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).     

Specificity of Ca2+-dependent protein interactions mediated by the C2A domains of synaptotagmins

Synaptotagmins represent a family of neuronal proteins thought to function in membrane traffic. The best characterized synaptotagmin, synaptotagmin I, is essential for fast Ca2+-dependent synaptic vesicle exocytosis, indicating a role in the Ca2+ triggering of membrane fusion. Synaptotagmins contain two C2 domains, the C2A and C2B domains, which bind Ca2+ and may mediate their functions by binding to specific targets. For synaptotagmin I, several putative targets have been identified, including the SNARE proteins syntaxin and SNAP-25. However, it is unclear which of the many binding proteins are physiologically relevant. Furthermore, more than 10 highly homologous synaptotagmins are expressed in brain, but it is unknown if they execute similar binding reactions. To address these questions, we have performed a systematic, unbiased study of proteins which bind to the C2A domains of synaptotagmins I-VII. Although the various C2A domains exhibit similar binding activities for phospholipids and syntaxin, we found that they differ greatly in their protein binding patterns. Surprisingly, none of the previously characterized binding proteins for synaptotagmin I are among the major interacting proteins identified. Instead, several proteins that were not known to interact with synaptotagmin I were bound tightly and stoichiometrically, most prominently the NSF homologue VCP, which is thought to be involved in membrane fusion, and an unknown protein of 40 kDa. Point mutations in the Ca2+ binding loops of the C2A domain revealed that the interactions of these proteins with synaptotagmin I were highly specific. Furthermore, a synaptotagmin I/VCP complex could be immunoprecipitated from brain homogenates in a Ca2+-dependent manner, and GST-VCP fusion proteins efficiently captured synaptotagmin I from brain. However, when we investigated the tissue distribution of VCP, we found that, different from synaptic proteins, VCP was not enriched in brain and exhibited no developmental increase paralleling synaptogenesis. Moreover, binding of VCP, which is an ATPase, to synaptotagmin I was inhibited by both ATP and ADP, indicating that the native, nucleotide-occupied state of VCP does not bind to synaptotagmin. Together our findings suggest that the C2A-domains of different synaptotagmins, despite their homology, exhibit a high degree of specificity in their protein interactions. This is direct evidence for diverse roles of the various synaptotagmins in brain, consistent with their differential subcellular localizations. Furthermore, our results indicate that traditional approaches, such as affinity chromatography and immunoprecipitations, are useful tools to evaluate the overall spectrum of binding activity for a protein but are not sufficient to estimate physiological relevance.     

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.     

Interactions between Presynaptic Calcium Channels and Proteins Implicated in Synaptic Vesicle Trafficking and Exocytosis

Monoclonal antibodies were generated by immunizing mice with chick brain synaptic membranes and screening for immunoprecipitation of solubilized conotoxin GVIA receptors (N-type calcium channels). Antibodies against two synaptic proteins (p35--syntaxin 1 and p58--synaptotagmin) were produced and used to purify and characterize a ternary complex containing N-type channels associated with these two proteins. These results provided the first evidence for a specific interaction between presynaptic calcium channels and SNARE proteins involved in synaptic vesicle docking and calcium-dependent exocytosis. Immunoprecipitation experiments supported the conclusion that syntaxin 1/SNAP-25/VAMP/synaptotagmin I or II complexes associate with N-type, P/Q-type, but not L-type calcium channels from rat brain nerve terminals. Immunofluorescent confocal microscopy at the frog neuromuscular junction was consistent with the co-localization of syntaxin 1, SNAP-25, and calcium channels, all of which are predominantly expressed at active zones of the presynaptic plasma membrane facing post-synaptic folds rich in acetylcholine receptors. The interaction of proteins implicated in calcium-dependent exocytosis with presynaptic calcium channels may locate the sensor(s) that trigger vesicle fusion within a microdomain of calcium entry.     

Association of neuronal calcium channels with modular adaptor proteins.

