Is synaptotagmin the calcium sensor?

After much debate, recent progress indicates that the synaptic vesicle protein synaptotagmin I probably functions as the calcium sensor for synchronous neurotransmitter release. Following calcium influx into presynaptic terminals, synaptotagmin I rapidly triggers the fusion of synaptic vesicles with the plasma membrane and underlies the fourth-order calcium cooperativity of release. Biochemical and genetic studies suggest that lipid and SNARE interactions underlie synaptotagmin's ability to mediate the incredible speed of vesicle fusion that is the hallmark of fast synaptic transmission.     

The Cytoplasmic Domain of Rat Synaptotagmin I Enhances Synaptic Transmission

Synaptotagmin, an integral membrane protein of synaptic vesicles, functions as a calcium sensor in the temporal control of neurotransmitter release. Although synaptotagmin facilitates lipid membrane fusion in biochemical experiments, overexpression of synaptotagmin inhibits neurotransmission. A facilitatory effect of synaptotagmin on synaptic transmission was never observed. To determine whether synaptotagmin may accelerate synaptic transmission in vivo, we injected the cytoplasmic domain of rat synaptotagmin I (CD-syt) into crayfish motor axons and tested the effect of CD-syt on synaptic response. We confirmed that CD-syt accelerates neuromuscular transmission. The injected preparation had larger synaptic potentials with shorter rise time. Experiments with varying calcium concentrations showed that CD-syt increased the maximum synaptic response of the neuromuscular synapses. Further tests on short-term plasticity of neuromuscular synapses revealed that CD-syt increases the release probability of the release-ready vesicles.     

Synaptotagmin interaction with the syntaxin/SNAP-25 dimer is mediated by an evolutionarily conserved motif and is sensitive to inositol hexakisphosphate

Synaptotagmins are membrane proteins that possess tandem C2 domains and play an important role in regulated membrane fusion in metazoan organisms. Here we show that both synaptotagmins I and II, the two major neuronal isoforms, can interact with the syntaxin/synaptosomal-associated protein of 25 kDa (SNAP-25) dimer, the immediate precursor of the soluble NSF attachment protein receptor (SNARE) fusion complex. A stretch of basic amino acids highly conserved throughout the animal kingdom is responsible for this calcium-independent interaction. Inositol hexakisphosphate modulates synaptotagmin coupling to the syntaxin/SNAP-25 dimer, which is mirrored by changes in chromaffin cell exocytosis. Our results shed new light on the functional importance of the conserved polybasic synaptotagmin motif, suggesting that synaptotagmin interacts with the t-SNARE dimer to up-regulate the probability of SNARE-mediated membrane fusion.     

Synaptotagmin activates membrane fusion through a Ca2+-dependent trans interaction with phospholipids

Synaptotagmin-1 is the calcium sensor for neuronal exocytosis, but the mechanism by which it triggers membrane fusion is not fully understood. Here we show that synaptotagmin accelerates SNARE-dependent fusion of liposomes by interacting with neuronal Q-SNARES in a Ca2+-independent manner. Ca2+-dependent binding of synaptotagmin to its own membrane impedes the activation. Preventing this cis interaction allows Ca2+ to trigger synaptotagmin binding in trans, accelerating fusion. However, when an activated SNARE acceptor complex is used, synaptotagmin has no effect on fusion kinetics, suggesting that synaptotagmin operates upstream of SNARE assembly in this system. Our results resolve major discrepancies concerning the effects of full-length synaptotagmin and its C2AB fragment on liposome fusion and shed new light on the interactions of synaptotagmin with SNAREs and membranes. However, our findings also show that the action of synaptotagmin on the fusion-arrested state of docked vesicles in vivo is not fully reproduced in vitro.     

SNAREpin Assembly by Munc18-1 Requires Previous Vesicle Docking by Synaptotagmin 1

Regulated exocytosis requires the general membrane fusion machinery-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and Sec1/Munc18 (SM) proteins. Using reconstituted giant unilamellar vesicles containing preassembled t-SNARE proteins (syntaxin 1·SNAP-25), we determined how Munc18-1 controls the docking, priming, and fusion of small unilamellar vesicles containing the v-SNARE VAMP2 and the Ca(2+) sensor synaptotagmin 1. In vitro assays allowed us to position Munc18-1 in the center of a sequential reaction cascade; vesicle docking by synaptotagmin 1 is a prerequisite for Munc18-1 to accelerate trans-SNARE complex (SNAREpin) assembly and membrane fusion. Complexin II stalls SNAREpin zippering at a late stage and, hence, contributes to synchronize membrane fusion in a Ca(2+)- and synaptotagmin 1-dependent manner. Thus, at the neuronal synapse, the priming factor Munc18-1 may accelerate the conversion of docked synaptic vesicles into a readily releasable pool by activating SNAREs for efficient membrane fusion.     

