Synaptotagmin interaction with SNAP-25 governs vesicle docking, priming, and fusion triggering

Ralf Mohrmann; Heidi de Wit; Emma Connell; Paulo César da Silva Pinheiro; Charlotte Leese; Dieter Bruns; Bazbek Davletov; Matthijs Verhage; Jakob Balslev Sørensen

doi:10.1523/JNEUROSCI.1236-13.2013

Synaptotagmin interaction with SNAP-25 governs vesicle docking, priming, and fusion triggering

Ralf Mohrmann, Heidi de Wit, Emma Connell, Paulo César da Silva Pinheiro, Charlotte Leese, Dieter Bruns, Bazbek Davletov, Matthijs Verhage, Jakob Balslev Sørensen

Neuronal Signalling

50 Citations (Scopus)

Abstract

SNARE complex assembly constitutes a key step in exocytosis that is rendered Ca(2+)-dependent by interactions with synaptotagmin-1. Two putative sites for synaptotagmin binding have recently been identified in SNAP-25 using biochemical methods: one located around the center and another at the C-terminal end of the SNARE bundle. However, it is still unclear whether and how synaptotagmin-1 × SNARE interactions at these sites are involved in regulating fast neurotransmitter release. Here, we have used electrophysiological techniques with high time-resolution to directly investigate the mechanistic ramifications of proposed SNAP-25 × synaptotagmin-1 interaction in mouse chromaffin cells. We demonstrate that the postulated central binding domain surrounding layer zero covers both SNARE motifs of SNAP-25 and is essential for vesicle docking, priming, and fast fusion-triggering. Mutation of this site caused no further functional alterations in synaptotagmin-1-deficient cells, indicating that the central acidic patch indeed constitutes a mechanistically relevant synaptotagmin-1 interaction site. Moreover, our data show that the C-terminal binding interface only plays a subsidiary role in triggering but is required for the full size of the readily releasable pool. Intriguingly, we also found that mutation of synaptotagmin-1 interaction sites led to more pronounced phenotypes in the context of the adult neuronal isoform SNAP-25B than in the embryonic isoform SNAP-25A. Further experiments demonstrated that stronger synaptotagmin-1 × SNAP-25B interactions allow for the larger primed vesicle pool supported by SNAP-25 isoform B. Thus, synaptotagmin-1 × SNARE interactions are not only required for multiple mechanistic steps en route to fusion but also underlie the developmental control of the releasable vesicle pool.

Original language	English
Journal	Journal of Neuroscience
Volume	33
Issue number	36
Pages (from-to)	14417-30
Number of pages	14
ISSN	0270-6474
DOIs	https://doi.org/10.1523/JNEUROSCI.1236-13.2013
Publication status	Published - 4 Sept 2013

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@article{b797e630e7044143b6cd30a8ad790fc1,

title = "Synaptotagmin interaction with SNAP-25 governs vesicle docking, priming, and fusion triggering",

abstract = "SNARE complex assembly constitutes a key step in exocytosis that is rendered Ca(2+)-dependent by interactions with synaptotagmin-1. Two putative sites for synaptotagmin binding have recently been identified in SNAP-25 using biochemical methods: one located around the center and another at the C-terminal end of the SNARE bundle. However, it is still unclear whether and how synaptotagmin-1 × SNARE interactions at these sites are involved in regulating fast neurotransmitter release. Here, we have used electrophysiological techniques with high time-resolution to directly investigate the mechanistic ramifications of proposed SNAP-25 × synaptotagmin-1 interaction in mouse chromaffin cells. We demonstrate that the postulated central binding domain surrounding layer zero covers both SNARE motifs of SNAP-25 and is essential for vesicle docking, priming, and fast fusion-triggering. Mutation of this site caused no further functional alterations in synaptotagmin-1-deficient cells, indicating that the central acidic patch indeed constitutes a mechanistically relevant synaptotagmin-1 interaction site. Moreover, our data show that the C-terminal binding interface only plays a subsidiary role in triggering but is required for the full size of the readily releasable pool. Intriguingly, we also found that mutation of synaptotagmin-1 interaction sites led to more pronounced phenotypes in the context of the adult neuronal isoform SNAP-25B than in the embryonic isoform SNAP-25A. Further experiments demonstrated that stronger synaptotagmin-1 × SNAP-25B interactions allow for the larger primed vesicle pool supported by SNAP-25 isoform B. Thus, synaptotagmin-1 × SNARE interactions are not only required for multiple mechanistic steps en route to fusion but also underlie the developmental control of the releasable vesicle pool.",

author = "Ralf Mohrmann and {de Wit}, Heidi and Emma Connell and {da Silva Pinheiro}, {Paulo C{\'e}sar} and Charlotte Leese and Dieter Bruns and Bazbek Davletov and Matthijs Verhage and S{\o}rensen, {Jakob Balslev}",

year = "2013",

month = sep,

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doi = "10.1523/JNEUROSCI.1236-13.2013",

language = "English",

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pages = "14417--30",

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T1 - Synaptotagmin interaction with SNAP-25 governs vesicle docking, priming, and fusion triggering

