Ca2+–Calmodulin regulates SNARE assembly and spontaneous neurotransmitter release via v-ATPase subunit V0a1

Most chemical neurotransmission occurs through Ca²⁺-dependent evoked or spontaneous vesicle exocytosis. In both cases, Ca²⁺ sensing is thought to occur shortly before exocytosis. In this paper, we provide evidence that the Ca²⁺ dependence of spontaneous vesicle release may partly result from an earlier requirement of Ca²⁺ for the assembly of soluble N-ethylmaleimide–sensitive fusion attachment protein receptor (SNARE) complexes. We show that the neuronal vacuolar-type H⁺-adenosine triphosphatase V0 subunit a1 (V100) can regulate the formation of SNARE complexes in a Ca²⁺–Calmodulin (CaM)-dependent manner. Ca²⁺–CaM regulation of V100 is not required for vesicle acidification. Specific disruption of the Ca²⁺-dependent regulation of V100 by CaM led to a >90% loss of spontaneous release but only had a mild effect on evoked release at Drosophila melanogaster embryo neuromuscular junctions. Our data suggest that Ca²⁺–CaM regulation of V100 may control SNARE complex assembly for a subset of synaptic vesicles that sustain spontaneous release.     

Munc18-1 binding to the neuronal SNARE complex controls synaptic vesicle priming

Munc18-1 and soluble NSF attachment protein receptors (SNAREs) are critical for synaptic vesicle fusion. Munc18-1 binds to the SNARE syntaxin-1 folded into a closed conformation and to SNARE complexes containing open syntaxin-1. Understanding which steps in fusion depend on the latter interaction and whether Munc18-1 competes with other factors such as complexins for SNARE complex binding is critical to elucidate the mechanisms involved. In this study, we show that lentiviral expression of Munc18-1 rescues abrogation of release in Munc18-1 knockout mice. We describe point mutations in Munc18-1 that preserve tight binding to closed syntaxin-1 but markedly disrupt Munc18-1 binding to SNARE complexes containing open syntaxin-1. Lentiviral rescue experiments reveal that such disruption selectively impairs synaptic vesicle priming but not Ca²⁺-triggered fusion of primed vesicles. We also find that Munc18-1 and complexin-1 bind simultaneously to SNARE complexes. These results suggest that Munc18-1 binding to SNARE complexes mediates synaptic vesicle priming and that the resulting primed state involves a Munc18-1–SNARE–complexin macromolecular assembly that is poised for Ca²⁺ triggering of fusion.     

Complexin 2 Modulates Vesicle-associated Membrane Protein (VAMP) 2-regulated Zymogen Granule Exocytosis in Pancreatic Acini

Complexins are soluble proteins that regulate the activity of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes necessary for vesicle fusion. Neuronal specific complexin 1 has inhibitory and stimulatory effects on exocytosis by clamping trans-SNARE complexes in a prefusion state and promoting conformational changes to facilitate membrane fusion following cell stimulation. Complexins are unable to bind to monomeric SNARE proteins but bind with high affinity to ternary SNARE complexes and with lower affinity to target SNARE complexes. Far less is understood about complexin function outside the nervous system. Pancreatic acini express the complexin 2 isoform by RT-PCR and immunoblotting. Immunofluorescence microscopy revealed complexin 2 localized along the apical plasma membrane consistent with a role in secretion. Accordingly, complexin 2 was found to interact with vesicle-associated membrane protein (VAMP) 2, syntaxins 3 and 4, but not with VAMP 8 or syntaxin 2. Introduction of recombinant complexin 2 into permeabilized acini inhibited Ca²⁺-stimulated secretion in a concentration-dependent manner with a maximal inhibition of nearly 50%. Mutations of the central α-helical domain reduced complexin 2 SNARE binding and concurrently abolished its inhibitory activity. Surprisingly, mutation of arginine 59 to histidine within the central α-helical domain did not alter SNARE binding and moreover, augmented Ca²⁺-stimulated secretion by 130% of control. Consistent with biochemical studies, complexin 2 colocalized with VAMP 2 along the apical plasma membrane following cholecystokinin-8 stimulation. These data demonstrate a functional role for complexin 2 outside the nervous system and indicate that it participates in the Ca²⁺-sensitive regulatory pathway for zymogen granule exocytosis.     

