scholarly journals Membrane bridging by Munc13-1 is crucial for neurotransmitter release

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Bradley Quade ◽  
Marcial Camacho ◽  
Xiaowei Zhao ◽  
Marta Orlando ◽  
Thorsten Trimbuch ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Neurotransmitter Release ◽  
Plasma Membranes ◽  
Central Function ◽  
Vesicle Docking ◽  
Physiological Relevance ◽  
Residue Substitution ◽  
Liposome Fusion ◽  
Membrane Binding Sites

Munc13-1 plays a crucial role in neurotransmitter release. We recently proposed that the C-terminal region encompassing the C1, C2B, MUN and C2C domains of Munc13-1 (C1C2BMUNC2C) bridges the synaptic vesicle and plasma membranes through interactions involving the C2C domain and the C1-C2B region. However, the physiological relevance of this model has not been demonstrated. Here we show that C1C2BMUNC2C bridges membranes through opposite ends of its elongated structure. Mutations in putative membrane-binding sites of the C2C domain disrupt the ability of C1C2BMUNC2C to bridge liposomes and to mediate liposome fusion in vitro. These mutations lead to corresponding disruptive effects on synaptic vesicle docking, priming, and Ca2+-triggered neurotransmitter release in mouse neurons. Remarkably, these effects include an almost complete abrogation of release by a single residue substitution in this 200 kDa protein. These results show that bridging the synaptic vesicle and plasma membranes is a central function of Munc13-1.

Sodium affinity of brain Na(+)-K(+)-ATPase is dependent on isozyme and environment of the pump

1990 ◽  
Vol 258 (5) ◽  
pp. C803-C811 ◽  
Author(s):  
J. L. Brodsky ◽  
G. Guidotti
Keyword(s):  
Plasma Membranes ◽  
Fibroblast Cell ◽  
Mouse Fibroblast ◽  
Synaptic Plasma Membranes ◽  
Apparent Dissociation Constant ◽  
Low Sodium ◽  
Physiological Relevance ◽  
Mouse Fibroblast Cell Line

The sodium affinities for the two forms of the Na(+)-K(+)-ATPase in brain were characterized. To mimic physiological conditions, synaptosomes, which are pinched off presynaptic nerve termini, were used. Examination of the pump in vitro was performed by preparing synaptic plasma membranes (SPMs). It was first shown that synaptosomes contain the two forms of the Na(+)-K(+)-ATPase, alpha 1 and alpha 2, and that these forms have markedly different affinities for the inhibitory cardiac glycoside ouabain. The apparent dissociation constant (K0.5) of alpha 1 for sodium changed from 12 to 9 mM when going from synaptosomes to membranes. For alpha 2, however, a shift from 36 to 12.5 mM was evident. The conclusion is that in vivo alpha 2 exists as a low sodium affinity species but can be altered to a high-affinity form simply by vesicle disruption. By comparison, the Na(+)-K(+)-ATPase from the mouse fibroblast cell line, 3T3-F442A cells, expressed only the alpha 1-isozyme, as shown by immunoblotting and by measurement of its ouabain and sodium affinities. The physiological relevance of these observations is also presented.

scholarly journals Reconstituted synaptotagmin I mediates vesicle docking, priming, and fusion

2011 ◽  
Vol 195 (7) ◽  
pp. 1159-1170 ◽  
Author(s):  
Zhao Wang ◽  
Huisheng Liu ◽  
Yiwen Gu ◽  
Edwin R. Chapman
Keyword(s):  
Synaptic Transmission ◽  
Membrane Fusion ◽  
Synaptic Vesicle ◽  
Cytoplasmic Domain ◽  
Snare Proteins ◽  
Antagonist Activity ◽  
Vesicle Docking ◽  
Synaptotagmin I ◽  
Target Membrane

The synaptic vesicle protein synaptotagmin I (syt) promotes exocytosis via its ability to penetrate membranes in response to binding Ca2+ and through direct interactions with SNARE proteins. However, studies using full-length (FL) membrane-embedded syt in reconstituted fusion assays have yielded conflicting results, including a lack of effect, or even inhibition of fusion, by Ca2+. In this paper, we show that reconstituted FL syt promoted rapid docking of vesicles (<1 min) followed by a priming step (3–9 min) that was required for subsequent Ca2+-triggered fusion between v- and t-SNARE liposomes. Moreover, fusion occurred only when phosphatidylinositol 4,5-bisphosphate was included in the target membrane. This system also recapitulates some of the effects of syt mutations that alter synaptic transmission in neurons. Finally, we demonstrate that the cytoplasmic domain of syt exhibited mixed agonist/antagonist activity during regulated membrane fusion in vitro and in cells. Together, these findings reveal further convergence of reconstituted and cell-based systems.

scholarly journals Inositol pyrophosphates inhibit synaptotagmin-dependent exocytosis

