Controlling synaptotagmin activity by electrostatic screening

Park, Yong Soo; Hernandez, Javier M.; Bogaart, Geert van den; Ahmed, Saheeb; Holt, Matthew; Riedel, Dietmar; Jahn, Reinhard

doi:10.1038/nsmb.2375

Cited by 77 publications

(263 citation statements)

References 53 publications

(78 reference statements)

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“…Our data imply that the inhibitory effect of 5-IP 7 is more critical for a subset of vesicles with higher fusion activities, because the singlevesicle assay selectively monitors higher-activity vesicles (i.e., those that react within the given 30-s time window). Our observations indicate that 5-IP 7 at tens of micromolar concentrations exhibits a significant inhibitory effect in the presence of 1 mM ATP, which is also a pyrophosphate molecule (41). Therefore, it appears that the inhibitory effect of 5-IP 7 does not arise from simple electrostatic screening.…”

Section: Resultsmentioning

confidence: 64%

“…Specifically, we used 3 mol percent (mol%) phosphatidylserine (PS) lipids for the v-vesicle membrane, while adding 15 mol% PS and 1 mol% phosphatidylinositol 4,5-bisphosphate (PIP 2 ) for the t vesicle. Moreover, we included 1 mM Mg 2+ ions and 1 mM ATP in the fusion reaction buffer to mimic the physiological concentrations of divalent ions and multivalent pyrophosphate molecules (41,44). Under these conditions, fusion kinetics, measured by the mixing of lipid dyes that constitute a FRET donor and acceptor pair [1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate (DiD)], was significantly accelerated at a Ca 2+ concentration of 100 μM (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…To dissect the effect of 5-IP 7 in the regulation of synaptic vesicle exocytosis, we used an in vitro vesicle fusion system that allowed monitoring of SNARE-and Syt1-mediated membrane fusion down to the single-vesicle level (34)(35)(36)(37)(38)(39)(40)(41) (Fig. 2).…”

Section: Resultsmentioning

confidence: 99%

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Inositol pyrophosphates inhibit synaptotagmin-dependent exocytosis

Lee

Kyung

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Inositol pyrophosphates such as 5-diphosphoinositol pentakisphosphate (5-IP7) are highly energetic inositol metabolites containing phosphoanhydride bonds. Although inositol pyrophosphates are known to regulate various biological events, including growth, survival, and metabolism, the molecular sites of 5-IP7 action in vesicle trafficking have remained largely elusive. We report here that elevated 5-IP7 levels, caused by overexpression of inositol hexakisphosphate (IP6) kinase 1 (IP6K1), suppressed depolarization-induced neurotransmitter release from PC12 cells. Conversely, IP6K1 depletion decreased intracellular 5-IP7 concentrations, leading to increased neurotransmitter release. Consistently, knockdown of IP6K1 in cultured hippocampal neurons augmented action potential-driven synaptic vesicle exocytosis at synapses. Using a FRET-based in vitro vesicle fusion assay, we found that 5-IP7, but not 1-IP7, exhibited significantly higher inhibitory activity toward synaptic vesicle exocytosis than IP6. Synaptotagmin 1 (Syt1), a Ca2+ sensor essential for synaptic membrane fusion, was identified as a molecular target of 5-IP7. Notably, 5-IP7 showed a 45-fold higher binding affinity for Syt1 compared with IP6. In addition, 5-IP7–dependent inhibition of synaptic vesicle fusion was abolished by increasing Ca2+ levels. Thus, 5-IP7 appears to act through Syt1 binding to interfere with the fusogenic activity of Ca2+. These findings reveal a role of 5-IP7 as a potent inhibitor of Syt1 in controlling the synaptic exocytotic pathway and expand our understanding of the signaling mechanisms of inositol pyrophosphates.

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Section: Resultsmentioning

confidence: 64%

Section: Resultsmentioning

confidence: 99%

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Inositol pyrophosphates inhibit synaptotagmin-dependent exocytosis