Palmitoylation of the synaptic vesicle fusion machinery

The fusion of synaptic vesicles with the pre-synaptic plasma membrane mediates the secretion of neurotransmitters at nerve terminals. This pathway is regulated by an array of protein–protein interactions. Of central importance are the soluble NSF ( N -ethylmaleimide-sensitive factor) attachment protein receptor (SNARE) proteins syntaxin 1 and SNAP25, which are associated with the pre-synaptic plasma membrane and vesicle-associated membrane protein (VAMP2), a synaptic vesicle SNARE. Syntaxin 1, SNAP25 and VAMP2 interact to form a tight complex bridging the vesicle and plasma membranes, which has been suggested to represent the minimal membrane fusion machinery. Synaptic vesicle fusion is stimulated by a rise in intraterminal Ca²⁺ levels, and a major Ca²⁺ sensor for vesicle fusion is synaptotagmin I. Synaptotagmin is likely to couple Ca²⁺ entry to vesicle fusion via Ca²⁺-dependent and independent interactions with membrane phospholipids and the SNARE proteins. Intriguingly, syntaxin 1, SNAP25, VAMP2 and synaptotagmin I have all been reported to be modified by palmitoylation in neurons. In this review, we discuss the mechanisms and dynamics of palmitoylation of these proteins and speculate on how palmitoylation might contribute to the regulation of synaptic vesicle fusion.     

Activation of store-operated calcium channels: assessment of the role of snare-mediated vesicular transport

Store-operated calcium channels (SOC) play a central role in cellular calcium homeostasis. Although it is well established that SOC are activated by depletion of the endoplasmic reticulum calcium stores, the molecular mechanism underlying this effect remains ill defined. It has been suggested that SOC activation requires fusion of endomembrane vesicles with the plasmalemma. In this model, SNARE-dependent exocytosis is proposed to deliver channels or their activators to the surface membrane to initiate calcium influx. To test this hypothesis, we studied the requirement for membrane fusion events in SOC activation, using a variety of dominant-negative constructs and toxins that interfere with SNARE function. Botulinum neurotoxin A (BotA), which cleaves SNAP-25, did not prevent SOC activation. Moreover, SNAP-25 was not detectable in the cells where BotA was reported earlier to inhibit SOC. Instead, the BotA-insensitive SNAP-23 was present. Impairment of VAMP function was similarly without effect on SOC opening. We also tested the role of N-ethylmaleimide-sensitive factor, a global regulator of SNARE-mediated membrane fusion. Expression of a mutated N-ethylmaleimide-sensitive factor construct inhibited all aspects of membrane traffic tested, including recycling of transferrin receptors to the plasma membrane, fusion of endosomes with lysosomes, and retrograde traffic to the Golgi complex. Despite this global inhibition of vesicular fusion, which was accompanied by gross alterations in cell morphology, SOC activation persisted. These observations cannot be easily reconciled with the vesicle-mediated coupling hypothesis of SOC activation. Our findings imply that the SOC and the machinery necessary to activate them exist in the plasma membrane or are associated with it prior to activation.     

Mechanism of calcium-independent synaptotagmin binding to target SNAREs

Synaptic vesicle exocytosis requires three SNARE (soluble N-ethylmaleimide-sensitive-factor attachment protein receptor) proteins: syntaxin and SNAP-25 on the plasma membrane (t-SNAREs) and synaptobrevin/VAMP on the synaptic vesicles (v-SNARE). Vesicular synaptotagmin 1 is essential for fast synchronous SNARE-mediated exocytosis and interacts with the SNAREs in brain material. To uncover the step at which synaptotagmin becomes linked to the three SNAREs, we purified all four proteins from brain membranes and analyzed their interactions. Our study reveals that, in the absence of calcium, native synaptotagmin 1 binds the t-SNARE heterodimer, formed from syntaxin and SNAP-25. This interaction is both stoichiometric and of high affinity. Synaptotagmin contains two divergent but conserved C2 domains that can act independently in calcium-triggered phospholipid binding. We now show that both C2 domains are strictly required for the calcium-independent interaction with the t-SNARE heterodimer, indicating that the double C2 domain structure of synaptotagmin may have evolved to acquire a function beyond calcium/phospholipid binding.     

