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Synaptotagmin-1 (Syt1) is a vesicular calcium sensor required for synchronous neurotransmitter release, composed of a single-pass transmembrane domain linked to two C2 domains (C2A and C2B) that bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. Despite its essential role, how Syt1 couples calcium entry to synchronous release is poorly understood. Calcium binding to C2B is critical for synchronous release, and C2B additionally binds the SNARE complex. The C2A domain is also required for Syt1 function, but it is not clear why. Here, we asked what critical feature of C2A may be responsible for its functional role and compared this to the analogous feature in C2B. We focused on highly conserved poly-lysine patches located on the sides of C2A (K189-192) and C2B (K324-327). We tested effects of charge-neutralization mutations in either region (Syt1(K189-192A) and Syt1(K326-327A)) side by side to determine their relative contributions to Syt1 function in cultured cortical neurons from mice of either sex and in single-molecule experiments. Combining electrophysiological recordings and optical tweezers measurements to probe dynamic single C2 domain–membrane interactions, we show that both C2A and C2B polybasic patches contribute to membrane binding, and both are required for evoked release. The size of the readily releasable vesicle pool and the rate of spontaneous release were unaffected, so both patches are likely required specifically for synchronization of release. We suggest these patches contribute to cooperative membrane binding, increasing the overall affinity of Syt1 for negatively charged membranes and facilitating evoked release. SIGNIFICANCE STATEMENT Synaptotagmin-1 is a vesicular calcium sensor required for synchronous neurotransmitter release. Its tandem cytosolic C2 domains (C2A and C2B) bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. How calcium binding to Synaptotagmin-1 leads to release and the relative contributions of the C2 domains are unclear. Combining electrophysiological recordings from cultured neurons and optical tweezers measurements of single C2 domain–membrane interactions, we show that conserved polybasic regions in both domains contribute to membrane binding cooperatively, and both are required for evoked release, likely by increasing the overall affinity of Synaptotagmin-1 for acidic membranes.

Granule-plasma membrane docking and fusion can only occur when proteins that enable these reactions are present at the granule-plasma membrane contact. Thus, the mobility of granule membrane proteins may influence docking, and membrane fusion. We measured the mobility of vesicle associated membrane protein 2 (VAMP2), synaptotagmin 1 (Syt1), and synaptotagmin 7 (Syt7) in chromaffin granule membranes in living chromaffin cells. We used a method that is not limited by standard optical resolution. A bright flash of strongly decaying evanescent field produced by total internal reflection (TIR) was used to photobleach GFP-labeled proteins in the granule membrane. Fluorescence recovery occurs as unbleached protein in the granule membrane distal from the glass interface diffuses into the more bleached proximal regions, enabling the measurement of diffusion coefficients. We found that VAMP2-EGFP and Syt7-EGFP are mobile with a diffusion coefficient of approximately 3 × 10-10 cm2/s. Syt1-EGFP mobility was below the detection limit. Utilizing these diffusion parameters, we estimated the time required for these proteins to arrive at docking and nascent fusion sites to be many tens of milliseconds. Our analyses raise the possibility that the diffusion characteristics of VAMP2 and Syt proteins could be a factor that influences the rate of exocytosis.

Synaptic vesicle (SV) exocytosis is mediated by SNARE proteins. Reconstituted SNAREs are constitutively active, so a major focus has been to identify fusion clamps that regulate their activity in synapses: the primary candidates are synaptotagmin (syt) 1 and complexin I/II. Syt1 is a Ca2+ sensor for SV release that binds Ca2+ via tandem C2-domains, C2A and C2B. Here, we first determined whether these C2-domains execute distinct functions. Remarkably, the C2B domain profoundly clamped all forms of SV fusion, despite synchronizing residual evoked release and rescuing the readily-releasable pool. Release was strongly enhanced by an adjacent C2A domain, and by the concurrent binding of complexin to trans-SNARE complexes. Knockdown of complexin had no impact on C2B-mediated clamping of fusion. We postulate that the C2B domain of syt1, independent of complexin, is the molecular clamp that arrests SVs prior to Ca2+-triggered fusion., The molecular identity of the clamp that arrests the fusion machinery such that synaptic vesicles are docked and primed to release neurotransmitters remains controversial. In this study, the authors use truncation mutants of synaptotagmin (syt) 1 and animal models to demonstrate that the C2B domain of syt1, and not complexin, is solely responsible for the reduction of the spontaneous release at the presynapse