Presynaptic voltage-gated calcium (Ca(2+)) channels mediate Ca(2+) influx into the presynaptic terminal that triggers synaptic vesicle fusion and neurotransmitter release. The immediate proximity of Ca(2+) channels to the synaptic vesicle release apparatus is critical for rapid and efficient synaptic transmission. In a series of biochemical experiments, we demonstrate a specific association of the cytosolic carboxyl terminus of the N-type Ca(2+) channel pore-forming alpha(1B) subunit with the modular adaptor proteins Mint1 and CASK. The carboxyl termini of alpha(1B) bind to the first PDZ domain of Mint1 (Mint1-1). The proline-rich region present in the carboxyl termini of alpha(1B) binds to the SH3 domain of CASK. Mint1-1 is specific for the E/D-X-W-C/S-COOH consensus, which defines a novel class of PDZ domains (class III). The Mint1-1 PDZ domain-binding motif is present only in the "long" carboxyl-terminal splice variants of N-type (alpha(1B)) and P/Q-type (alpha(1A)) Ca(2+) channels, but not in R-type (alpha(1E)) or L-type (alpha(1C)) Ca(2+) channels. Our results directly link presynaptic Ca(2+) channels to a macromolecular complex formed by modular adaptor proteins at synaptic junction and advance our understanding of coupling between cell adhesion and synaptic vesicle exocytosis.     

The subcellular localizations of atypical synaptotagmins III and VI. Synaptotagmin III is enriched in synapses and synaptic plasma membranes but not in synaptic vesicles.

Multiple synaptotagmins are expressed in brain, but only synaptotagmins I and II have known functions in fast, synchronous Ca2+-triggered neurotransmitter release. Synaptotagmin III was proposed to regulate other aspects of synaptic vesicle exocytosis, particularly its slow component. Such a function predicts that synaptotagmin III should be an obligatory synaptic vesicle protein, as would also be anticipated from its high homology to synaptotagmins I and II. To test this hypothesis, we studied the distribution, developmental expression, and localization of synaptotagmin III and its closest homolog, synaptotagmin VI. We find that synaptotagmins III and VI are present in all brain regions in heterogeneous distributions and that their levels increase during development in parallel with synaptogenesis. Furthermore, we show by immunocytochemistry that synaptotagmin III is concentrated in synapses, as expected. Surprisingly, however, we observed that synaptotagmin III is highly enriched in synaptic plasma membranes but not in synaptic vesicles. Synaptotagmin VI was also found to be relatively excluded from synaptic vesicles. Our data suggest that synaptotagmins III and VI perform roles in neurons that are not linked to synaptic vesicle exocytosis but to other Ca2+-related nerve terminal events, indicating that the functions of synaptotagmins are more diverse than originally thought.     

Ca2+-dependent synaptotagmin binding to SNAP-25 is essential for Ca2+-triggered exocytosis

Synaptotagmin is a proposed Ca2+ sensor on the vesicle for regulated exocytosis and exhibits Ca2+-dependent binding to phospholipids, syntaxin, and SNAP-25 in vitro, but the mechanism by which Ca2+ triggers membrane fusion is uncertain. Previous studies suggested that SNAP-25 plays a role in the Ca2+ regulation of secretion. We found that synaptotagmins I and IX associate with SNAP-25 during Ca2+-dependent exocytosis in PC12 cells, and we identified C-terminal amino acids in SNAP-25 (Asp179, Asp186, Asp193) that are required for Ca2+-dependent synaptotagmin binding. Replacement of SNAP-25 in PC12 cells with SNAP-25 containing C-terminal Asp mutations led to a loss-of-function in regulated exocytosis at the Ca2+-dependent fusion step. These results indicate that the Ca2+-dependent interaction of synaptotagmin with SNAP-25 is essential for the Ca2+-dependent triggering of membrane fusion.     