Vesicle dynamics: how synaptic proteins regulate different modes of neurotransmission

Central synapses operate neurotransmission in several modes: synchronous/fast neurotransmission (neurotransmitters release is tightly coupled to action potentials and fast), asynchronous neurotransmission (neurotransmitter release is slower and longer lasting), and spontaneous neurotransmission (where small amounts of neurotransmitter are released without being evoked by an action potential). A substantial body of evidence from the past two decades suggests that seemingly identical synaptic vesicles possess distinct propensities to fuse, thus selectively serving different modes of neurotransmission. In efforts to better understand the mechanism(s) underlying the different modes of synaptic transmission, many research groups found that synaptic vesicles used in different modes of neurotransmission differ by a number of synaptic proteins. Synchronous transmission with higher temporal fidelity to stimulation seems to require synaptotagmin 1 and complexin for its Ca²⁺ sensitivity, RIM proteins for closer location of synaptic vesicles (SV) to the voltage operated calcium channels (VGCC), and dynamin for SV retrieval. Asynchronous release does not seem to require functional synaptotagmin 1 as a calcium sensor or complexins, but the activity of dynamin is indispensible for its maintenance. On the other hand, the control of spontaneous neurotransmission remains less clear as deleting a number of essential synaptic proteins does not abolish this type of synaptic vesicle fusion. VGCC distance from the SV seems to have little control on spontaneous transmission, while there is an involvement of functional synaptic proteins including synaptotagmins and complexin. Recently, presynaptic deficits have been proposed to contribute to a number of pathological conditions including cognitive and mental disorders. In this review, we evaluate recent advances in understanding the regulatory mechanisms of synaptic vesicle dynamics and in understanding how different molecular substrates maintain selective modes of neurotransmission. We also highlight the implications of these studies in understanding pathological conditions.     

Extracellular Caper se inhibits quantal size of catecholamine release in adrenal slice chromaffin cells

Classic calcium hypothesis states that depolarization-induced increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) triggers vesicle exocytosis by increasing vesicle release probability in neurons and neuroendocrine cells. The extracellular Ca²⁺, in this calcium hypothesis, serves as a reservoir of Ca²⁺ source. Recently we find that extracellular Ca²⁺per se inhibits the [Ca²⁺]_i dependent vesicle exocytosis, but it remains unclear whether quantal size is regulated by extracellular, or intracellular Ca²⁺ or both [1]. In this work we showed that, in physiological condition, extracellular Ca²⁺per se specifically inhibited the quantal size of single vesicle release in rat adrenal slice chromaffin cells. The extracellular Ca²⁺ in physiological concentration (2.5 mM) directly regulated fusion pore kinetics of spontaneous quantal release of catecholamine. In addition, removal of extracellular Ca²⁺ directly triggered vesicle exocytosis without eliciting intracellular Ca²⁺. We propose that intracellular Ca²⁺ and extracellular Ca²⁺per se cooperately regulate single vesicle exocytosis. The vesicle release probability was jointly modulated by both intracellular and extracellular Ca²⁺, while the vesicle quantal size was mainly determined by extracellular Ca²⁺ in chromaffin cells physiologically.     

A complexin/synaptotagmin 1 switch controls fast synaptic vesicle exocytosis

Ca(2+) binding to synaptotagmin 1 triggers fast exocytosis of synaptic vesicles that have been primed for release by SNARE-complex assembly. Besides synaptotagmin 1, fast Ca(2+)-triggered exocytosis requires complexins. Synaptotagmin 1 and complexins both bind to assembled SNARE complexes, but it is unclear how their functions are coupled. Here we propose that complexin binding activates SNARE complexes into a metastable state and that Ca(2+) binding to synaptotagmin 1 triggers fast exocytosis by displacing complexin from metastable SNARE complexes. Specifically, we demonstrate that, biochemically, synaptotagmin 1 competes with complexin for SNARE-complex binding, thereby dislodging complexin from SNARE complexes in a Ca(2+)-dependent manner. Physiologically, increasing the local concentration of complexin selectively impairs fast Ca(2+)-triggered exocytosis but retains other forms of SNARE-dependent fusion. The hypothesis that Ca(2+)-induced displacement of complexins from SNARE complexes triggers fast exocytosis accounts for the loss-of-function and gain-of-function phenotypes of complexins and provides a molecular explanation for the high speed and synchronicity of fast Ca(2+)-triggered neurotransmitter release.     