Exocytosis mediates the release of neurotransmitters and hormones from neurons and neuroendocrine cells. Tandem C2 domain proteins in the synaptotagmin (syt) and double C2 domain (Doc2) families regulate exocytotic membrane fusion via direct interactions with Ca(2+) and phospholipid bilayers. Syt1 is a fast-acting, low-affinity Ca(2+) sensor that penetrates membranes upon binding Ca(2+) to trigger synchronous vesicle fusion. The closely related Doc2β is a slow-acting, high-affinity Ca(2+) sensor that triggers spontaneous and asynchronous vesicle fusion, but whether it also penetrates membranes is unknown. Both syt1 and Doc2β bind the dynamically regulated plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP(2)), but it is unclear whether PIP(2) serves only as a membrane contact or enables specialized membrane-binding modes by these Ca(2+) sensors. Furthermore, it has been shown that PIP(2) uncaging can trigger rapid, syt1-dependent exocytosis in the absence of Ca(2+) influx, suggesting that current models for the action of these Ca(2+) sensors are incomplete. Here, using a series of steady-state and time-resolved fluorescence measurements, we show that Doc2β, like syt1, penetrates membranes in a Ca(2+)-dependent manner. Unexpectedly, we observed that PIP(2) can drive membrane penetration by both syt1 and Doc2β in the absence of Ca(2+), providing a plausible mechanism for Ca(2+)-independent, PIP(2)-dependent exocytosis. Quantitative measurements of penetration depth revealed that, in the presence of Ca(2+), PIP(2) drives Doc2β, but not syt1, substantially deeper into the membrane, defining a biophysical regulatory mechanism specific to this high-affinity Ca(2+) sensor. Our results provide evidence of a novel role for PIP(2) in regulating, and under some circumstances triggering, exocytosis.

Significance Synaptotagmin 1 (syt1) is a synaptic vesicle (SV) protein that is rapidly activated by Ca2+ influx into presynaptic nerve terminals, triggering SV exocytosis. Syt1 also inhibits exocytosis, prior to Ca2+ influx, and thus helps synchronize evoked exocytosis upon Ca2+ binding. Herein, we identified a cluster of lysine residues, in the oft-ignored juxtamembrane linker region of syt1, that governs homo-multimerization in an anionic lipid-dependent manner. Neutralization of this positively charged region abolished syt1 self-association on phospholipid bilayers in vitro. Subsequently, in neurons, we found mutations that disrupted syt1 self-association were correlated with defects in clamping spontaneous SV release and in triggering and synchronizing evoked exocytosis. Thus, syt1 regulates SV exocytosis as an oligomer via charged residues in the juxtamembrane linker., Synaptotagmin 1 (syt1) is a Ca2+ sensor that regulates synaptic vesicle exocytosis. Cell-based experiments suggest that syt1 functions as a multimer; however, biochemical and electron microscopy studies have yielded contradictory findings regarding putative self-association. Here, we performed dynamic light scattering on syt1 in solution, followed by electron microscopy, and we used atomic force microscopy to study syt1 self-association on supported lipid bilayers under aqueous conditions. Ring-like multimers were clearly observed. Multimerization was enhanced by Ca2+ and required anionic phospholipids. Large ring-like structures (∼180 nm) were reduced to smaller rings (∼30 nm) upon neutralization of a cluster of juxtamembrane lysine residues; further substitution of residues in the second C2-domain completely abolished self-association. When expressed in neurons, syt1 mutants with graded reductions in self-association activity exhibited concomitant reductions in 1) clamping spontaneous release and 2) triggering and synchronizing evoked release. Thus, the juxtamembrane linker of syt1 plays a crucial role in exocytosis by mediating multimerization.

Synaptotagmin-1 (Syt1) is a vesicular calcium sensor required for synchronous neurotransmitter release. It is composed of a single-pass transmembrane domain linked to two tandem C2 domains (C2A and C2B) that bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. Despite its essential role, how Syt1 couples calcium entry to synchronous release is not well understood. Calcium binding to C2B, but not to C2A, is critical for synchronous release and C2B additionally binds the SNARE complex. The C2A domain is also required for Syt1 function, but it is not clear why. Here we asked what critical feature of C2A may be responsible for its functional role, and compared this to the analogous feature in C2B. We focused on highly conserved poly-lysine patches located on the sides of C2A (K189-192) and C2B (K324-327). We tested effects of charge-neutralization mutations in either region (Syt1K189-192A and Syt1K326-327A) side-by-side to determine their relative contributions to Syt1 function in cultured cortical mouse neurons and in single-molecule experiments. Combining electrophysiological recordings and optical tweezers measurements to probe dynamic single C2 domain-membrane interactions, we show that both C2A and C2B polybasic patches contribute to membrane binding, and both are required for evoked release. The readily releasable vesicle pool or spontaneous release were not affected, so both patches are specifically required for synchronization of release. We suggest these patches contribute to cooperative binding to membranes, increasing the overall affinity of Syt1 for negatively charged membranes and facilitating evoked release.Significance StatementSynaptotagmin-1 is a vesicular calcium sensor required for synchronous neurotransmitter release. Its tandem cytosolic C2 domains (C2A and C2B) bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. How calcium-binding to Synaptotagmin-1 leads to release and the relative contributions of the two C2 domains is not clear: unlike C2B, calcium-binding to C2A is not critical for evoked release, yet both domains are needed for Syt1 function. Combining electrophysiological recordings from cultured neurons and optical tweezers measurements that probe single C2 domain-membrane interactions, we show that conserved polybasic regions in both domains contribute to membrane binding cooperatively, and both are required for evoked release, likely by increasing the overall affinity of Synaptotagmin-1 for negatively charged membranes.

