title: Munc18-1 tuning of vesicle merger and fusion pore properties.
abstract: The release of hormones and neurotransmitters, mediated by regulated exocytosis, can be modified by regulation of the fusion pore. The fusion pore is considered stable and narrow initially, eventually leading to the complete merger of the vesicle and the plasma membranes. By using the high-resolution patch-clamp capacitance technique, we studied single vesicles and asked whether the Sec1/Munc18 proteins, interacting with the membrane fusion-mediating SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, affect fusion pore properties. Munc18-1 mutants were transfected into lactotrophs to affect the interaction of Munc18-1 with syntaxin1 (Synt1) (R39C), Rab3A (E466K), and Mints (P242S). Compared with wild-type, Munc18-1 E466K increased the frequency of the fusion event. The latter two mutants increased the fusion pore dwell-time. All the mutants stabilized narrow fusion pores and increased the amplitude of fusion events, likely via preferential fusion of larger vesicles, since overexpression of Munc18-1 R39C did not affect the average size of vesicles, as determined by stimulated emission depletion (STED) microscopy. Single-molecule atomic force microscopy experiments revealed that wild-type Munc18-1, but not Munc18-1 R39C, abrogates the interaction between synaptobrevin2 (Syb2) and Synt1 binary trans-complexes. However, neither form of Munc18-1 affected the interaction of Syb2 with the preformed binary cis-Synt1A-SNAP25B complexes. This indicates that Munc18-1 performs a proofing function by inhibiting tethering of Syb2-containing vesicles solely to Synt1 at the plasmalemma and favoring vesicular tethering to the preformed binary cis-complex of Synt1A-SNAP25B. The association of Munc18-1 with the ternary SNARE complex leads to tuning of fusion pores via multiple and converging mechanisms involving Munc18-1 interactions with Synt1A, Rab3A, and Mints.
authors.affiliation: Laboratory of Neuroendocrinology-Molecular Cell Physiology, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia.
authors.forename: Jernej
authors.initials: J
authors.lastname: Jorgacevski
authors.forename: Maja
authors.initials: M
authors.lastname: Potokar
authors.forename: Sonja
authors.initials: S
authors.lastname: Grilc
authors.forename: Marko
authors.initials: M
authors.lastname: Kreft
authors.forename: Wei
authors.initials: W
authors.lastname: Liu
authors.forename: Jeff W
authors.initials: JW
authors.lastname: Barclay
authors.forename: Johanna
authors.initials: J
authors.lastname: Bückers
authors.forename: Rebecca
authors.initials: R
authors.lastname: Medda
authors.forename: Stefan W
authors.initials: SW
authors.lastname: Hell
authors.forename: Vladimir
authors.initials: V
authors.lastname: Parpura
authors.forename: Robert D
authors.initials: RD
authors.lastname: Burgoyne
authors.forename: Robert
authors.initials: R
authors.lastname: Zorec
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D002478:Cells, Cultured; D022162:Cytoplasmic Vesicles / Q000502:physiology*; D025802:Electric Capacitance; D005973:Glutamine / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D052682:Lactotrophs / Q000166:cytology; D008239:Lysine / Q000235:genetics; D008297:Male; D008561:Membrane Fusion / Q000235:genetics / Q000502:physiology*; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics; D027541:Mentha / Q000235:genetics / Q000378:metabolism; D018625:Microscopy, Atomic Force / Q000379:methods; D018613:Microscopy, Confocal; D008954:Models, Biological; D051938:Munc18 Proteins / Q000235:genetics* / Q000502:physiology; D009154:Mutation / Q000235:genetics*; D018408:Patch-Clamp Techniques; D051381:Rats; D017208:Rats, Wistar; D018709:Statistics, Nonparametric; D050825:Synaptosomal-Associated Protein 25 / Q000235:genetics / Q000378:metabolism; D050827:Syntaxin 1 / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000379:methods; D020826:rab3A GTP-Binding Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D051938:Munc18 Proteins; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; C485184:enhanced green fluorescent protein; D005973:Glutamine; D049452:Green Fluorescent Proteins; D020826:rab3A GTP-Binding Protein; D008239:Lysine