Ca-regulated secretory granule exocytosis in pancreatic and parotid acinar cells

Protein secretion from acinar cells of the pancreas and parotid glands is controlled by G-protein coupled receptor activation and generation of the cellular messengers Ca²⁺, diacylglycerol and cAMP. Secretory granule (SG) exocytosis shares some common characteristics with nerve, neuroendocrine and endocrine cells which are regulated mainly by elevated cell Ca²⁺. However, in addition to diverse signaling pathways, acinar cells have large ∼1 μm diameter SGs (∼30 fold larger diameter than synaptic vesicles), respond to stimulation at slower rates (seconds versus milliseconds), demonstrate significant constitutive secretion, and in isolated acini, undergo sequential compound SG–SG exocytosis at the apical membrane. Exocytosis proceeds as an initial rapid phase that peaks and declines over 3 min followed by a prolonged phase that decays to near basal levels over 20–30 min. Studies indicate the early phase is triggered by Ca²⁺ and involves the SG proteins VAMP2 (vesicle associated membrane protein2), Ca²⁺-sensing protein synatotagmin 1 (syt1) and the accessory protein complexin 2. The molecular details for regulation of VAMP8-mediated SG exocytosis and the prolonged phase of secretion are still emerging. Here we review the known regulatory molecules that impact the sequential exocytic process of SG tethering, docking, priming and fusion in acinar cells.     

Lipid-anchored Synaptobrevin Provides Little or No Support for Exocytosis or Liposome Fusion

SNARE proteins catalyze many forms of biological membrane fusion, including Ca²⁺-triggered exocytosis. Although fusion mediated by SNAREs generally involves proteins anchored to each fusing membrane by a transmembrane domain (TMD), the role of TMDs remains unclear, and previous studies diverge on whether SNAREs can drive fusion without a TMD. This issue is important because it relates to the question of the structure and composition of the initial fusion pore, as well as the question of whether SNAREs mediate fusion solely by creating close proximity between two membranes versus a more active role in transmitting force to the membrane to deform and reorganize lipid bilayer structure. To test the role of membrane attachment, we generated four variants of the synaptic v-SNARE synaptobrevin-2 (syb2) anchored to the membrane by lipid instead of protein. These constructs were tested for functional efficacy in three different systems as follows: Ca²⁺-triggered dense core vesicle exocytosis, spontaneous synaptic vesicle exocytosis, and Ca²⁺-synaptotagmin-enhanced SNARE-mediated liposome fusion. Lipid-anchoring motifs harboring one or two lipid acylation sites completely failed to support fusion in any of these assays. Only the lipid-anchoring motif from cysteine string protein-α, which harbors many lipid acylation sites, provided support for fusion but at levels well below that achieved with wild type syb2. Thus, lipid-anchored syb2 provides little or no support for exocytosis, and anchoring syb2 to a membrane by a TMD greatly improves its function. The low activity seen with syb2-cysteine string protein-α may reflect a slower alternative mode of SNARE-mediated membrane fusion.     

Etomidate evokes synaptic vesicle exocytosis without increasing miniature endplate potentials frequency at the mice neuromuscular junction

Etomidate is an intravenous anesthetic used during anesthesia induction. This agent induces spontaneous movements, especially myoclonus after its administration suggesting a putative primary effect at the central nervous system or the periphery. Therefore, the aim of this study was to investigate the presynaptic and postsynaptic effects of etomidate at the mouse neuromuscular junction (NMJ). Diaphragm nerve muscle preparations were isolated and stained with the styryl dye FM1-43, a fluorescent tool that tracks synaptic vesicles exo-endocytosis that are key steps for neurotransmission. We observed that etomidate induced synaptic vesicle exocytosis in a dose-dependent fashion, an effect that was independent of voltage-gated Na⁺ channels. By contrast, etomidate-evoked exocytosis was dependent on extracellular Ca²⁺ because its effect was abolished in Ca²⁺-free medium and also inhibited by omega-Agatoxin IVA (30 and 200 nM) suggesting the participation of P/Q-subtype Ca²⁺ channels. Interestingly, even though etomidate induced synaptic vesicle exocytosis, we did not observe any significant difference in the frequency and amplitude of miniature end-plate potentials (MEPPs) in the presence of the anesthetic. We therefore investigated whether etomidate could act on nicotinic acetylcholine receptors labeled with α-bungarotoxin-Alexa 594 and we observed less fluorescence in preparations exposed to the anesthetic. In conclusion, our results suggest that etomidate exerts a presynaptic effect at the NMJ inducing synaptic vesicle exocytosis, likely through the activation of P-subtype voltage gated Ca²⁺ channels without interfering with MEPPs frequency. The present data contribute to a better understanding about the effect of etomidate at the neuromuscular synapse and may help to explain some clinical effects of this agent.     