This study provides novel mechanistic insights into how Syt-1 and Syt-7 C2B domains regulate the later stages of exocytosis. We show that small structural differences within this region exert significant effects on fusion pore properties. We propose that these effects may arise from differences in C2B domain affinity for the plasma membrane., In chromaffin cells, the kinetics of fusion pore expansion vary depending on which synaptotagmin isoform (Syt-1 or Syt-7) drives release. Our recent studies have shown that fusion pores of granules harboring Syt-1 expand more rapidly than those harboring Syt-7. Here we sought to define the structural specificity of synaptotagmin action at the fusion pore by manipulating the Ca2+-binding C2B module. We generated a chimeric Syt-1 in which its C2B Ca2+-binding loops had been exchanged for those of Syt-7. Fusion pores of granules harboring a Syt-1 C2B chimera with all three Ca2+-binding loops of Syt-7 (Syt-1:7C2B123) exhibited slower rates of fusion pore expansion and neuropeptide cargo release relative to WT Syt-1. After fusion, this chimera also dispersed more slowly from fusion sites than WT protein. We speculate that the Syt-1:7 C2B123 and WT Syt-1 are likely to differ in their interactions with Ca2+ and membranes. Subsequent in vitro and in silico data demonstrated that the chimera exhibits a higher affinity for phospholipids than WT Syt-1. We conclude that the affinity of synaptotagmin for the plasma membrane, and the rate at which it releases the membrane, contribute in important ways to the rate of fusion pore expansion.

Summary Neurotransmission is a tightly regulated Ca 2+ -dependent process. Upon Ca 2+ influx, Synaptotagmin1 (Syt1) promotes fusion of synaptic vesicles (SVs) with the plasma membrane. This requires regulation at multiple levels, but the role of metabolites in SV release is unclear. Here, we uncover a role for isocitrate dehydrogenase 3a ( idh3a ), a Krebs cycle enzyme, in neurotransmission. Loss of idh3a leads to a reduction of the metabolite, alpha-ketoglutarate (αKG), causing defects in synaptic transmission similar to the loss of syt1. Supplementing idh3a flies with αKG suppresses these defects through an ATP or neurotransmitter-independent mechanism. Indeed, αKG, but not glutamate, enhances Syt1-dependent fusion in a reconstitution assay. αKG promotes interaction between the C2-domains of Syt1 and phospholipids. The data reveal conserved metabolic regulation of synaptic transmission via αKG. Our studies provide a synaptic role for αKG, a metabolite that has been proposed as a treatment for aging and neurodegenerative disorders.

Synaptotagmin (Syt)-7, a major component of the exocytotic machinery in neurons, is also the major Syt in rodent pancreatic β-cells shown to mediate glucose-stimulated insulin secretion (GSIS). However, Syt-7’s precise exocytotic actions in β-cells remain unknown. We show that Syt-7 is abundant in human β-cells. Adenovirus–short hairpin RNA knockdown (KD) of Syt-7 in human islets reduced first- and second-phase GSIS attributed to the reduction of exocytosis of predocked and newcomer insulin secretory granules (SGs). Glucose stimulation expectedly induced Syt-7 association in a Ca2+-dependent manner with syntaxin-3 and syntaxin-1A soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complexes known to mediate exocytosis of newcomer and predocked SGs, respectively. However, Syt-7-KD did not disrupt SNARE complex assembly. Instead, electron microscopy analysis showed that Syt-7-KD reduced the recruitment of SGs to the plasma membrane after glucose-stimulated depletion, which could not be rescued by glucagon-like peptide 1 pretreatment. To assess the possibility that this new action of Syt-7 on SG recruitment may involve calmodulin (CaM), pretreatment of islets with CaM blocker calmidazolium showed effects very similar to those of Syt-7-KD. Syt-7 therefore plays a novel more dominant function in the replenishment of releasable SG pools in human β-cells than its previously purported role in exocytotic fusion per se.

SNARE proteins catalyze many forms of biological membrane fusion, including Ca(2+)-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: Ca(2+)-triggered dense core vesicle exocytosis, spontaneous synaptic vesicle exocytosis, and Ca(2+)-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.

Various forms of synaptic plasticity underlie aspects of learning and memory. Synaptic augmentation is a form of short-term plasticity characterized by synaptic enhancement that persists for seconds following specific patterns of stimulation. The mechanisms underlying this form of plasticity are unclear but are thought to involve residual presynaptic Ca2+. Here, we report that augmentation was reduced in cultured mouse hippocampal neurons lacking the Ca2+ sensor, Doc2; other forms of short-term enhancement were unaffected. Doc2 binds Ca2+ and munc13 and translocates to the plasma membrane to drive augmentation. The underlying mechanism was not associated with changes in readily releasable pool size or Ca2+ dynamics, but rather resulted from superpriming a subset of synaptic vesicles. Hence, Doc2 forms part of the Ca2+-sensing apparatus for synaptic augmentation via a mechanism that is molecularly distinct from other forms of short-term plasticity.

Spontaneous neurotransmitter release (mini) is an important form of Ca(2+)-dependent synaptic transmission that occurs in the absence of action potentials. A molecular understanding of this process requires an identification of the underlying Ca(2+)-sensors. Here, we address the roles of the relatively low- and high-affinity Ca(2+) sensors, synapotagmin-1 (syt1) and Doc2α/β, respectively. We found that both syt1 and Doc2 regulate minis but, surprisingly, their relative contributions depend on whether release was from excitatory or inhibitory neurons. Doc2α promoted glutamatergic minis, while Doc2β and syt1 both regulated GABAergic minis. We identified Ca(2+) ligand mutations in Doc2 that either disrupted or constitutively activated the regulation of minis. Finally, Ca(2+) entry via voltage-gated Ca(2+) channels triggered miniature GABA release by activating syt1, but had no effect on Doc2-driven minis. This work reveals an unexpected divergence in the regulation of spontaneous excitatory and inhibitory transmission in terms of both Ca(2+) sensors and sources.