2016 ◽  
Vol 113 (29) ◽  
pp. 8314-8319 ◽  
Author(s):  
Tae-Sun Lee ◽  
Joo-Young Lee ◽  
Jae Won Kyung ◽  
Yoosoo Yang ◽  
Seung Ju Park ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Neurotransmitter Release ◽  
Hippocampal Neurons ◽  
Vesicle Fusion ◽  
Synaptic Membrane ◽  
Synaptic Vesicle Exocytosis ◽  
Dependent Inhibition ◽  
Vesicle Exocytosis ◽  
Inositol Pyrophosphates

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.

scholarly journals Ring-like oligomers of Synaptotagmins and related C2 domain proteins

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Maria N Zanetti ◽  
Oscar D Bello ◽  
Jing Wang ◽  
Jeff Coleman ◽  
Yiying Cai ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Synaptic Transmission ◽  
Synaptic Vesicle ◽  
Neurotransmitter Release ◽  
Structural Aspect ◽  
C2 Domain ◽  
Early Step ◽  
Vesicle Docking ◽  
Ring Assembly ◽  
Synaptotagmin 1

We recently reported that the C2AB portion of Synaptotagmin 1 (Syt1) could self-assemble into Ca2+-sensitive ring-like oligomers on membranes, which could potentially regulate neurotransmitter release. Here we report that analogous ring-like oligomers assemble from the C2AB domains of other Syt isoforms (Syt2, Syt7, Syt9) as well as related C2 domain containing protein, Doc2B and extended Synaptotagmins (E-Syts). Evidently, circular oligomerization is a general and conserved structural aspect of many C2 domain proteins, including Synaptotagmins. Further, using electron microscopy combined with targeted mutations, we show that under physiologically relevant conditions, both the Syt1 ring assembly and its rapid disruption by Ca2+ involve the well-established functional surfaces on the C2B domain that are important for synaptic transmission. Our data suggests that ring formation may be triggered at an early step in synaptic vesicle docking and positions Syt1 to synchronize neurotransmitter release to Ca2+ influx.

scholarly journals Synaptic Vesicle Protein 2 (SV2) does not hydrolyze ATP

F1000Research ◽  
2013 ◽  
Vol 2 ◽  
pp. 209
Author(s):  
Jia Yao ◽  
Sandra M. Bajjalieh
Keyword(s):  
Atpase Activity ◽  
Synaptic Vesicle ◽  
Neurotransmitter Release ◽  
Endocrine Cells ◽  
Essential Role ◽  
Adenine Nucleotides ◽  
Regulated Secretion ◽  
Synaptic Vesicle Protein ◽  
Vesicle Protein

Synaptic vesicle protein 2 (SV2) is a transporter-like protein specifically expressed in endocrine cells and neurons, where it is localized to vesicles that undergo regulated secretion and plays an essential role in regulating neurotransmitter release. SV2 binds adenine nucleotides including ATP. Analysis of ATP transport revealed that SV2 is not an ATP transporter, nor does it affect ATP transport. As a further step toward understanding how ATP binding contributes to SV2 function, we investigated whether SV2 is an ATPase using an in vitro measure of ATPase activity. The study reported here indicates that SV2 does not have ATPase activity. Thus, binding to adenine nucleotides likely modulates other actions of SV2.

A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion

Cell ◽  
1993 ◽  
Vol 75 (3) ◽  
pp. 409-418 ◽  
Author(s):  
Thomas Söllner ◽  
Mark K. Bennett ◽  
Sidney W. Whiteheart ◽  
Richard H. Scheller ◽  
James E. Rothman
Keyword(s):  
Synaptic Vesicle ◽  
Protein Assembly ◽  
Vesicle Docking

scholarly journals A novel region in the CaV2.1 α1 subunit C-terminus regulates fast synaptic vesicle fusion and vesicle docking at the mammalian presynaptic active zone

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Matthias Lübbert ◽  
R Oliver Goral ◽  
Rachel Satterfield ◽  
Travis Putzke ◽  
Arn MJM van den Maagdenberg ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Active Zone ◽  
Neurotransmitter Release ◽  
Physical Distance ◽  
Subunit C ◽  
Vesicle Docking ◽  
C Terminus ◽  
Synaptic Vesicle Release ◽  
Synaptic Vesicle Fusion ◽  
Α1 Subunit

In central nervous system (CNS) synapses, action potential-evoked neurotransmitter release is principally mediated by CaV2.1 calcium channels (CaV2.1) and is highly dependent on the physical distance between CaV2.1 and synaptic vesicles (coupling). Although various active zone proteins are proposed to control coupling and abundance of CaV2.1 through direct interactions with the CaV2.1 α1 subunit C-terminus at the active zone, the role of these interaction partners is controversial. To define the intrinsic motifs that regulate coupling, we expressed mutant CaV2.1 α1 subunits on a CaV2.1 null background at the calyx of Held presynaptic terminal. Our results identified a region that directly controlled fast synaptic vesicle release and vesicle docking at the active zone independent of CaV2.1 abundance. In addition, proposed individual direct interactions with active zone proteins are insufficient for CaV2.1 abundance and coupling. Therefore, our work advances our molecular understanding of CaV2.1 regulation of neurotransmitter release in mammalian CNS synapses.

scholarly journals Multiple factors maintain assembled trans-SNARE complexes in the presence of NSF and αSNAP

