Open syntaxin overcomes synaptic transmission defects in diverseC. elegansexocytosis mutants

Abstract

Assembly of SNARE complexes that mediate neurotransmitter release requires opening of a 'closed' conformation of UNC-64/syntaxin. Rescue of unc-13/Munc13 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 exhibited enhanced spontaneous and evoked exocytosis compared to wild-type animals. Unexpectedly, the open syntaxin KI partially suppressed exocytosis defects of various mutants, including snt-1/synaptotagmin, unc-2/P/Q/N-type Ca 2+ 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 aggravated 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. Keywords: exocytosis, vesicle priming, SNARE, synaptotagmin, Munc18/UNC-18, Munc13/UNC-13 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 Ca 2+ influx [reviewed in 4 ]. Ca 2+ , acting through synaptotagmin-1 5,6 , triggers full zippering of the SNARE complex, and ultimately results in fusion of the vesicle with the plasma membrane. A biochemical step preceding the full fusion is often referred to as "priming". It involves formation of the fusion-competent state of synaptic vesicles just prior to Ca 2+ entry. The priming process is tightly controlled by multiple proteins, including Munc18-1/UNC-18 7-9 , Munc13-1/2/UNC-13 10-12 , Tomosyn 13-15 and RIM/UNC-10 16,17 . However, the precise mechanisms through which these proteins mediate priming is still unclear. 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-18 18 . The binding of closed syntaxin and Munc18-1/UNC-18 has been shown to be critical for the level and plasmalemmal localization of syntaxin 7,19-22 . However, when held closed by Munc18-1/UNC-18, syntaxin is unable to interact with synaptobrevin and SNAP-25 to form SNARE complexes 23,24 . Munc13-1/UNC-13 was postulated to open syntaxin and consequently promote SNARE complex assembly and subsequent exocytosis at axon terminals. This important hypothesis originated from studies in which multi-copy over-expression of a constitutively open form of syntaxin/UNC-64 in C. elegans partially restored motility and strongly rescued synaptic vesicle exocytosis defects of unc-13 null worms 25 . The rescue was suggested to be due to a specific genetic interaction between unc-13 and syntaxin, as expression of the open syntaxin in unc-64 null (js21) mutant 26 did not enhance exocytosis compar...