SUMMARY Syntaphilin (SNPH) is a major mitochondrial anchoring protein targeted to axons and excluded from dendrites. In this study, we provide in vivo evidence that this spatial specificity is lost in Shiverer (Shi) mice, a model for progressive multiple sclerosis (MS), resulting in inappropriate intrusion of SNPH into dendrites of cerebellar Purkinje cells with neurodegenerative consequences. Thus, reconstituting dendritic SNPH intrusion in SNPH-KO mice by viral transduction greatly sensitizes Purkinje cells to excitotoxicity when the glutamatergic climbing fibers are stimulated. Finally, we demonstrate in vitro that overexpression of SNPH in dendrites compromises neuronal viability by inducing N-methyl-D-aspartate (NMDA) excitotoxicity, reducing mitochondrial calcium uptake, and interfering with quality control of mitochondria by blocking somal mitophagy. Collectively, we propose that inappropriate immobilization of dendritic mitochondria by SNPH intrusion produces excitotoxicity and suggest that interception of dendritic SNPH intrusion is a therapeutic strategy to combat neurodegeneration., In Brief Joshi et al. show that an axon-specific mitochondrial anchoring protein, Syntaphilin, invades dendrites in the progressive multiple sclerosis (MS) mouse model Shiverer. Dendritic SNPH intrusion sensitizes neurons to excitotoxicity, alters mitochondrial Ca2+ buffering, and impedes somal mitophagy, suggesting interception of dendritic SNPH intrusion as a therapeutic strategy in MS., Graphical Abstract

In 1992, Brose et al. showed that the synaptic vesicle (SV) protein p65/synaptotagmin (syt) 1 binds - in a mutually dependent manner - Ca(2+) and anionic phospholipids, prompting the idea that it functions as a Ca(2+) sensor for exocytosis. These findings now define two key aspects of excitation-secretion coupling.

Adrenal chromaffin cells express two synaptotagmin isoforms, Syt-1 and Syt-7. Isoforms are usually sorted to separate secretory granules, are differentially activated by depolarizing stimuli, and favor discrete modes of exocytosis. It is proposed that stimulus/Ca+-dependent secretion in the chromaffin cell relies on selective Syt isoform activation., Adrenal chromaffin cells release hormones and neuropeptides that are essential for physiological homeostasis. During this process, secretory granules fuse with the plasma membrane and deliver their cargo to the extracellular space. It was once believed that fusion was the final regulated step in exocytosis, resulting in uniform and total release of granule cargo. Recent evidence argues for nonuniform outcomes after fusion, in which cargo is released with variable kinetics and selectivity. The goal of this study was to identify factors that contribute to the different outcomes, with a focus on the Ca2+-sensing synaptotagmin (Syt) proteins. Two Syt isoforms are expressed in chromaffin cells: Syt-1 and Syt-7. We find that overexpressed and endogenous Syt isoforms are usually sorted to separate secretory granules and are differentially activated by depolarizing stimuli. In addition, overexpressed Syt-1 and Syt-7 impose distinct effects on fusion pore expansion and granule cargo release. Syt-7 pores usually fail to expand (or reseal), slowing the dispersal of lumenal cargo proteins and granule membrane proteins. On the other hand, Syt-1 diffuses from fusion sites and promotes the release of lumenal cargo proteins. These findings suggest one way in which chromaffin cells may regulate cargo release is via differential activation of synaptotagmin isoforms.

Synaptotagmin (syt)-1 is a Ca2+ sensor that triggers rapid synaptic vesicle exocytosis. Mutations that disrupt physical interactions between the tandem Ca2+-sensing modules (C2 domains) of syt-1 disrupt regulated membrane fusion in reconstituted fusion reactions and in neurons. Hence contacts between these domains are important for function., C2 domains are widespread motifs that often serve as Ca2+-binding modules; some proteins have more than one copy. An open issue is whether these domains, when duplicated within the same parent protein, interact with one another to regulate function. In the present study, we address the functional significance of interfacial residues between the tandem C2 domains of synaptotagmin (syt)-1, a Ca2+ sensor for neuronal exocytosis. Substitution of four residues, YHRD, at the domain interface, disrupted the interaction between the tandem C2 domains, altered the intrinsic affinity of syt-1 for Ca2+, and shifted the Ca2+ dependency for binding to membranes and driving membrane fusion in vitro. When expressed in syt-1 knockout neurons, the YHRD mutant yielded reductions in synaptic transmission, as compared with the wild-type protein. These results indicate that physical interactions between the tandem C2 domains of syt-1 contribute to excitation–secretion coupling.

