Open syntaxin overcomes exocytosis defects of diverse mutants in C. elegans

Chi-Wei Tien,Mengjia Huang,Shuzo Sugita,Karolina P Stepien,Liping Han,Bin Yu,Kyoko Sugita,Mei Zhen,Philippe P Monnier,Shangbang Gao,Josep Rizo,Xiaoyu Xie

doi:10.1038/s41467-020-19178-x

Abstract

Assembly of SNARE complexes that mediate neurotransmitter release requires opening of a ‘closed’ conformation of UNC-64/syntaxin. Rescue of unc-13/Munc13 mutant phenotypes by overexpressed open UNC-64/syntaxin suggested a specific function of UNC-13/Munc13 in opening UNC-64/ syntaxin. Here, we revisit the effects of open unc-64/syntaxin by generating knockin (KI) worms. The KI animals exhibit enhanced spontaneous and evoked exocytosis compared to WT animals. Unexpectedly, the open syntaxin KI partially suppresses exocytosis defects of various mutants, including snt-1/synaptotagmin, unc-2/P/Q/N-type Ca2+ channel alpha-subunit and unc-31/CAPS, in addition to unc-13/Munc13 and unc-10/RIM, and enhanced exocytosis in tom-1/Tomosyn mutants. However, open syntaxin aggravates the defects of unc-18/Munc18 mutants. Correspondingly, open syntaxin partially bypasses the requirement of Munc13 but not Munc18 for liposome fusion. Our results show that facilitating opening of syntaxin enhances exocytosis in a wide range of genetic backgrounds, and may provide a general means to enhance synaptic transmission in normal and disease states.

Highlights

Of SNARE complexes that mediate neurotransmitter release requires opening of a ‘closed’ conformation of UNC-64/syntaxin
The signal at ~80 kDa disappeared after repeated boiling of the samples. These results suggest that open syntaxin accelerates SNARE complex assembly as previously suggested[31]
Great advances have been made in the past three decades in our understanding of the mechanism of neurotransmitter release, showing that release depends on SNARE complexes that bridge the vesicle and plasma membranes, and that SNARE complex assembly is orchestrated by Munc18-1/UNC-18 and Munc13/ UNC-13

Summary

Introduction

Of SNARE complexes that mediate neurotransmitter release requires opening of a ‘closed’ conformation of UNC-64/syntaxin. Attachment receptor) proteins syntaxin, synaptobrevin/VAMP, and SNAP-25 play a central role in exocytosis by forming a tight SNARE complex[1] that consists of a four-helix bundle[2,3]. This four-helix bundle is believed to be partially formed before Ca2+ influx[4]. Syntaxin/UNC-64 is a key SNARE protein that is crucial for exocytosis It exists in an open and a closed conformation, the latter forming a tight complex with Munc18-1/UNC-1818.

Methods

Results

Conclusion