; neurotransmitter transport; northern blottings; protein purification; southern blotting; synaptic vesicles; tissue /cell culture; transmission electron microscopy

The aim of this application is the elucidation of the role of the synaptic vesicle protein, Synaptotagmin, in neurotransmitter release. This will be accomplished by molecularly identifying and phenotypically characterizing mutations in the synaptotagmin (syt) gene of Drosophila. Synaptotagmin has been identified as a potential docking and fusion protein based on its calcium dependent lipid binding and potential association with presynaptic proteins, the latrotoxin receptor and voltage-gated calcium channel. As such, mutations in Synaptotagmin should directly affect docking and fusion in vivo. In order to test this, we have cloned the Drosophila Synaptotagmin cDNA and generated antibodies specific for Drosophila Synaptotagmin. We have also investigated the developmental expression of message and protein. syt message is neuron specific and present in all PNS and most if not all CNS neurons of the embryo. Protein expressed coincident with axonogenesis and is rapidly transported to the synapses of CNS and muscles. We have localized the syt gene to cytological bands 23A6-A7-B1. In addition, we have identified and mapped four essential genes to 23A4-7-23B1-2. Mutations in one complementation group do not complement the Curly (Cy) mutation (cy maps at 23 B1-2). Embryos homozygous for Cy mutations are morphologically normal and some lack muscle contractions suggesting a direct defect in neurotransmission. We will generate addition syt mutations using EMS, P-element enhancer detectors and gamma-rays to revert Cy. We will molecularly characterize syt mutations using genomic Southerns, Northerns and direct sequencing of PCRed exons. We will phenotypically characterize syt mutants by immunocytochemical and electron microscopy localization of Synaptotagmin, and by electrophysiological recordings of the excitatory junctional potential in larval muscle. Together these two approaches should allow determination of specific defects of neurotransmission. Finally we will initiate studies to identify Drosophila proteins that interact with Synaptotagmin in order to expand this genetic and phenotypic approach to other proteins involved in neurotransmitter release.

该应用的目的是阐明 神经递质释放中的突触囊泡蛋白，突触毒素。 这将通过分子识别和表型来实现 表征果蝇突触（SYT）基因中突变。 Synaptotagmin已被确定为潜在的对接和融合 蛋白质基于其钙依赖性脂质结合和电势 与突触前蛋白，latrotoxin受体和 电压门控钙通道。 因此，突触中的突变 应直接影响体内对接和融合。 为了测试 这，我们已经克隆了果蝇突触蛋白cDNA并产生 果蝇突触特异的抗体。 我们也有 研究了信息和蛋白质的发育表达。 Syt 消息是特定于神经元的，并且在所有PN中都存在，并且大多数CNS（如果不是全部） 胚胎的神经元。 蛋白质与轴突发生一致 并迅速运输到中枢神经系统和肌肉的突触中。 我们有 将SYT基因局部到细胞学频段23A6-A7-B1。 另外，我们 已经确定并映射了四个基本基因至23A4-7-23B1-2。 一个互补组的突变不补充卷曲（CY） 突变（在23 b1-2处的CY地图）。 纯合的胚胎是Cy突变的 形态上正常，有些缺乏肌肉收缩，表明 神经传递的直接缺陷。 我们将生成添加的SYT 使用EMS，P元素增强子检测器和伽马射线的突变 恢复CY。 我们将使用基因组分子表征SYT突变 南方人，北部和直接测序PCRED外显子。 我们将 表型通过免疫细胞化学和 Electron显微镜的定位突触，并通过 兴奋性连接电位的电生理记录 在幼虫肌肉中。 这两种方法应该允许 确定神经传递的特定缺陷。 最后我们会的 开始研究以鉴定果蝇蛋白与 为了将这种遗传和表型方法扩展到 其他参与神经递质释放的蛋白质。