In vitro reconstitution fusion assays incorporating full-length membrane-anchored synaptotagmin I clarify its role in several steps in the secretory pathway., 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 (

Synaptotagmin-1 is a Ca(2+) sensor that triggers synchronous neurotransmitter release. The first documented biochemical property of synaptotagmin-1 was its ability to aggregate membranes in response to Ca(2+). However, the mechanism and function of this process were poorly understood. Here we show that synaptotagmin-1-mediated vesicle aggregation is driven by trans interactions between synaptotagmin-1 molecules bound to different membranes. We found a strong correlation between the ability of Ca(2+)-bound synaptotagmin-1 to aggregate vesicles and to stimulate SNARE-mediated membrane fusion. Moreover, artificial aggregation of membranes-using non-synaptotagmin proteins-also efficiently promoted fusion of SNARE-bearing liposomes. Finally, using a modified fusion assay, we observed that synaptotagmin-1 drove the assembly of otherwise non-fusogenic individual t-SNARE proteins into fusion-competent heterodimers, independently of aggregation. Thus, membrane aggregation and t-SNARE assembly appear to be two key aspects of fusion reactions that are regulated by Ca(2+)-bound synaptotagmin-1 and catalyzed by SNAREs.

The vesicle protein synaptotagmin I is the Ca2+sensor that triggers fast, synchronous release of neurotransmitter. Specifically, Ca2+binding by the C2B domain of synaptotagmin is required at intact synapses, yet the mechanism whereby Ca2+binding results in vesicle fusion remains controversial. Ca2+-dependent interactions between synaptotagmin and SNARE (solubleN-ethylmaleimide-sensitive fusion protein attachment receptor) complexes and/or anionic membranes are possible effector interactions. However, no effector-interaction mutations to date impact synaptic transmission as severely as mutation of the C2B Ca2+-binding motif, suggesting that these interactions are facilitatory rather than essential. Here we useDrosophilato show the functional role of a highly conserved, hydrophobic residue located at the tip of each of the two Ca2+-binding pockets of synaptotagmin. Mutation of this residue in the C2A domain (F286) resulted in a ∼50% decrease in evoked transmitter release at an intact synapse, again indicative of a facilitatory role. Mutation of this hydrophobic residue in the C2B domain (I420), on the other hand, blocked all locomotion, was embryonic lethal even in syt I heterozygotes, and resulted in less evoked transmitter release than that insytnullmutants, which is more severe than the phenotype of C2B Ca2+-binding mutants. Thus, mutation of a single, C2B hydrophobic residue required for Ca2+-dependent penetration of anionic membranes results in the most severe disruption of synaptotagmin functionin vivoto date. Our results provide direct support for the hypothesis that plasma membrane penetration, specifically by the C2B domain of synaptotagmin, is the critical effector interaction for coupling Ca2+binding with vesicle fusion.

Synaptotagmin isoforms and mutants altered fusion event frequency and fusion pore transitions. These effects showed a strong correlation with PS binding, but not with SNARE binding. Synaptotagmin-PS interaction thus function in two distinct kinetic steps in Ca2+ triggered exocytosis, and stabilize open fusion pores., Synaptotagmin (syt) serves as a Ca2+ sensor in the release of neurotransmitters and hormones. This function depends on the ability of syt to interact with other molecules. Syt binds to phosphatidylserine (PS)-containing lipid bilayers as well as to soluble N-ethylmaleimide sensitive factor receptors (SNAREs) and promotes SNARE assembly. All these interactions are regulated by Ca2+, but their specific roles in distinct kinetic steps of exocytosis are not well understood. To explore these questions we used amperometry recording from PC12 cells to investigate the kinetics of exocytosis. Syt isoforms and syt I mutants were overexpressed to perturb syt-PS and syt-SNARE interactions to varying degrees and evaluate the effects on fusion event frequency and the rates of fusion pore transitions. Syt I produced more rapid dilation of fusion pores than syt VII or syt IX, consistent with its role in synchronous synaptic release. Stronger syt-PS interactions were accompanied by a higher frequency of fusion events and more stable fusion pores. By contrast, syt-SNARE interactions and syt-induced SNARE assembly were uncorrelated with rates of exocytosis. This associates the syt-PS interaction with two distinct kinetic steps in Ca2+ triggered exocytosis and supports a role for the syt-PS interaction in stabilizing open fusion pores.

The synaptic vesicle protein synaptotagmin I (Syt I) binds phosphatidylserine (PS) in a Ca2+-dependent manner. This interaction is thought to play a role in exocytosis, but its precise functions remain unclear. To determine potential roles for Syt I-PS binding, we varied the PS content in PC12 cells and liposomes and studied the effects on the kinetics of exocytosis and Syt I binding in parallel. Raising PS produced a steeply nonlinear, saturating increase in Ca2+-triggered fusion, and a graded slowing of the rate of fusion pore dilation. Ca2+-Syt I bound liposomes more tightly as PS content was raised, with a steep increase in binding at low PS, and a further gradual increase at higher PS. These two phases in the PS dependence of Ca2+-dependent Syt I binding to lipid may correspond to the two distinct and opposing kinetic effects of PS on exocytosis. PS influences exocytosis in two ways, enhancing an early step leading to fusion pore opening, and slowing a later step when fusion pores dilate. The possible relevance of these results to Ca2+-triggered Syt I binding is discussed along with other possible roles of PS.