Interactions between proteins implicated in exocytosis and voltage-gated calcium channels

Neurotransmitter release from synaptic vesicles is triggered by voltage-gated calcium influx through P/Q-type or N-type calcium channels. Purification of N-type channels from rat brain synaptosomes initially suggested molecular interactions between calcium channels and two key proteins implicated in exocytosis: synaptotagmin I and syntaxin 1. Co-immunoprecipitation experiments were consistent with the hypothesis that both N- and P/Q-type calcium channels, but not L-type channels, are associated with the 7S complex containing syntaxin 1, SNAP-25, VAMP and synaptotagmin I or II. Immunofluorescence confocal microscopy at the frog neuromuscular junction confirmed that calcium channels, syntaxin 1 and SNAP-25 are co-localized at active zones of the presynaptic plasma membrane where transmitter release occurs. Experiments with recombinant proteins were performed to map synaptic protein interaction sites on the alpha 1A subunit, which forms the pore of the P/Q-type calcium channel. In vitro-translated 35S-synaptotagmin I bound to a site located on the cytoplasmic loop linking homologous domains II and III of the alpha 1A subunit. This direct link would target synaptotagmin, a putative calcium sensor for exocytosis, to a microdomain of calcium influx close to the channel mouth. Cysteine string proteins (CSPs) contain a J-domain characteristic of molecular chaperones that cooperate with Hsp70. They are located on synaptic vesicles and thought to be involved in modulating the activity of presynaptic calcium channels. CSPs were found to bind to the same domain of the calcium channel as synaptotagmin, and also to associate with VAMP. CSPs may act as molecular chaperones in association with Hsp70 to direct assembly or dissociation of multiprotein complexes at the calcium channel.     

Synaptotagmin VII as a plasma membrane Ca(2+) sensor in exocytosis

Synaptotagmins I and II are Ca(2+) binding proteins of synaptic vesicles essential for fast Ca(2+)-triggered neurotransmitter release. However, central synapses and neuroendocrine cells lacking these synaptotagmins still exhibit Ca(2+)-evoked exocytosis. We now propose that synaptotagmin VII functions as a plasma membrane Ca(2+) sensor in synaptic exocytosis complementary to vesicular synaptotagmins. We show that alternatively spliced forms of synaptotagmin VII are expressed in a developmentally regulated pattern in brain and are concentrated in presynaptic active zones of central synapses. In neuroendocrine PC12 cells, the C(2)A and C(2)B domains of synaptotagmin VII are potent inhibitors of Ca(2+)-dependent exocytosis, but only when they bind Ca(2+). Our data suggest that in synaptic vesicle exocytosis, distinct synaptotagmins function as independent Ca(2+) sensors on the two fusion partners, the plasma membrane (synaptotagmin VII) versus synaptic vesicles (synaptotagmins I and II).     

Immunocytochemical Analysis of Axonal Outgrowth in Synaptotagmin Mutations

Abstract: Synaptotagmin is a synaptic vesicle specific protein that binds calcium and phospholipids in vitro and is required for calcium-regulated fusion of synaptic vesicles with the presynaptic membrane. We have examined the possible requirement for synaptotagmin in axonal outgrowth by following neuronal development in Drosophila embryos deficient for the synaptotagmin gene. We find that synaptotagmin is expressed abundantly in axons and growth cones before synapse formation in wild-type embryos. Using antibodies to the intravesicular domain of synaptotagmin to label live embryos, we demonstrate that vesicle populations containing synaptotagmin actively undergo exocytosis during axonogenesis. We have used immunocytochemical techniques to examine the distribution of the axonal protein Fasciclin II, the presynaptic membrane protein syntaxin, and the synaptic vesicle protein cysteine string protein, in synaptotagmin null mutations. The distribution of these proteins is similar in wild-type and synaptotagmin mutant embryos, suggesting that synaptotagmin is not required for axonogenesis in the CNS or PNS. Based on these findings, we suggest that the molecular mechanisms underlying vesicular-mediated membrane expansion during axonal outgrowth are distinct from those required for synaptic vesicle fusion during neurotransmitter release.     