Lee

Kyung

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Though EPR data agreed with this model of membrane bridging (24), and increasing evidence supports the notion that the membrane-bridging activity of synaptotagmin-1 is critical for its function (7,(25)(26)(27)(28)(29)(30)(31), a recent study concluded that the bridging occurs by a fundamentally different mechanism that requires trans interactions between synaptotagmin-1 oligomers bound to each membrane, without binding of the bottom of the C 2 B domain to the lipids (29). This mechanism, which we refer to as the oligomerization model, is compared in Fig.…”

mentioning

confidence: 77%

Prevalent mechanism of membrane bridging by synaptotagmin-1

Seven¹,

Brewer²,

Shi

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Synaptotagmin-1 functions as a Ca 2+ sensor in neurotransmitter release through its two C 2 domains (the C 2 A and C 2 B domain). The ability of synaptotagmin-1 to bridge two membranes is likely crucial for its function, enabling cooperation with the soluble Nethylmaleimide sensitive factor adaptor protein receptors (SNAREs) in membrane fusion, but two bridging mechanisms have been proposed. A highly soluble synaptotagmin-1 fragment containing both domains (C 2 AB) was shown to bind simultaneously to two membranes via the Ca 2+ -binding loops at the top of both domains and basic residues at the bottom of the C 2 B domain (direct bridging mechanism). In contrast, a longer fragment including a linker sequence (lnC 2 AB) was found to aggregate in solution and was proposed to bridge membranes through trans interactions between lnC 2 AB oligomers bound to each membrane via the Ca 2+ -binding loops, with no contact of the bottom of the C 2 B domain with the membranes. We now show that lnC 2 AB containing impurities indeed aggregates in solution, but properly purified lnC 2 AB is highly soluble. Moreover, cryo-EM images reveal that a majority of lnC 2 AB molecules bridge membranes directly. Fluorescence spectroscopy indicates that the bottom of the C 2 B domain contacts the membrane in a sizeable population of molecules of both membranebound C 2 AB and membrane-bound lnC 2 AB. NMR data on nanodiscs show that a fraction of C 2 AB molecules bind to membranes with antiparallel orientations of the C 2 domains. Together with previous studies, these results show that direct bridging constitutes the prevalent mechanism of membrane bridging by both C 2 AB and lnC 2 AB, suggesting that this mechanism underlies the function of synaptotagmin-1 in neurotransmitter release.membrane bending | Ca2+ sensing | exocytosis | synaptic transmission N eurotransmitter release is a key event in interneuronal communication that is acutely triggered by Ca 2+ influx into a presynaptic terminal (1). The synaptic vesicle protein synaptotagmin-1 acts as a Ca 2+ sensor in fast release through the two C 2 domains that form most of its cytoplasmic region (the C 2 A and C 2 B domains) (2-5). This function is coupled to membrane fusion through the neuronal soluble N-ethylmaleimide sensitive factor adaptor protein receptor (SNARE) proteins (6, 7), which bring the synaptic vesicle and plasma membranes together by forming SNARE complexes (2, 3). Synaptotagmin-1 function also depends on a tight interplay with complexins (8-10) and other key proteins of the release machinery (11, 12). The synaptotagmin-1 C 2 domains bind three or two Ca 2+ ions through loops at the top of β-sandwich structures (13-15) (Fig. 1A). These top loops also mediate Ca 2+ -dependent phospholipid binding (15-17), which is crucial for synaptotagmin-1 function (5, 18). Moreover, the synaptotagmin-1 cytoplasmic region clusters chromaffin granules and liposomes (19), and a fragment containing its two C 2 domains was shown by cryo-EM to bind simultaneously to two membranes, bringing them i...

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“…The clustering of C2 domains would act to localize this effect, perhaps enhancing curvature changes. The ability of Syt1 to promote curvature changes may in part be due to its ability to demix PS (54) and interact with lipids such as PIP 2 (52,55). By interacting with these charged lipids, Syt1 oligomerization would also influence the local composition of the bilayer, as well as the SNAREs, which have been observed to bind PIP 2 (56 -58).…”

Section: Discussionmentioning

confidence: 99%

The Juxtamembrane Linker of Full-length Synaptotagmin 1 Controls Oligomerization and Calcium-dependent Membrane Binding

Lü

Kiessling

Tamm

et al. 2014

Journal of Biological Chemistry

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Background: Synaptotagmin 1 is the Ca 2ϩ sensor for neuronal exocytosis. Results: The juxtamembrane linker of synaptotagmin 1 is oligomerized through a glycine zipper motif, and this interaction controls the activity of synaptotagmin 1. Conclusion: Structural modifications in the linker alter the activity of synaptotagmin 1. Significance: The oligomerization of synaptotagmin 1 may control the organization at the focal site of fusion.

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Controlling synaptotagmin activity by electrostatic screening

Cited by 77 publications

References 53 publications

Inositol pyrophosphates inhibit synaptotagmin-dependent exocytosis

Inositol pyrophosphates inhibit synaptotagmin-dependent exocytosis

Prevalent mechanism of membrane bridging by synaptotagmin-1

The Juxtamembrane Linker of Full-length Synaptotagmin 1 Controls Oligomerization and Calcium-dependent Membrane Binding

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