GENETIC AND FUNCTIONAL ANALYSIS OF SYNAPTIC TRANSMISSION

了解神经元电信号传导机制的经典方法是使用果蝇温度敏感（TS）麻痹突变体。 这些突变体在允许的温度下正常发育，并且可以转移到限制性温度以检查特定基因产物的生理功能。 果蝇是用于此目的的理想动物，因为它是放热的并且适合复杂的遗传、分子、生物化学、电生理学和行为分析。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 基因产物 dNSF1 在启动对接囊泡以实现快速、钙触发的胞吐作用中发挥作用。 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，包括筛选 comt 的遗传修饰剂。 comt 的一个增强子是钙通道 α1 亚基基因的 TS 麻痹等位基因，即不和谐音 (cac)。 该突变体的突触生理学称为 cacTS2，表明 cac 基因产物代表负责神经肌肉突触递质释放的主要钙通道 α1 亚基。拟议的实验将利用在 cacTS2 修饰基因的遗传筛选中发现的许多新突变体。 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，为扩展我们对突触传递的遗传分析提供了另一个中心起点。 我们对 cacTS2 遗传修饰剂的筛选非常成功，导致新的 cac 等位基因和在突触传递中起作用的其他基因的外源突变得到恢复。 拟议的实验将利用该筛选的成功，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。

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.