Complexin arrests a pool of docked vesicles for fast Ca(2+)-dependent release

Regulated exocytosis requires that the assembly of the basic membrane fusion machinery is temporarily arrested. Synchronized membrane fusion is then caused by a specific trigger-a local rise of the Ca(2+) concentration. Using reconstituted giant unilamellar vesicles (GUVs), we have analysed the role of complexin and membrane-anchored synaptotagmin 1 in arresting and synchronizing fusion by lipid-mixing and cryo-electron microscopy. We find that they mediate the formation and consumption of docked small unilamellar vesicles (SUVs) via the following sequence of events: Synaptotagmin 1 mediates v-SNARE-SUV docking to t-SNARE-GUVs in a Ca(2+)-independent manner. Complexin blocks vesicle consumption, causing accumulation of docked vesicles. Together with synaptotagmin 1, complexin synchronizes and stimulates rapid fusion of accumulated docked vesicles in response to physiological Ca(2+) concentrations. Thus, the reconstituted assay resolves both the stimulatory and inhibitory function of complexin and mimics key aspects of synaptic vesicle fusion.     

Direct interaction of the calcium sensor protein synaptotagmin I with a cytoplasmic domain of the alpha1A subunit of the P/Q-type calcium channel.

Synaptotagmins are synaptic vesicle proteins containing two calcium-binding C2 domains which are involved in coupling calcium influx through voltage-gated channels to vesicle fusion and exocytosis of neurotransmitters. The interaction of synaptotagmins with native P/Q-type calcium channels was studied in solubilized synaptosomes from rat cerebellum. Antibodies against synaptotagmins I and II, but not IV co-immunoprecipitated [125I]omega-conotoxin MVIIC-labelled calcium channels. Direct interactions were studied between in vitro-translated [35S]synaptotagmin I and fusion proteins containing cytoplasmic loops of the alpha1A subunit (BI isoform). Gel overlay revealed the association of synaptotagmin I with a single region (residues 780-969) located in the intracellular loop connecting homologous domains II and III. Saturable calcium-independent binding occurred with equilibrium dissociation constants of 70 nM and 340 nM at 4 degrees C and pH 7.4, and association was blocked by addition of excess recombinant synaptotagmin I. Direct synaptotagmin binding to the pore-forming subunit of the P/Q-type channel may optimally locate the calcium-binding sites that initiate exocytosis within a zone of voltage-gated calcium entry.     

Intermembrane docking reactions are regulated by membrane curvature

The polymorphism of eukaryotic cellular membranes is a tightly regulated and well-conserved phenotype. Recent data have revealed important regulatory roles of membrane curvature on the spatio-temporal localization of proteins and in membrane fusion. Here we quantified the influence of membrane curvature on the efficiency of intermembrane docking reactions. Using fluorescence microscopy, we monitored the docking of single vesicle–vesicle pairs of different diameter (30–200 nm) and therefore curvature, as mediated by neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and streptavidin-biotin. Surprisingly, the intermembrane docking efficiency exhibited an ∼30–60 fold enhancement as a function of curvature. In comparison, synaptotagmin and calcium accelerate SNARE-mediated fusion in vitro by a factor of 2–10. To explain this finding, we formulated a biophysical model. On the basis of our findings, we propose that membrane curvature can regulate intermembrane tethering reactions and consequently any downstream process, including the fusion of vesicles and possibly viruses with their target membranes.     

Synaptic vesicles are constitutively active fusion machines that function independently of Ca2+

BACKGROUND: In neurons, release of neurotransmitter occurs through the fusion of synaptic vesicles with the plasma membrane. Many proteins required for this process have been identified, with the SNAREs syntaxin 1, SNAP-25, and synaptobrevin thought to constitute the core fusion machinery. However, there is still a large gap between our understanding of individual protein-protein interactions and the functions of these proteins revealed by perturbations in intact synaptic preparations. To bridge this gap, we have used purified synaptic vesicles, together with artificial membranes containing core-constituted SNAREs as reaction partners, in fusion assays. RESULTS: By using complementary experimental approaches, we show that synaptic vesicles fuse constitutively, and with high efficiency, with proteoliposomes containing the plasma membrane proteins syntaxin 1 and SNAP-25. Fusion is inhibited by clostridial neurotoxins and involves the formation of SNARE complexes. Despite the presence of endogenous synaptotagmin, Ca(2+) does not enhance fusion, even if phosphatidylinositol 4,5-bisphosphate is present in the liposome membrane. Rather, fusion kinetics are dominated by the availability of free syntaxin 1/SNAP-25 acceptor sites for synaptobrevin. CONCLUSIONS: Synaptic vesicles are constitutively active fusion machines, needing only synaptobrevin for activity. Apparently, the final step in fusion does not involve the regulatory activities of other vesicle constituents, although these may be involved in regulating earlier processes. This is particularly relevant for the calcium-dependent regulation of exocytosis, which, in addition to synaptotagmin, requires other factors not present in the vesicle membrane. The in vitro system described here provides an ideal starting point for unraveling of the molecular details of such regulatory events.     

