UNC-18 function in C. elegans synaptic transmission

描述（由申请人提供）： 神经元通过胞吐作用将神经递质释放到突触间隙中， 突触囊泡介导胞吐作用的蛋白质是 参与一般细胞内融合事件的保守蛋白质家族。 这个过程中的关键蛋白质包括SNARE蛋白、突触融合蛋白、SNAP-25 和小突触泡蛋白，以及β-18和β-13。尽管我们知道， 和β-13与必需的SNARE蛋白突触融合蛋白相互作用， 这些蛋白质调节融合的顺序和顺序还有待于研究。 测定在我的实验室里，我们将联合收割机遗传学和分子学方法结合起来， 新开发的电生理技术来研究胞吐作用， 线虫线虫使用这些技术，我们已经证明 在UNC-13突变体中胞吐作用在晚期被阻止。我们现建议 研究了β-18在C.突触传递β-18结合 突触融合蛋白的闭合构象，排除了SNARE的形成 复杂.目前已提出了几种不同的模型来描述18函数：（1）18函数 解离可以促进或维持突触融合蛋白处于开放状态， 复杂的组装和融合。(2)β-18可能在封闭的细胞中维持突触融合蛋白， 构象以防止SNARE复合物形成。(3)β-18和突触融合蛋白 直接介导融合步骤。为了测试这些模型，我们建议： 1)确定在囊泡停靠到细胞外基质后，β-18是否促进胞吐作用。 质膜。 2)测试β-18-syntaxin相互作用是否在 胞吐作用 3)测试组成性开放的syntaxin是否绕过了 -18。 4)通过a）鉴定与α-18下游或平行作用的其他蛋白质 定位和鉴定最近分离的unc-18突变抑制子，和B） 进行基因筛选以鉴定其他UNC-18抑制因子。 这些实验可能有助于我们了解阿尔茨海默病， 中风和囊泡运输障碍。

DESCRIPTION (provided by applicant): Neurons release neurotransmitter into the synaptic cleft by exocytosis of synaptic vesicles. The proteins that mediate exocytosis are members of conserved protein families involved in general intracellular fusion events. Critical proteins in this process include the SNARE proteins, syntaxin, SNAP-25 and synaptobrevin, as well as UNC-18 and UNC-13. Although we know that UNC-18 and UNC-13 interact with the essential SNARE protein syntaxin, the precise role and sequential order in which these proteins regulate fusion have yet to be determined. In my laboratory we combine genetic and molecular approaches with a newly developed electrophysiological technique to study exocytosis in the nematode Caenorhabditis elegans. Using these techniques we have demonstrated that exocytosis is arrested at a late stage in unc-13 mutants. We now propose to examine the role of UNC-18 in C. elegans synaptic transmission. UNC-18 binds to a closed conformation of syntaxin, which excludes the formation of the SNARE complex. Several models have been proposed for UNC-18 function: (1) UNC-18 dissociation may promote or maintain syntaxin in an open state, enabling SNARE complex assembly and fusion. (2) UNC-18 may maintain syntaxin in the closed conformation to prevent SNARE complex formation. (3) UNC- 18 and syntaxin may mediate the fusion step directly. To test these models, we propose to: 1) Determine whether UNC-18 promotes exocytosis after vesicles have docked to the plasma membrane. 2) Test whether the UNC-18-syntaxin interaction plays an inhibitory role in exocytosis. 3) Test whether constitutively open syntaxin bypasses the requirement for UNC-18. 4) Identify other proteins that act downstream or in parallel with UNC-18 by a) mapping and identifying a recently isolated unc-18 mutant suppressor and b) conducting a genetic screen to identify other unc-18 suppressors. These experiments may contribute to our understanding of Alzheimer's disease, stroke and vesicle trafficking disorders.