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Eric A Prinslow ◽  
Karolina P Stepien ◽  
Yun-Zu Pan ◽  
Junjie Xu ◽  
Josep Rizo
Keyword(s):  
Complex Formation ◽  
Synaptic Vesicle ◽  
Neurotransmitter Release ◽  
Plasma Membranes ◽  
Snare Complex ◽  
Complex Assembly ◽  
Multiple Factors ◽  
Synaptotagmin 1 ◽  
Snare Complex Formation

Neurotransmitter release requires formation of trans-SNARE complexes between the synaptic vesicle and plasma membranes, which likely underlies synaptic vesicle priming to a release-ready state. It is unknown whether Munc18-1, Munc13-1, complexin-1 and synaptotagmin-1 are important for priming because they mediate trans-SNARE complex assembly and/or because they prevent trans-SNARE complex disassembly by NSF-αSNAP, which can lead to de-priming. Here we show that trans-SNARE complex formation in the presence of NSF-αSNAP requires both Munc18-1 and Munc13-1, as proposed previously, and is facilitated by synaptotagmin-1. Our data also show that Munc18-1, Munc13-1, complexin-1 and likely synaptotagmin-1 contribute to maintaining assembled trans-SNARE complexes in the presence of NSF-αSNAP. We propose a model whereby Munc18-1 and Munc13-1 are critical not only for mediating vesicle priming but also for precluding de-priming by preventing trans-SNARE complex disassembly; in this model, complexin-1 also impairs de-priming, while synaptotagmin-1 may assist in priming and hinder de-priming.

scholarly journals Synaptotagmin rings as high sensitivity regulators of synaptic vesicle docking and fusion

2021 ◽  
Author(s):  
Jie Zhu ◽  
Zachary A McDargh ◽  
Feng Li ◽  
Shyam Krishnakumar ◽  
James E Rothman ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Plasma Membranes ◽  
Calcium Influx ◽  
High Sensitivity ◽  
Bulk Solution ◽  
Membrane Composition ◽  
Ring Conformation ◽  
Anionic Phospholipid ◽  
Vesicle Docking ◽  
Phospholipid Monolayers

Synchronous release at neuronal synapses is accomplished by a machinery that senses calcium influx and fuses the synaptic vesicle and plasma membranes to release neurotransmitters. Previous studies suggested the calcium sensor Synaptotagmin (Syt) is a facilitator of vesicle docking and both a facilitator and inhibitor of fusion. On phospholipid monolayers, the Syt C2AB domain spontaneously oligomerized into rings that are disassembled by Ca2+, suggesting Syt rings may clamp fusion as membrane-separating "washers" until Ca2+-mediated disassembly triggers fusion and release (Wang et al., 2014). Here we combined mathematical modeling with experiment to measure mechanical properties of Syt rings and to test this mechanism. Consistent with experiment, the model quantitatively recapitulates observed Syt ring-induced dome and volcano shapes on phospholipid monolayers, and predicts rings are stabilized by anionic phospholipid bilayers or bulk solution with ATP. The selected ring conformation is highly sensitive to membrane composition and bulk ATP levels, a property that may regulate vesicle docking and fusion in ATP-rich synaptic terminals. We find the Syt molecules hosted by a synaptic vesicle oligomerize into a halo, unbound from the vesicle, but in proximity to sufficiently PIP2-rich plasma membrane (PM) domains the PM-bound trans Syt ring conformation is preferred. Thus, the Syt halo serves as landing gear for spatially directed docking at PIP2-rich sites that define the active zones of exocytotic release, positioning the Syt ring to clamp fusion and await calcium. Our results suggest the Syt ring is both a Ca2+-sensitive fusion clamp and a high-fidelity sensor for directed docking.

scholarly journals Reconstructing essential active zone functions within a synapse

2021 ◽  
Author(s):  
Chao Tan ◽  
Shan Shan H Wang ◽  
Giovanni de Nola ◽  
Pascal S Kaeser
Keyword(s):  
Fusion Protein ◽  
Synaptic Vesicle ◽  
Active Zone ◽  
Neurotransmitter Release ◽  
Molecular Machines ◽  
Vesicle Docking ◽  
Active Zones ◽  
Ca2 Channels

Active zones are molecular machines that control neurotransmitter release through synaptic vesicle docking and priming, and through coupling of these vesicles to Ca2+ entry. The complexity of active zone machinery has made it challenging to determine which mechanisms drive these roles in release. Here, we induce RIM+ELKS knockout to eliminate active zone scaffolding networks, and then reconstruct each active zone function. Re-expression of RIM1-Zn fingers positioned Munc13 on undocked vesicles and rendered them release-competent. Reconstitution of release-triggering required docking of these vesicles to Ca2+ channels. Fusing RIM1-Zn to CaVbeta4-subunits sufficed to restore docking, priming and release-triggering without reinstating active zone scaffolds. Hence, exocytotic activities of the 80 kDa CaVbeta4-Zn fusion protein bypassed the need for megadalton-sized secretory machines. These data define key mechanisms of active zone function, establish that fusion competence and docking are mechanistically separable, and reveal that active zone scaffolding networks are not required for release.

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