Calcium control of neurotransmitter release

Upon entering a presynaptic terminal, an action potential opens Ca(2+) channels, and transiently increases the local Ca(2+) concentration at the presynaptic active zone. Ca(2+) then triggers neurotransmitter release within a few hundred microseconds by activating synaptotagmins Ca(2+). Synaptotagmins bind Ca(2+) via two C2-domains, and transduce the Ca(2+) signal into a nanomechanical activation of the membrane fusion machinery; this activation is mediated by the Ca(2+)-dependent interaction of the synaptotagmin C2-domains with phospholipids and SNARE proteins. In triggering exocytosis, synaptotagmins do not act alone, but require an obligatory cofactor called complexin, a small protein that binds to SNARE complexes and simultaneously activates and clamps the SNARE complexes, thereby positioning the SNARE complexes for subsequent synaptotagmin action. The conserved function of synaptotagmins and complexins operates generally in most, if not all, Ca(2+)-regulated forms of exocytosis throughout the body in addition to synaptic vesicle exocytosis, including in the degranulation of mast cells, acrosome exocytosis in sperm cells, hormone secretion from endocrine cells, and neuropeptide release.     

How to make a synaptic ribbon: RIBEYE deletion abolishes ribbons in retinal synapses and disrupts neurotransmitter release

Synaptic ribbons are large proteinaceous scaffolds at the active zone of ribbon synapses that are specialized for rapid sustained synaptic vesicles exocytosis. A single ribbon‐specific protein is known, RIBEYE, suggesting that ribbons may be constructed from RIBEYE protein. RIBEYE knockdown in zebrafish, however, only reduced but did not eliminate ribbons, indicating a more ancillary role. Here, we show in mice that full deletion of RIBEYE abolishes all presynaptic ribbons in retina synapses. Using paired recordings in acute retina slices, we demonstrate that deletion of RIBEYE severely impaired fast and sustained neurotransmitter release at bipolar neuron/AII amacrine cell synapses and rendered spontaneous miniature release sensitive to the slow Ca²⁺‐buffer EGTA, suggesting that synaptic ribbons mediate nano‐domain coupling of Ca²⁺ channels to synaptic vesicle exocytosis. Our results show that RIBEYE is essential for synaptic ribbons as such, and may organize presynaptic nano‐domains that position release‐ready synaptic vesicles adjacent to Ca²⁺ channels.     

Point Mutation in Syntaxin-1A Causes Abnormal Vesicle Recycling,Behaviors, and Short Term Plasticity

Syntaxin-1A is a t-SNARE that is involved in vesicle docking and vesicle fusion; it is important in presynaptic exocytosis in neurons because it interacts with many regulatory proteins. Previously, we found the following: 1) that autophosphorylated Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), an important modulator of neural plasticity, interacts with syntaxin-1A to regulate exocytosis, and 2) that a syntaxin missense mutation (R151G) attenuated this interaction. To determine more precisely the physiological importance of this interaction between CaMKII and syntaxin, we generated mice with a knock-in (KI) syntaxin-1A (R151G) mutation. Complexin is a molecular clamp involved in exocytosis, and in the KI mice, recruitment of complexin to the SNARE complex was reduced because of an abnormal CaMKII/syntaxin interaction. Nevertheless, SNARE complex formation was not inhibited, and consequently, basal neurotransmission was normal. However, the KI mice did exhibit more enhanced presynaptic plasticity than wild-type littermates; this enhanced plasticity could be associated with synaptic response than did wild-type littermates; this pronounced response included several behavioral abnormalities. Notably, the R151G phenotypes were generally similar to previously reported CaMKII mutant phenotypes. Additionally, synaptic recycling in these KI mice was delayed, and the density of synaptic vesicles was reduced. Taken together, our results indicated that this single point mutation in syntaxin-1A causes abnormal regulation of neuronal plasticity and vesicle recycling and that the affected syntaxin-1A/CaMKII interaction is essential for normal brain and synaptic functions in vivo.     