Lambert-Eaton myasthenic syndrome as an autoimmune calcium channelopathy

Lambert-Eaton myasthenic syndrome, often associated with small-cell lung carcinoma, is a disease of neuromuscular transmission in which antibodies directed against voltage-gated calcium channel (VGCC)(P/Q-type) in the motor nerve terminal play a crucial role in causing a deficient quantal release of acetylcholine. The motor nerve terminal and carcinoma cell may share a common antigen. The study using synthetic peptides and recombinant protein specified the extracellular S5-S6 linker regions in 3 of 4 domains as immunodominant sites in the molecular structure of P/Q-type VGCC alpha1 subunit. Also, the study by use of peptides and recombinant protein corresponding to synaptotagmin I suggested that in this functionally VGCC-associated presynaptic protein, the segment which exposes extracellularly during exocytosis can be immunogenic for the syndrome.     

Voltage-dependent P/Q-type calcium channels at the frog neuromuscular junction

It is well known that antagonists of N-type voltage-gated calcium channels inhibit the evoked quantal release of acetylcholine in amphibian neuromuscular synapses. This, however, does not exclude the functional expression of other types of voltage-gated calcium channels in these nerve terminals. Using immunocytochemistry, we detected the expression of the alpha1A subunit of P/Q-type calcium channels (that is otherwise typical of mammalian motor nerve endings) in the frog neuromuscular junction. In addition, we demonstrated that the P/Q-type channel blocker omega-agatoxin IVA (20 nM) reduced the action potential-induced calcium transient and significantly decreased both spontaneous and evoked mediator release. Our data indicates the functional expression of P/Q-type calcium channels in the frog motor nerve ending which participate in acetylcholine release.     

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.     

Kinetics of synaptotagmin responses to Ca2+ and assembly with the core SNARE complex onto membranes

The synaptic vesicle protein synaptotagmin I binds Ca2+ and is required for efficient neurotransmitter release. Here, we measure the response time of the C2 domains of synaptotagmin to determine whether synaptotagmin is fast enough to function as a Ca2+ sensor for rapid exocytosis. We report that synaptotagmin is "tuned" to sense Ca2+ concentrations that trigger neuronal exocytosis. The speed of response is unique to synaptotagmin I and readily satisfies the kinetic constraints of synaptic vesicle membrane fusion. We further demonstrate that Ca2+ triggers penetration of synaptotagmin into membranes and simultaneously drives assembly of synaptotagmin onto the base of the ternary SNARE (soluble N-ethylmaleimide-sensitive fusion protein [NSF] attachment receptor) complex, near the transmembrane anchor of syntaxin. These data support a molecular model in which synaptotagmin triggers exocytosis through its interactions with membranes and the SNARE complex.     

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.     

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.     

Physical Link and Functional Coupling of Presynaptic Calcium Channels and the Synaptic Vesicle Docking/Fusion Machinery

N- and P/Q-type calcium channels are localized in high density in presynaptic nerve terminals and are crucial elements in neuronal excitation–secretion coupling. In addition to mediating Ca²⁺ entry to initiate transmitter release, they are thought to interact directly with proteins of the synaptic vesicle docking/fusion machinery. As outlined in the preceding article, these calcium channels can be purified from brain as a complex with SNARE proteins which are involved in exocytosis. In addition, N-type and P/Q-type calcium channels are co-localized with syntaxin in high-density clusters in nerve terminals. Here we review the role of the synaptic protein interaction (synprint) sites in the intracellular loop II–III (L_II–III) of both _1B and _1A subunits of N-type and P/Q-type calcium channels, which bind to syntaxin, SNAP-25, and synaptotagmin. Calcium has a biphasic effect on the interactions of N-type calcium channels with SNARE complexes, stimulating optimal binding in the range of 10–20 M. PKC or CaM KII phosphorylation of the N-type synprint peptide inhibits interactions with native brain SNARE complexes containing syntaxin and SNAP-25. Introduction of the synprint peptides into presynaptic superior cervical ganglion neurons reversibly inhibits EPSPs from synchronous transmitter release by 42%. At physiological Ca²⁺ concentrations, synprint peptides cause an approximate 25% reduction in transmitter release of injected frog neuromuscular junction in cultures, consistent with detachment of 70% of the docked vesicles from calcium channels based on a theoretical model. Together, these studies suggest that presynaptic calcium channels not only provide the calcium signal required by the exocytotic machinery, but also contain structural elements that are integral to vesicle docking, priming, and fusion processes.     