Many members of the synaptotagmin (Syt) protein family bind Ca(2+) and trigger exocytosis, but some Syt proteins appear to have no Ca(2+)-dependent actions and their biological functions remain obscure. Syt IV is an activity-induced brain protein with no known Ca(2+)-dependent interactions and its subcellular localization and biological functions have sparked considerable controversy. We found Syt IV on both micro- and dense-core vesicles in posterior pituitary nerve terminals in mice. In terminals from Syt IV knockout mice compared with those from wild types, low Ca(2+) entry triggered more exocytosis, high Ca(2+) entry triggered less exocytosis and endocytosis was accelerated. In Syt IV knockouts, dense-core and microvesicle fusion was enhanced in cell-attached patches and dense-core vesicle fusion pores had conductances that were half as large as those in wild types. Given the neuroendocrine functions of the posterior pituitary, changes in Syt IV levels could be involved in endocrine transitions involving alterations in the release of the neuropeptides oxytocin and vasopressin.

Synaptotagmin I is the Ca2+sensor for fast, synchronous release of neurotransmitter; however, the molecular interactions that couple Ca2+binding to membrane fusion remain unclear. The structure of synaptotagmin is dominated by two C2domains that interact with negatively charged membranes after binding Ca2+.In vitrowork has implicated a conserved basic residue at the tip of loop 3 of the Ca2+-binding pocket in both C2domains in coordinating this electrostatic interaction with anionic membranes. Although results from cultured cells suggest that the basic residue of the C2A domain is functionally significant, such studies provide contradictory results regarding the importance of the C2B basic residue during vesicle fusion. To directly test the functional significance of each of these residues at an intact synapsein vivo, we neutralized either the C2A or the C2B basic residue and assessed synaptic transmission at theDrosophilaneuromuscular junction. The conserved basic residues at the tip of the Ca2+-binding pocket of both the C2A and C2B domains mediate Ca2+-dependent interactions with anionic membranes and are required for efficient evoked transmitter release. Our results directly support the hypothesis that the interactions between synaptotagmin and the presynaptic membrane, which are mediated by the basic residues at the tip of both the C2A and C2B Ca2+-binding pockets, are critical for coupling Ca2+influx with vesicle fusion during synaptic transmissionin vivo. Our model for synaptotagmin's direct role in coupling Ca2+binding to vesicle fusion incorporates this finding with results from multiplein vitroandin vivostudies.

Neurotransmitter release at synapses involves a highly specialized form of membrane fusion that is triggered by Ca2+ ions and is optimized for speed. These observations were established decades ago, but only recently have the molecular mechanisms that underlie this process begun to come into view. Here, we summarize findings obtained from genetically modified neurons and neuroendocrine cells, as well as from reconstituted systems, which are beginning to reveal the molecular mechanism by which Ca2+—acting on the synaptic vesicle (SV) protein synaptotagmin I (syt)—triggers rapid exocytosis. This work sheds light not only on presynaptic aspects of synaptic transmission, but also on the fundamental problem of membrane fusion, which has remained a puzzle that has yet to be solved in any biological system.

Ca(2+)-triggered exocytosis in neurons and neuroendocrine cells is regulated by the Ca(2+)-binding protein synaptotagmin (syt) I. Sixteen additional isoforms of syt have been identified, but little is known concerning their biochemical or functional properties. Here, we assessed the abilities of fourteen syt isoforms to directly regulate SNARE (soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor)-catalyzed membrane fusion. One group of isoforms stimulated neuronal SNARE-mediated fusion in response to Ca(2+), while another set inhibited SNARE catalyzed fusion in both the absence and presence of Ca(2+). Biochemical analysis revealed a strong correlation between the ability of syt isoforms to bind 1,2-dioleoyl phosphatidylserine (PS) and t-SNAREs in a Ca(2+)-promoted manner with their abilities to enhance fusion, further establishing PS and SNAREs as critical effectors for syt action. The ability of syt I to efficiently stimulate fusion was specific for certain SNARE pairs, suggesting that syts might contribute to the specificity of intracellular membrane fusion reactions. Finally, a subset of inhibitory syts down-regulated the ability of syt I to activate fusion, demonstrating that syt isoforms can modulate the function of each other.

During exocytosis, fusion pores form the first aqueous connection that allows escape of neurotransmitters and hormones from secretory vesicles. Although it is well established that SNARE proteins catalyze fusion, the structure and composition of fusion pores remain unknown. Here, we exploited the rigid framework and defined size of nanodiscs to interrogate the properties of reconstituted fusion pores, using the neurotransmitter glutamate as a content-mixing marker. Efficient Ca(2+)-stimulated bilayer fusion, and glutamate release, occurred with approximately two molecules of mouse synaptobrevin 2 reconstituted into ∼6-nm nanodiscs. The transmembrane domains of SNARE proteins assumed distinct roles in lipid mixing versus content release and were exposed to polar solvent during fusion. Additionally, tryptophan substitutions at specific positions in these transmembrane domains decreased glutamate flux. Together, these findings indicate that the fusion pore is a hybrid structure composed of both lipids and proteins.

