Reexamination of N-terminal domains of syntaxin-1 in vesicle fusion from central murine synapses

Abstract

Syntaxin-1 (STX1) and Munc18-1 are two requisite components of synaptic vesicular release machinery, so much so synaptic transmission cannot proceed in their absence. They form a tight complex through two major binding modes: through STX1's N-peptide and through STX's closed conformation driven by its Habc- domain. However, physiological roles of these two reportedly different binding modes in synapses are still controversial. Here we characterized the roles of STX1's N-peptide, Habc-domain, and open conformation with and without N-peptide deletion using our STX1-null mouse model system and exogenous reintroduction of STX1A mutants. We show, on the contrary to the general view, that the Habc-domain is absolutely required and N-peptide is dispensable for synaptic transmission. However, STX1A's N-peptide plays a regulatory role, particularly in the Ca2+-sensitivity and the short-term plasticity of vesicular release, whereas STX1's open-conformation governs the vesicle fusogenicity. Strikingly, we also show neurotransmitter release still proceeds when the two interaction modes between STX1A and Munc18-1 are presumably intervened, necessitating a refinement of the conceptualization of STX1A-Munc18-1 interaction.

Syntaxin-1A modulates vesicle fusion in mammalian neurons via juxtamembrane domain dependent palmitoylation of its transmembrane domain

“…To test whether loss of palmitoylation by STX1A K260E alone and loss of cysteine residues and their palmitoylation would affect the Ca 2+ -channel activity, we monitored Ca 2+ -influx in the presynapse in the neurons additionally transduced with the Ca 2+ -sensor SynGCampf-6 by stimulating them with different numbers of APs ( Figure 5A-C ). As we have previously reported ( Vardar et al, 2021 ), loss of STX1 reduced the global Ca 2+ -influx ( Figure 5A and B ). On the other hand, neither STX1A CVCV nor STX1A K260E showed significantly different SynGCampf-6 signal where the former trended towards an increase for 1AP and the latter trended towards a decrease for 2, 5, 10, and 20 APs ( Figure 5A and C ).…”

“…We inserted three AAs, glycine-serine-glycine (GSG), into the JMD of STX1A either at the junction of its SNARE domain and JMD (STX1A GSG259 ) or of its JMD and TMD (STX1A GSG265 ) ( Figure 1A ), similar to the previous studies ( Hu et al, 2021 ; Kesavan et al, 2007 ; McNew et al, 1999 ; Mostafavi et al, 2017 ; Zhou et al, 2013 ). Using our lentiviral expression system in STX1-null neurons ( Vardar et al, 2016 ; Vardar et al, 2021 ) and electrophysiological assessment, we surprisingly found that the insertion of one extra helical turn into the JMD of STX1A led to position-specific physiological phenotypes ( Figure 1 ).…”

Interspecies complementation identifies a pathway to assemble SNAREs

“…The functions of the chimeric proteins were determined by locomotion assays ( Figures 1 A and 1B) and sensitivity to the acetylcholine—esterase inhibitor aldicarb ( Figure 1 C)—resistance to the drug implies a reduced level of acetylcholine release ( Mahoney et al., 2006 ). The N-peptide swap only exhibited subtle changes to locomotion, consistent with some previous experiments ( Meijer et al., 2012 ; Park et al., 2016 ; Vardar et al., 2021 ), but not all ( Hu et al., 2007 ; Shen et al., 2007 , 2010 ; Zhou et al., 2013 ). The replacement of the linker domain resulted in significant defects in locomotion and aldicarb sensitivity.…”

“…In agreement, the deletion of the Habc domain from Sso1 increases SNARE complex assembly over 2000-fold ( Nicholson et al., 1998 ). However, the deletion of the yeast Vam3 Habc domain ( Lürick et al., 2015 ), the mouse syntaxin Habc domain ( Vardar et al., 2021 ; Zhou et al., 2013 ), and the worm syntaxin Habc domain ( Rathore et al., 2010 ) all decreased fusion. The decrease in fusion could be attributed to poor trafficking ( Fan et al., 2007 ; Medine et al., 2007 ; Yang et al., 2006 ) or poor expression ( Vardar et al., 2021 ; Zhou et al., 2013 ).…”

“…However, the deletion of the yeast Vam3 Habc domain ( Lürick et al., 2015 ), the mouse syntaxin Habc domain ( Vardar et al., 2021 ; Zhou et al., 2013 ), and the worm syntaxin Habc domain ( Rathore et al., 2010 ) all decreased fusion. The decrease in fusion could be attributed to poor trafficking ( Fan et al., 2007 ; Medine et al., 2007 ; Yang et al., 2006 ) or poor expression ( Vardar et al., 2021 ; Zhou et al., 2013 ). We observed some reduced trafficking of syntaxin to axons in our yeast-Habc chimera ( Figure S2 A).…”