Dominant role of lipid rafts L-type calcium channel in activity-dependent potentiation of large dense-core vesicle exocytosis

Calcium influx triggers exocytosis by promoting vesicle fusion with the plasma membrane. However, different subtypes of voltage-gated calcium channel (VGCC) have distinct roles in exocytosis. We previously reported that repetitive stimulation induces activity-dependent potentiation (ADP) which represents the increase of neurotransmitter release. Here, we show that L-type VGCC have a dominant role in ADP of large dense-core vesicle (LDCV) exocytosis. Repetitive stimulation activating VGCC can induce ADP, whereas activation of bradykinin (BK) G protein-coupled receptors or purinergic P2X cation channels can not. L-type VGCC has the dominant role in ADP of LDCV exocytosis by regulating Protein Kinase C (PKC)-epsilon translocation and phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS), a target molecule of PKC-epsilon. We provide evidence that L-type VGCC, PKC-epsilon, and MARCKS, but not Q-type VGCC, are selectively located in lipid rafts. Also, PKC-epsilon translocation induced by L-type VGCC activation occurs in lipid rafts. Disruption of lipid rafts abolishes ADP of LDCV exocytosis and changes the fusion pore kinetics without affecting the first stimulation-induced exocytosis, showing that lipid rafts are involved in the potentiation process. Taken together, we suggest that L-type VGCC in lipid rafts selectively mediates ADP of LDCV exocytosis by regulating PKC-epsilon translocation and MARCKS phosphorylation.     

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.     

Synaptotagmins in membrane traffic: which vesicles do the tagmins tag?

The aim of this review is to give a broad picture of what is actually known about the synaptotagmin family. Synaptotagmin I is an abundant synaptic vesicle and secretory granule protein in neurons and endocrine cells which plays a key role in Ca(2+)-induced exocytosis. It belongs to the large family of C2 domain-proteins as it contains two internal repeats that have homology to the C2 domain of protein kinase C. Eleven synaptotagmin genes have been described in rat and mouse. Except for synaptotagmin I, and by analogy synaptotagmin II, the functions of these proteins are unknown. In this review we focus on data obtained on the various isoforms without exhaustively discussing the role of synaptotagmin I in neurotransmission. Numerous in vitro interactions of synaptotagmin I with key components of the exocytosis-endocytosis machinery have been reported. Additional data concerning the other synaptotagmins are now becoming available and are reviewed here. Only interactions which have been described for several synaptotagmins, are mentioned. It is unlikely that a single isoform displays all of these potential interactions in vivo and probably the subcellular distribution of the protein will favor some of them and preclude others. Therefore, to discuss the putative role of the various synaptotagmins we have examined in detail published data concerning their localization.     

Molecular regulation of membrane resealing in 3T3 fibroblasts

Membrane resealing in mammalian cells after injury depends on Ca(2+)-dependent fusion of intracellular vesicles with the plasma membrane. When cells are wounded twice, the subsequent resealing is generally faster. Physiological and biochemical studies have shown the initiation of two different repair signaling pathways, which are termed facilitated and potentiated responses. The facilitated response is dependent on the generation and recruitment of new vesicles, whereas the potentiated response is not. Here, we report that the two responses can be differentially defined molecularly. Using recombinant fragments of synaptobrevin-2 and synaptotagmin C2 domains we were able to dissociate the molecular requirements of vesicle exocytosis for initial membrane resealing and the facilitated and potentiated responses. The initial resealing response was blocked by fragments of synaptobrevin-2 and the C2B domain of synaptotagmin VII. Both the facilitated and potentiated responses were also blocked by the C2B domain of synaptotagmin VII. Although the initial resealing response was not blocked by the C2AB domain of synaptotagmin I or the C2A domain of synaptotagmin VII, recruitment of new vesicles for the facilitated response was inhibited. We also used Ca2+ binding mutant studies to show that the effects of synaptotagmins on membrane resealing are Ca(2+)-dependent. The pattern of inhibition by synaptotagmin C2 fragments that we observed cannot be used to specify a vesicle compartment, such as lysosomes, in membrane repair.