Synaptic Vesicle Exocytosis

Presynaptic nerve terminals release neurotransmitters by synaptic vesicle exocytosis. Membrane fusion mediating synaptic exocytosis and other intracellular membrane traffic is affected by a universal machinery that includes SNARE (for “soluble NSF-attachment protein receptor”) and SM (for “Sec1/Munc18-like”) proteins. During fusion, vesicular and target SNARE proteins assemble into an α-helical trans-SNARE complex that forces the two membranes tightly together, and SM proteins likely wrap around assembling trans-SNARE complexes to catalyze membrane fusion. After fusion, SNARE complexes are dissociated by the ATPase NSF (for “N-ethylmaleimide sensitive factor”). Fusion-competent conformations of SNARE proteins are maintained by chaperone complexes composed of CSPα, Hsc70, and SGT, and by nonenzymatically acting synuclein chaperones; dysfunction of these chaperones results in neurodegeneration. The synaptic membrane-fusion machinery is controlled by synaptotagmin, and additionally regulated by a presynaptic protein matrix (the “active zone”) that includes Munc13 and RIM proteins as central components.Synaptic vesicles are uniform organelles of ∼40 nm diameter that constitute the central organelle for neurotransmitter release. Each presynaptic nerve terminal contains hundreds of synaptic vesicles that are filled with neurotransmitters. When an action potential depolarizes the presynaptic plasma membrane, Ca²⁺-channels open, and Ca²⁺ flows into the nerve terminal to trigger the exocytosis of synaptic vesicles, thereby releasing their neurotransmitters into the synaptic cleft (Fig. 1). Ca²⁺ triggers exocytosis by binding to synaptotagmin; after exocytosis, vesicles are re-endocytosed, recycled, and refilled with neurotransmitters. Recycling can occur by multiple parallel pathways, either by fast recycling via local reuse of vesicles (“kiss-and-run” and “kiss-and-stay”), or by slower recycling via an endosomal intermediate (Fig. 1).Open in a separate windowFigure 1.The synaptic vesicle cycle. A presynaptic nerve terminal is depicted schematically as it contacts a postsynaptic neuron. The synaptic vesicle cycle consists of exocytosis (red arrows) followed by endocytosis and recycling (yellow arrows). Synaptic vesicles (green circles) are filled with neurotransmitters (NT; red dots) by active transport (neurotransmitter uptake) fueled by an electrochemical gradient established by a proton pump that acidifies the vesicle interior (vesicle acidification; green background). In preparation to synaptic exocytosis, synaptic vesicles are docked at the active zone, and primed by an ATP-dependent process that renders the vesicles competent to respond to a Ca²⁺-signal. When an action potential depolarizes the presynaptic membrane, Ca²⁺-channels open, causing a local increase in intracellular Ca²⁺ at the active zone that triggers completion of the fusion reaction. Released neurotransmitters then bind to receptors associated with the postsynaptic density (PSD). After fusion pore opening, synaptic vesicles probably recycle via three alternative pathways: local refilling with neurotransmitters without undocking (“kiss-and-stay”), local recycling with undocking (“kiss-and-run”), and full recycling of vesicles with passage through an endosomal intermediate. (Adapted from Südhof 2004.)Due to their small size, synaptic vesicles contain a limited complement of proteins that have been described in detail (Südhof 2004; Takamori et al. 2006). Although the functions of several vesicle components remain to be identified, most vesicle components participate in one of three processes: neurotransmitter uptake and storage, vesicle exocytosis, and vesicle endocytosis and recycling. In addition, it is likely that at least some vesicle proteins are involved in the biogenesis of synaptic vesicles and the maintenance of their exquisite uniformity and stability, but little is known about how vesicles are made, and what determines their size.     

