title: Interactions Between SNAP-25 and Synaptotagmin-1 Are Involved in Vesicle Priming, Clamping Spontaneous and Stimulating Evoked Neurotransmission.
abstract: Whether interactions between synaptotagmin-1 (syt-1) and the soluble NSF attachment protein receptors (SNAREs) are required during neurotransmission is debated. We examined five SNAP-25 mutations designed to interfere with syt-1 interactions. One mutation, D51/E52/E55A, targeted negative charges within region II of the primary interface (Zhou et al., 2015); two mutations targeted region I (D166A and D166/E170A) and one mutation targeted both (D51/E52/E55/D166A). The final mutation (D186/D193A) targeted C-terminal residues not expected to interact with syt-1. An in vitro assay showed that the region I, region II, and region I+II (D51/E52/E55/D166A) mutants markedly reduced the attachment between syt-1 and t-SNARE-carrying vesicles in the absence of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. In the presence of PI(4,5)P2, vesicle attachment was unaffected by mutation. When expressed in Snap-25-null mouse autaptic neurons, region I mutations reduced the size of the readily releasable pool of vesicles, whereas the region II mutation reduced vesicular release probability. Combining both in the D51/E52/E55/D166A mutation abrogated evoked release. These data point to a division of labor between region I (vesicle priming) and region II (evoked release). Spontaneous release was disinhibited by region I mutations and found to correlate with defective complexin (Cpx) clamping in an in vitro fusion assay, pointing to an interdependent role of synaptotagmin and Cpx in release clamping. Mutation in region II (D51/E52/E55A) also unclamped release, but this effect could be overcome by synaptotagmin overexpression, arguing against an obligatory role in clamping. We conclude that three synaptic release functions of syt-1, vesicle priming, spontaneous release clamping, and evoked release triggering, depend on direct SNARE complex interaction.
  
  
  
  The function of synaptotagmin-1 (syt-1):soluble NSF attachment protein receptor (SNARE) interactions during neurotransmission remains unclear. We mutated SNAP-25 within the recently identified region I and region II of the primary synaptotagmin:SNARE interface. Using in vitro assays and rescue experiments in autaptic neurons, we show that interactions within region II of the primary interface are necessary for synchronized calcium-triggered release, whereas region I is involved in vesicle priming. Spontaneous release was disinhibited by region I mutation and found to correlate with defective complexin (Cpx) clamping in vitro, pointing to an interdependent role of synaptotagmin and Cpx in release clamping. Therefore, vesicle priming, clamping spontaneous release, and eliciting evoked release are three different functions of syt-1 that involve different interaction modes with the SNARE complex.
authors.affiliation: Neurosecretion Group, Department of Neuroscience and Pharmacology, University of Copenhagen, DK-2200 Copenhagen, Denmark; and.
authors.forename: Melanie
authors.identifier: 0000-0002-5631-3589
authors.initials: M
authors.lastname: Schupp
authors.affiliation: Heidelberg University Biochemistry Center, D-69120 Heidelberg, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Malsam
authors.affiliation: Neurosecretion Group, Department of Neuroscience and Pharmacology, University of Copenhagen, DK-2200 Copenhagen, Denmark; and.
authors.forename: Marvin
authors.identifier: 0000-0002-3267-4785
authors.initials: M
authors.lastname: Ruiter
authors.affiliation: Heidelberg University Biochemistry Center, D-69120 Heidelberg, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Scheutzow
authors.affiliation: Neurosecretion Group, Department of Neuroscience and Pharmacology, University of Copenhagen, DK-2200 Copenhagen, Denmark; and.
authors.forename: Keimpe D B
authors.identifier: 0000-0002-8784-9490
authors.initials: KD
authors.lastname: Wierda
authors.affiliation: Heidelberg University Biochemistry Center, D-69120 Heidelberg, Germany.
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Söllner
authors.affiliation: Neurosecretion Group, Department of Neuroscience and Pharmacology, University of Copenhagen, DK-2200 Copenhagen, Denmark; and jakobbs@sund.ku.dk.
authors.forename: Jakob B
authors.identifier: 0000-0001-5465-3769
authors.initials: JB
authors.lastname: Sørensen
mesh_terms: D000200:Action Potentials / Q000502:physiology*; D000818:Animals; D001665:Binding Sites; D020013:Calcium Signaling / Q000502:physiology; D005260:Female; D051379:Mice; D018345:Mice, Knockout; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D015398:Signal Transduction / Q000502:physiology*; D013329:Structure-Activity Relationship; D009435:Synaptic Transmission / Q000502:physiology*; D013572:Synaptic Vesicles / Q000502:physiology*; D050825:Synaptosomal-Associated Protein 25 / Q000235:genetics / Q000378:metabolism*; D050863:Synaptotagmin I / Q000235:genetics / Q000378:metabolism*
chemical_list: C494209:Snap25 protein, mouse; D050825:Synaptosomal-Associated Protein 25; D050863:Synaptotagmin I; C494311:Syt1 protein, mouse
keywords: SNAP-25; SNARE; autaptic neuron; glutamatergic synapse; patch-clamp; synaptotagmin