AU - Mohrmann, Ralf

AU - de Wit, Heidi

AU - Connell, Emma

AU - da Silva Pinheiro, Paulo César

AU - Leese, Charlotte

AU - Bruns, Dieter

AU - Davletov, Bazbek

AU - Verhage, Matthijs

AU - Sørensen, Jakob Balslev

PY - 2013/9/4

Y1 - 2013/9/4

N2 - SNARE complex assembly constitutes a key step in exocytosis that is rendered Ca(2+)-dependent by interactions with synaptotagmin-1. Two putative sites for synaptotagmin binding have recently been identified in SNAP-25 using biochemical methods: one located around the center and another at the C-terminal end of the SNARE bundle. However, it is still unclear whether and how synaptotagmin-1 × SNARE interactions at these sites are involved in regulating fast neurotransmitter release. Here, we have used electrophysiological techniques with high time-resolution to directly investigate the mechanistic ramifications of proposed SNAP-25 × synaptotagmin-1 interaction in mouse chromaffin cells. We demonstrate that the postulated central binding domain surrounding layer zero covers both SNARE motifs of SNAP-25 and is essential for vesicle docking, priming, and fast fusion-triggering. Mutation of this site caused no further functional alterations in synaptotagmin-1-deficient cells, indicating that the central acidic patch indeed constitutes a mechanistically relevant synaptotagmin-1 interaction site. Moreover, our data show that the C-terminal binding interface only plays a subsidiary role in triggering but is required for the full size of the readily releasable pool. Intriguingly, we also found that mutation of synaptotagmin-1 interaction sites led to more pronounced phenotypes in the context of the adult neuronal isoform SNAP-25B than in the embryonic isoform SNAP-25A. Further experiments demonstrated that stronger synaptotagmin-1 × SNAP-25B interactions allow for the larger primed vesicle pool supported by SNAP-25 isoform B. Thus, synaptotagmin-1 × SNARE interactions are not only required for multiple mechanistic steps en route to fusion but also underlie the developmental control of the releasable vesicle pool.

AB - SNARE complex assembly constitutes a key step in exocytosis that is rendered Ca(2+)-dependent by interactions with synaptotagmin-1. Two putative sites for synaptotagmin binding have recently been identified in SNAP-25 using biochemical methods: one located around the center and another at the C-terminal end of the SNARE bundle. However, it is still unclear whether and how synaptotagmin-1 × SNARE interactions at these sites are involved in regulating fast neurotransmitter release. Here, we have used electrophysiological techniques with high time-resolution to directly investigate the mechanistic ramifications of proposed SNAP-25 × synaptotagmin-1 interaction in mouse chromaffin cells. We demonstrate that the postulated central binding domain surrounding layer zero covers both SNARE motifs of SNAP-25 and is essential for vesicle docking, priming, and fast fusion-triggering. Mutation of this site caused no further functional alterations in synaptotagmin-1-deficient cells, indicating that the central acidic patch indeed constitutes a mechanistically relevant synaptotagmin-1 interaction site. Moreover, our data show that the C-terminal binding interface only plays a subsidiary role in triggering but is required for the full size of the readily releasable pool. Intriguingly, we also found that mutation of synaptotagmin-1 interaction sites led to more pronounced phenotypes in the context of the adult neuronal isoform SNAP-25B than in the embryonic isoform SNAP-25A. Further experiments demonstrated that stronger synaptotagmin-1 × SNAP-25B interactions allow for the larger primed vesicle pool supported by SNAP-25 isoform B. Thus, synaptotagmin-1 × SNARE interactions are not only required for multiple mechanistic steps en route to fusion but also underlie the developmental control of the releasable vesicle pool.

U2 - 10.1523/JNEUROSCI.1236-13.2013

DO - 10.1523/JNEUROSCI.1236-13.2013

M3 - Journal article

C2 - 24005294

SN - 0270-6474

VL - 33

SP - 14417

EP - 14430

JO - The Journal of neuroscience : the official journal of the Society for Neuroscience

JF - The Journal of neuroscience : the official journal of the Society for Neuroscience

IS - 36

ER -

Synaptotagmin interaction with SNAP-25 governs vesicle docking, priming, and fusion triggering

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