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PMID: 27881774 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Interactions Between SNAP-25 and Synaptotagmin-1 Are Involved in Vesicle Priming, Clamping Spontaneous and Stimulating Evoked Neurotransmission.

The Journal of neuroscience : the official journal of the Society for Neuroscience ·Vol. 36 ·No. 47 ·2016-00-23 ·Pages 11865-11880

Schupp M, Malsam J, Ruiter M, Scheutzow A, Wierda KD, Söllner TH, Sørensen JB

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.

Keywords
SNAP-25 SNARE autaptic neuron glutamatergic synapse patch-clamp synaptotagmin
MeSH Terms
Action Potentials/physiology Animals Binding Sites Calcium Signaling/physiology Female Mice Mice, Knockout Mutagenesis, Site-Directed Protein Binding Signal Transduction/physiology Structure-Activity Relationship Synaptic Transmission/physiology Synaptic Vesicles/physiology Synaptosomal-Associated Protein 25/genetics,metabolism Synaptotagmin I/genetics,metabolism
Chemicals
Snap25 protein, mouse Synaptosomal-Associated Protein 25 Synaptotagmin I Syt1 protein, mouse
Authors & Affiliations
7 authors, click to expand affiliations / ORCID
Schupp Melanie ORCID
Neurosecretion Group, Department of Neuroscience and Pharmacology, University of Copenhagen, DK-2200 Copenhagen, Denmark; and.
Malsam Jörg
Heidelberg University Biochemistry Center, D-69120 Heidelberg, Germany.
Ruiter Marvin ORCID
Neurosecretion Group, Department of Neuroscience and Pharmacology, University of Copenhagen, DK-2200 Copenhagen, Denmark; and.
Scheutzow Andrea
Heidelberg University Biochemistry Center, D-69120 Heidelberg, Germany.
Wierda Keimpe D B ORCID
Neurosecretion Group, Department of Neuroscience and Pharmacology, University of Copenhagen, DK-2200 Copenhagen, Denmark; and.
Söllner Thomas H
Heidelberg University Biochemistry Center, D-69120 Heidelberg, Germany.
Sørensen Jakob B ORCID
Neurosecretion Group, Department of Neuroscience and Pharmacology, University of Copenhagen, DK-2200 Copenhagen, Denmark; and [email protected].
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Article Info
Journal
The Journal of neuroscience : the official journal of the Society for Neuroscience
Abbr.
J Neurosci
ISSN
1529-2401
DOI
10.1523/JNEUROSCI.1011-16.2016
Email
[email protected]
Published
2016-00-23
Pages
11865-11880
Language
English
Region
United States
NLM ID
8102140
PMCID
PMC6604916
Subset
IM
External Links
PubMed source DOI: 10.1523/JNEUROSCI.1011-16.2016 PMC Full Text View journal details
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