The anterior pituitary releases six different hormones that control virtually all aspects of vertebrate physiology, yet the molecular mechanisms underlying their Ca2+-triggered release remain unknown. A subset of the synaptotagmin (syt) family of proteins serve as Ca2+ sensors for exocytosis in neurons and neuroendocrine cells, and are thus likely to regulate pituitary hormone secretion. Here we show that numerous syt isoforms are highly expressed in the pituitary gland in a lobe, and sex-specific manner. We further investigated a Ca2+-activated isoform, syt-9, and found that it is expressed in a subpopulation of anterior pituitary cells, the gonadotropes. Follicle-stimulating hormone (FSH) and syt-9 are highly co-localized in female, but not male, mice. Loss of syt-9 results in diminished basal and stimulated FSH secretion only in females, resulting in alterations in the oestrus cycle. This work uncovers a new function for syt-9 and reveals a novel sex difference in reproductive hormone secretion., Pituitary gonadotropes secrete follicle-stimulating hormone (FSH) in both male and female mice. Roper et al. show that the synaptotagmin isoform syt-9 is specifically required for FSH secretion, but only in females, revealing a mechanism by which specificity can be encoded in complex secretory cells.

Significance Evoked synaptic transmission is mediated by synchronous and asynchronous phases of neurotransmitter release. Synaptotagmin 1 (syt1) serves as the Ca 2+ sensor for synchronous release. Recently, we proposed that double C2-like domain-containing protein alpha and beta (Doc2α and Doc2β), cytosolic proteins with tandem C2 domains homologous to syt1, function as Ca 2+ sensors for asynchronous release, but this idea remains controversial. Here, we systematically analyzed the functional significance of each Ca 2+ ligand in Doc2β and found a correlation between the Ca 2+ -dependent translocation activity of these mutants (to the plasma membrane) and changes in asynchronous release. Moreover, we show that syt1–Doc2β chimeras exhibit altered kinetics in vitro and change the rates of synaptic transmission in cultured neurons. These results establish Doc2β as a Ca 2+ sensor for the slow phase of neurotransmission.

Synaptotagmin (Syt) VII is a ubiquitously expressed member of the Syt family of Ca2+ sensors. It is present on lysosomes in several cell types, where it regulates Ca2+-dependent exocytosis. Because [Ca2+]i and exocytosis have been associated with phagocytosis, we investigated the phagocytic ability of macrophages from Syt VII−/− mice. Syt VII−/− macrophages phagocytose normally at low particle/cell ratios but show a progressive inhibition in particle uptake under high load conditions. Complementation with Syt VII rescues this phenotype, but only when functional Ca2+-binding sites are retained. Reinforcing a role for Syt VII in Ca2+-dependent phagocytosis, particle uptake in Syt VII−/− macrophages is significantly less dependent on [Ca2+]i. Syt VII is concentrated on peripheral domains of lysosomal compartments, from where it is recruited to nascent phagosomes. Syt VII recruitment is rapidly followed by the delivery of Lamp1 to phagosomes, a process that is inhibited in Syt VII−/− macrophages. Thus, Syt VII regulates the Ca2+-dependent mobilization of lysosomes as a supplemental source of membrane during phagocytosis.

We show that activation of postsynaptic inositol 1,4,5-tris-phosphate receptors (IP3Rs) with the IP3R agonist adenophostin A (AdA) produces large increases in AMPA receptor (AMPAR) excitatory postsynaptic current (EPSC) amplitudes at hippocampal CA1 synapses. Co-perfusion of the Ca2+chelator bis-( o-aminophenoxy)- N,N,N′,N′-tetraacetic acid strongly inhibited AdA-enhanced increases in EPSC amplitudes. We examined the role of AMPAR insertion/anchoring in basal synaptic transmission. Perfusion of an inhibitor of synaptotagmin-soluble n-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor SNARE-mediated exocytosis depressed basal EPSC amplitudes, whereas a peptide that inhibits GluR2/3 interactions with postsynaptic density-95 (PDZ) domain proteins glutamate receptor interacting protein (GRIP)/protein interacting with C-kinase-1 (PICK1) enhanced basal synaptic transmission. These results suggest that constitutive trafficking and anchoring of AMPARs help maintain basal synaptic transmission. The regulation of postsynaptic AMPAR trafficking involves synaptotagmin-SNARE-mediated vesicle exocytosis and interactions between AMPARs and the PDZ domains in GRIP/PICK1. We show that inhibitors of synaptotagmin-SNARE-mediated exocytosis, or interactions between AMPARs and GRIP/PICK1, attenuated AdA-enhanced increases in EPSC amplitudes. These results suggest that IP3R-mediated Ca2+release can enhance AMPAR EPSC amplitudes through mechanisms that involve AMPAR-PDZ interactions and/or synaptotagmin-SNARE-mediated receptor trafficking.

Rapid signaling between neurons relies on the Ca(2+)-triggered exocytosis of neurotransmitters. Release is mediated by 'kiss-and-run' or complete fusion of secretory organelles with the plasma membrane. Current models indicate that exocytosis is regulated by synaptotagmin I (syt) and mediated by SNARE (soluble NSF-attachment protein receptor) proteins. Syt senses Ca(2+) via two conserved motifs, C2A and C2B. C2B engages a wider array of effector molecules than C2A and appears to play a more crucial role in synaptic transmission. However, it has recently become clear that the tandem C2 domains of syt influence each another in unexpected ways. Here, we focus on recent structure-function studies that are beginning to provide insights into the mechanism through which the C2 domains of syt trigger exocytosis.