Suppression of Ca2+ syntillas increases spontaneous exocytosis in mouse adrenal chromaffin cells

A central concept in the physiology of neurosecretion is that a rise in cytosolic [Ca²⁺] in the vicinity of plasmalemmal Ca²⁺ channels due to Ca²⁺ influx elicits exocytosis. Here, we examine the effect on spontaneous exocytosis of a rise in focal cytosolic [Ca²⁺] in the vicinity of ryanodine receptors (RYRs) due to release from internal stores in the form of Ca²⁺ syntillas. Ca²⁺ syntillas are focal cytosolic transients mediated by RYRs, which we first found in hypothalamic magnocellular neuronal terminals. (scintilla, Latin for spark; found in nerve terminals, normally synaptic structures.) We have also observed Ca²⁺ syntillas in mouse adrenal chromaffin cells. Here, we examine the effect of Ca²⁺ syntillas on exocytosis in chromaffin cells. In such a study on elicited exocytosis, there are two sources of Ca²⁺: one due to influx from the cell exterior through voltage-gated Ca²⁺ channels, and that due to release from intracellular stores. To eliminate complications arising from Ca²⁺ influx, we have examined spontaneous exocytosis where influx is not activated. We report here that decreasing syntillas leads to an increase in spontaneous exocytosis measured amperometrically. Two independent lines of experimentation each lead to this conclusion. In one case, release from stores was blocked by ryanodine; in another, stores were partially emptied using thapsigargin plus caffeine, after which syntillas were decreased. We conclude that Ca²⁺ syntillas act to inhibit spontaneous exocytosis, and we propose a simple model to account quantitatively for this action of syntillas.     

Direct interaction of SNARE complex binding protein synaphin/complexin with calcium sensor synaptotagmin 1

Although the binding of synaphin (also called complexin) to the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is critical for synaptic vesicle exocytosis, the exact role of synaphin remains unclear. Here, we show that synaphin directly binds to synaptotagmin 1, a major Ca²⁺ sensor for fast neurotransmitter release, in a 1:1 stoichiometry. Mapping of the synaphin site involved in synaptotagmin 1 binding revealed that the C-terminal region is essential for the interaction between these two proteins. Binding was sensitive to ionic strength, suggesting the involvement of charged residues in the C-terminus region. Mutation of the seven consecutive glutamic acid residues (residues 108–114) at the C-terminal region of synaphin to alanines or glutamines resulted in a dramatic reduction in synaptotagmin 1 binding activity. Furthermore, a peptide from the C-terminus of synaphin (residues 91–124) blocked the binding of synaptotagmin 1 to synaphin, an effect that was abolished by mutating the consecutive glutamic acid residues to alanine. Immunoprecipitation experiments with brain membrane extracts showed the presence of a complex consisting of synaphin, synaptotagmin 1, and SNAREs. We propose that synaphin recruits synaptotagmin 1 to the SNARE-based fusion complex and synergistically functions with synaptotagmin 1 in mediating fast synaptic vesicle exocytosis.     

A quaternary SNARE-synaptotagmin-Ca2+-phospholipid complex in neurotransmitter release

The function of synaptotagmin as a Ca²⁺ sensor in neurotransmitter release involves Ca²⁺-dependent phospholipid binding to its two C₂ domains, but this activity alone does not explain why Ca²⁺ binding to the C₂B domain is more critical for release than Ca²⁺ binding to the C₂A domain. Synaptotagmin also binds to SNARE complexes, which are central components of the membrane fusion machinery, and displaces complexins from the SNAREs. However, it is unclear how phospholipid binding to synaptotagmin is coupled to SNARE binding and complexin displacement. Using supported lipid bilayers deposited within microfluidic channels, we now show that Ca²⁺ induces simultaneous binding of synaptotagmin to phospholipid membranes and SNARE complexes, resulting in an intimate quaternary complex that we name SSCAP complex. Mutagenesis experiments show that Ca²⁺ binding to the C₂B domain is critical for SSCAP complex formation and displacement of complexin, providing a clear rationale for the preponderant role of the C₂B domain in release. This and other correlations between the effects of mutations on SSCAP complex formation and their functional effects in vivo suggest a key role for this complex in release. We propose a model whereby the highly positive electrostatic potential at the tip of the SSCAP complex helps to induce membrane fusion during release.     

