Huang Mengjia, Wang Ya, Chow Chun Hin, Stepien Karolina P, Indrawinata Karen, Xu Junjie, Argiropoulos Peter, Xie Xiaoyu, Sugita Kyoko, Tien Chi-Wei, Lee Soomin, Monnier Philippe P, Rizo Josep, Gao Shangbang, Sugita Shuzo
Division of Experimental & Translational Neuroscience, Krembil Brain Institute, University Health Network, Ontario, M5T 0S8, Canada.
Department of Physiology, Temerty Faculty of Medicine, University of Toronto, Toronto, Ontario, M5S 1A8, Canada.
bioRxiv. 2023 Nov 10:2023.08.18.553709. doi: 10.1101/2023.08.18.553709.
SNARE and Sec/Munc18 proteins are essential in synaptic vesicle exocytosis. Open form t-SNARE syntaxin and UNC-18 P334A are well-studied exocytosis-enhancing mutants. Here we investigate the interrelationship between the two mutations by generating double mutants in various genetic backgrounds in . While each single mutation rescued the motility of and mutants significantly, double mutations unexpectedly worsened motility or lost their rescuing effects. Electrophysiological analyses revealed that simultaneous mutations of open syntaxin and gain-of-function P334A UNC-18 induces a strong imbalance of excitatory over inhibitory transmission. In liposome fusion assays performed with mammalian proteins, the enhancement of fusion caused by the two mutations individually was abolished when the two mutations were introduced simultaneously, consistent with what we observed in . We conclude that open syntaxin and P334A UNC-18 do not have additive beneficial effects, and this extends to ' characteristics such as motility, growth, offspring bared, body size, and exocytosis, as well as liposome fusion in vitro. Our results also reveal unexpected differences between the regulation of exocytosis in excitatory versus inhibitory synapses.
SNARE蛋白和Sec/Munc18蛋白在突触小泡胞吐作用中至关重要。开放形式的t-SNARE蛋白Syntaxin和UNC-18 P334A是经过充分研究的增强胞吐作用的突变体。在此，我们通过在不同遗传背景下产生双突变体来研究这两种突变之间的相互关系。虽然每个单突变都能显著挽救 和 突变体的运动能力，但双突变却意外地使运动能力恶化或失去了挽救效果。电生理分析表明，开放型Syntaxin和功能获得型P334A UNC-18的同时突变会导致兴奋性与抑制性传递的强烈失衡。在用哺乳动物蛋白进行的脂质体融合试验中，当同时引入这两种突变时，由这两种突变单独引起的融合增强作用被消除，这与我们在 中观察到的结果一致。我们得出结论，开放型Syntaxin和P334A UNC-18没有累加的有益作用，并且这延伸到了诸如运动能力、生长、后代产出、体型和胞吐作用等 的特征，以及体外脂质体融合。我们的结果还揭示了兴奋性突触与抑制性突触中胞吐作用调节之间意想不到的差异。
