GENETIC AND FUNCTIONAL ANALYSIS OF SYNAPTIC TRANSMISSION

突触传递的遗传和功能分析

• 批准号: 6266947
• 负责人: RICHARD W ORDWAY
• 金额: $ 24.51万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-15 至 2005-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6266947
• 关键词: Drosophilidae; calcium channel; electron microscopy; electrophysiology; gene targeting; genetic mapping; genetic regulation; genetically modified animals; intermolecular interaction; invertebrate embryology; invertebrate locomotion; morphometry; neural plasticity; neural transmission; neurogenetics; neuromuscular junction; neurophysiology; neuropsychology; neurotransmitter transport; paralysis; protein structure function; suppressor mutations; synaptic vesicles; temperature sensitive mutant; voltage gated channel; western blottings

A classical approach to understanding the mechanisms of electrical signaling in neurons has been the use of temperature- sensitive (TS) paralytic mutants of Drosophila. These mutants develop normally at permissive temperature and can be shifted to restrictive temperatures to examine the physiological function of a specific gene product. Drosophila is ideal for this purpose given that it is exothermic and amenable to sophisticated genetic, molecular, biochemical, electrophysiological, and behavioral analysis. Our previous work has extended some early studies on TS paralytic mutants by examining the role of the comatose (comt) gene product in synaptic transmission. Our analysis has shown that the comt gene product, dNSF1, functions in the priming of docked vesicles for fast, calcium-triggered exocytosis. To broaden our investigation of synaptic mechanisms, we have conducted genetic screens for new mutations affecting synaptic transmission, including a screen for genetic modifiers of comt. One enhancer of comt is a TS paralytic allele of a calcium channel alpha1 subunit gene, cacophony (cac). Synaptic physiology in this mutant, termed cacTS2, shows that the cac gene product represents the primary calcium channel alpha1 subunit responsible for transmitter release at neuromuscular synapses. The proposed experiments will advantage of a number of new mutants recovered in a genetic screen for modifiers of cacTS2. The rapid TS paralytic phenotype of cacTS2, as well as the central role of voltage-gated calcium channels in neurotransmitter release, have provided another central starting point from which to expand our genetic analysis of synaptic transmission. Our screen for genetic modifiers of cacTS2 has been highly successful, resulting in the recovery of both new cac alleles and extragenic mutations in other genes functioning in synaptic transmission. The proposed experiments will capitalize on the success of this screen to pursue further genetic analysis of the functions and interactions of cac-encoded calcium channels in synaptic transmission.

理解神经元中电信号机制的经典方法是使用果蝇的温度敏感（TS）麻痹突变体。 这些突变体在允许温度下正常发育，并且可以转移到限制温度以检查特定基因产物的生理功能。 果蝇是这一目的的理想选择，因为它是放热的，并且适合复杂的遗传、分子、生物化学、电生理和行为分析。我们之前的工作通过检查昏迷（comt）基因产物在突触传递中的作用，扩展了对TS麻痹突变体的一些早期研究。 我们的分析表明，comt基因的产物，dNSF1，功能在启动对接囊泡快速，钙触发胞吐。 为了扩大我们对突触机制的研究，我们对影响突触传递的新突变进行了遗传筛选，包括对comt基因修饰剂的筛选。 comt的一个增强子是钙通道α 1亚基基因cacophony（cac）的TS麻痹等位基因。 在这种突变体中的突触生理学，称为cacTS2，表明cac基因产物代表主要的钙通道α 1亚基负责神经肌肉突触的递质释放。所提出的实验将有利于在cacTS2修饰剂的遗传筛选中回收的许多新突变体。cacTS2的快速TS麻痹表型，以及电压门控钙通道在神经递质释放中的核心作用，提供了另一个中心起点，从中扩展我们对突触传递的遗传分析。 我们对cacTS2的遗传修饰剂的筛选是非常成功的，导致在突触传递中起作用的其他基因中的新的cac等位基因和基因外突变的恢复。 拟议中的实验将利用这一筛选的成功，对突触传递中cac编码的钙通道的功能和相互作用进行进一步的遗传分析。