A novel dual Ca2+ sensor system regulates Ca2+-dependent neurotransmitter release

Ca²⁺-dependent neurotransmitter release requires synaptotagmins as Ca²⁺ sensors to trigger synaptic vesicle (SV) exocytosis via binding of their tandem C2 domains—C2A and C2B—to Ca²⁺. We have previously demonstrated that SNT-1, a mouse synaptotagmin-1 (Syt1) homologue, functions as the fast Ca²⁺ sensor in Caenorhabditis elegans. Here, we report a new Ca²⁺ sensor, SNT-3, which triggers delayed Ca²⁺-dependent neurotransmitter release. snt-1;snt-3 double mutants abolish evoked synaptic transmission, demonstrating that C. elegans NMJs use a dual Ca²⁺ sensor system. SNT-3 possesses canonical aspartate residues in both C2 domains, but lacks an N-terminal transmembrane (TM) domain. Biochemical evidence demonstrates that SNT-3 binds both Ca²⁺ and the plasma membrane. Functional analysis shows that SNT-3 is activated when SNT-1 function is impaired, triggering SV release that is loosely coupled to Ca²⁺ entry. Compared with SNT-1, which is tethered to SVs, SNT-3 is not associated with SV. Eliminating the SV tethering of SNT-1 by removing the TM domain or the whole N terminus rescues fast release kinetics, demonstrating that cytoplasmic SNT-1 is still functional and triggers fast neurotransmitter release, but also exhibits decreased evoked amplitude and release probability. These results suggest that the fast and slow properties of SV release are determined by the intrinsically different C2 domains in SNT-1 and SNT-3, rather than their N-termini–mediated membrane tethering. Our findings therefore reveal a novel dual Ca²⁺ sensor system in C. elegans and provide significant insights into Ca²⁺-regulated exocytosis.     

Postsynaptic regulation of synaptic plasticity by synaptotagmin 4 requires both C2 domains

Ca²⁺ influx into synaptic compartments during activity is a key mediator of neuronal plasticity. Although the role of presynaptic Ca²⁺ in triggering vesicle fusion though the Ca²⁺ sensor synaptotagmin 1 (Syt 1) is established, molecular mechanisms that underlie responses to postsynaptic Ca²⁺ influx remain unclear. In this study, we demonstrate that fusion-competent Syt 4 vesicles localize postsynaptically at both neuromuscular junctions (NMJs) and central nervous system synapses in Drosophila melanogaster. Syt 4 messenger RNA and protein expression are strongly regulated by neuronal activity, whereas altered levels of postsynaptic Syt 4 modify synaptic growth and presynaptic release properties. Syt 4 is required for known forms of activity-dependent structural plasticity at NMJs. Synaptic proliferation and retrograde signaling mediated by Syt 4 requires functional C2A and C2B Ca²⁺–binding sites, as well as serine 284, an evolutionarily conserved substitution for a key Ca²⁺-binding aspartic acid found in other synaptotagmins. These data suggest that Syt 4 regulates activity-dependent release of postsynaptic retrograde signals that promote synaptic plasticity, similar to the role of Syt 1 as a Ca²⁺ sensor for presynaptic vesicle fusion.     

Complexin clamps asynchronous release by blocking a secondary Ca(2+) sensor via its accessory α helix

Complexin activates and clamps neurotransmitter release; impairing complexin function decreases synchronous, but increases spontaneous and asynchronous synaptic vesicle exocytosis. Here, we show that complexin-different from the Ca(2+) sensor synaptotagmin-1-activates synchronous exocytosis by promoting synaptic vesicle priming, but clamps spontaneous and asynchronous exocytosis-similar to synaptotagmin-1-by blocking a secondary Ca(2+) sensor. Activation and clamping functions of complexin depend on distinct, autonomously acting sequences, namely its N-terminal region and accessory α helix, respectively. Mutations designed to test whether the accessory α helix of complexin clamps exocytosis by inserting into SNARE-complexes support this hypothesis, suggesting that the accessory α helix blocks completion of trans-SNARE-complex assembly until Ca(2+) binding to synaptotagmin relieves this block. Moreover, a juxtamembranous mutation in the SNARE-protein synaptobrevin-2, which presumably impairs force transfer from nascent trans-SNARE complexes onto fusing membranes, also unclamps spontaneous fusion by disinhibiting a secondary Ca(2+) sensor. Thus, complexin performs mechanistically distinct activation and clamping functions that operate in conjunction with synaptotagmin-1 by controlling trans-SNARE-complex assembly.