SNAREs in mammalian sperm: possible implications for fertilization

Soluble N-ethylmalameide-sensitive factor attachment protein receptor (SNARE) proteins are present in mammalian sperm and could be involved in critical membrane fusion events during fertilization, namely the acrosome reaction. Vesicle-associated membrane protein/synaptobrevin, a SNARE on the membrane of a vesicular carrier, and syntaxin 1, a SNARE on the target membrane, as well as the calcium sensor synaptotagmin I, are present in the acrosome of mammalian sperm (human, rhesus monkey, bull, hamster, mouse). Sperm SNAREs are sloughed off during the acrosome reaction, paralleling the release of sperm membrane vesicles and acrosomal contents, and SNARE antibodies inhibit both the acrosome reaction and fertilization, without inhibiting sperm-egg binding. In addition, sperm SNAREs may be responsible, together with other sperm components, for the asynchronous male DNA decondensation that occurs following intracytoplasmic sperm injection, an assisted reproduction technique that bypasses normal sperm-egg surface interactions. The results suggest the participation of sperm SNAREs during membrane fusion events at fertilization in mammals.     

Synaptotagmin VI participates in the acrosome reaction of human spermatozoa.

Acrosomal exocytosis is a calcium-dependent secretion event causing the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. The synaptotagmins are a family of calcium-binding proteins that participate in the exocytosis of synaptic vesicles. The ubiquitous synaptotagmin VI isoform was found in human sperm cells by Western blot analysis. Immunocytochemistry at the optical and electron microscopy levels localized the protein to the outer acrosomal membrane. Calcium-triggered acrosomal exocytosis in permeabilized sperm cells was abrogated by a specific anti-synaptotagmin VI antibody, indicating that the protein is required for the process. Moreover, a recombinant fusion protein between glutathione S-transferase and the two calcium and phospholipid binding domains of synaptotagmin VI completely inhibited calcium-triggered exocytosis. Interestingly, phorbol ester-dependent in vitro phosphorylation of this recombinant protein abolished its inhibitory effect. We previously showed that, in permeabilized spermatozoa, addition of active Rab3A triggers acrosomal exocytosis at very low calcium concentration. Rab3A-promoted exocytosis was inhibited by the cytosolic domain of synaptotagmin VI and by the anti-synaptotagmin VI antibody, indicating that synaptotagmin is also necessary for Rab-mediated acrosomal content release. In conclusion, the results strongly indicate that synaptotagmin VI is a key component of the secretory machinery involved in acrosomal exocytosis.     

C2B polylysine motif of synaptotagmin facilitates a Ca2+-independent stage of synaptic vesicle priming in vivo

Synaptotagmin I, a synaptic vesicle protein required for efficient synaptic transmission, contains a highly conserved polylysine motif necessary for function. Using Drosophila, we examined in which step of the synaptic vesicle cycle this motif functions. Polylysine motif mutants exhibited an apparent decreased Ca2+ affinity of release, and, at low Ca2+, an increased failure rate, increased facilitation, and increased augmentation, indicative of a decreased release probability. Disruption of Ca2+ binding, however, cannot account for all of the deficits in the mutants; rather, the decreased release probability is probably due to a disruption in the coupling of synaptotagmin to the release machinery. Mutants exhibited a major slowing of recovery from synaptic depression, which suggests that membrane trafficking before fusion is disrupted. The disrupted process is not endocytosis because the rate of FM 1-43 uptake was unchanged in the mutants, and the polylysine motif mutant synaptotagmin was able to rescue the synaptic vesicle depletion normally found in syt(null) mutants. Thus, the polylysine motif functions after endocytosis and before fusion. Finally, mutation of the polylysine motif inhibits the Ca2+-independent ability of synaptotagmin to accelerate SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-mediated fusion. Together, our results demonstrate that the polylysine motif is required for efficient Ca2+-independent docking and/or priming of synaptic vesicles in vivo.     

How SNARE molecules mediate membrane fusion: recent insights from molecular simulations

SNARE molecules are the core constituents of the protein machinery that facilitate fusion of synaptic vesicles with the presynaptic plasma membrane, resulting in the release of neurotransmitter. On a molecular level, SNARE complexes seem to play a quite versatile and involved role during all stages of fusion. In addition to merely triggering fusion by forcing the opposing membranes into close proximity, SNARE complexes are now seen to also overcome subsequent fusion barriers and to actively guide the fusion reaction up to the expansion of the fusion pore. Here, we review recent advances in the understanding of SNARE-mediated membrane fusion by molecular simulations.