The secretory vesicle protein synaptotagmin I (syt) plays a critical role in Ca2+-triggered exocytosis. Its cytoplasmic domain is composed of tandem C2 domains, C2A and C2B; each C2 domain binds Ca2+. Upon binding Ca2+, positively charged residues within the Ca2+-binding loops are thought to interact with negatively charged phospholipids in the target membrane to mediate docking of the cytoplasmic domain of syt onto lipid bilayers. The C2 domains of syt also interact with syntaxin and SNAP-25, two components of a conserved membrane fusion complex. Here, we have neutralized single positively charged residues at the membrane-binding interface of C2A (R233Q) and C2B (K366Q). Either of these mutations shifted the Ca2+ requirements for syt-liposome interactions from approximately 20 to approximately 40 microm Ca2+. Kinetic analysis revealed that the reduction in Ca2+-sensing activity was associated with a decrease in affinity for membranes. These mutations did not affect sytsyntaxin interactions but resulted in an approximately 50% loss in SNAP-25 binding activity, suggesting that these residues lie at an interface between membranes and SNAP-25. Expression of full-length versions of syt that harbored these mutations reduced the rate of exocytosis in PC12 cells. In both biochemical and functional assays, effects of the R233Q and K366Q mutations were not additive, indicating that mutations in one domain affect the activity of the adjacent domain. These findings indicate that the tandem C2 domains of syt cooperate with one another to trigger release via loop-mediated electrostatic interactions with effector molecules.

Exocytosis-the release of the contents of a vesicle--proceeds by two mechanisms. Full fusion occurs when the vesicle and plasma membranes merge. Alternatively, in what is termed kiss-and-run, vesicles can release transmitter during transient contacts with the plasma membrane. Little is known at the molecular level about how the choice between these two pathways is regulated. Here we report amperometric recordings of catecholamine efflux through individual fusion pores. Transfection with synaptotagmin (Syt) IV increased the frequency and duration of kiss-and-run events, but left their amplitude unchanged. Endogenous Syt IV, induced by forskolin treatment, had a similar effect. Full fusion was inhibited by mutation of a Ca2+ ligand in the C2A domain of Syt I; kiss-and-run was inhibited by mutation of a homologous Ca2+ ligand in the C2B domain of Syt IV. The Ca2+ sensitivity for full fusion was 5-fold higher with Syt I than Syt IV, but for kiss-and-run the Ca2+ sensitivities differed by a factor of only two. Syt thus regulates the choice between full fusion and kiss-and-run, with Ca2+ binding to the C2A and C2B domains playing an important role in this choice.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by an expanded CAG repeat in the HD gene. We reported recently that complexin II, a protein involved in neurotransmitter release, is depleted from both the brains of mice carrying the HD mutation and from the striatum of post mortem HD brains. Here we show that this loss of complexin II is recapitulated in PC12 cells expressing the HD mutation and is accompanied by a dramatic decline in Ca2+-triggered exocytosis of neurotransmitter. Overexpression of complexin II (but not complexin I) rescued exocytosis, demonstrating that the decline in neurotransmitter release is a direct consequence of complexin II depletion. Complexin II depletion in the brain may account for some of the abnormalities in neurotransmission associated with HD.

T he synaptotagmins (syts) are a family of membrane proteins proposed to regulate membrane traffic in neuronal and nonneuronal cells. In neurons, the Ca2+-sensing ability of syt I is critical for fusion of docked synaptic vesicles with the plasma membrane in response to stimulation. Several putative Ca2+–syt effectors have been identified, but in most cases the functional significance of these interactions remains unknown. Here, we have used recombinant C2 domains derived from the cytoplasmic domains of syts I–XI to interfere with endogenous syt–effector interactions during Ca2+-triggered exocytosis from cracked PC12 cells. Inhibition was closely correlated with syntaxin–SNAP-25 and phosphatidylinositol 4,5-bisphosphate (PIP2)–binding activity. Moreover, we measured the expression levels of endogenous syts in PC12 cells; the major isoforms are I and IX, with trace levels of VII. As expected, if syts I and IX function as Ca2+ sensors, fragments from these isoforms blocked secretion. These data suggest that syts trigger fusion via their Ca2+-regulated interactions with t-SNAREs and PIP2, target molecules known to play critical roles in exocytosis.

The Ca2+-binding synaptic-vesicle protein synaptotagmin I has attracted considerable interest as a potential Ca2+ sensor that regulates exocytosis from neurons and neuroendocrine cells. Recent studies have shed new light on the structure, biochemical/biophysical properties and function of synaptotagmin, and the emerging view is that it plays an important role in both exocytosis and endocytosis. At least a dozen additional isoforms exist, some of which are expressed outside of the nervous system, suggesting that synaptotagmins might regulate membrane traffic in a variety of cell types. Here we provide an overview of the members of this gene family, with particular emphasis on the question of whether and how synaptotagmin I functions during the final stages of membrane fusion: does it regulate the Ca2+-triggered opening and dilation of fusion pores?

Botulinum neurotoxins cleave synaptic SNAREs and block exocytosis, demonstrating that these proteins function in neurosecretion. However, the function of the SNARE syntaxin remains less clear because no neurotoxin cleaves it selectively. Starting with a botulinum neurotoxin which cleaves both syntaxin and SNAP-25, we engineered a version that retains activity against syntaxin but spares SNAP-25. These mutants block synaptic release in neurons and norepinephrine release in neuroendocrine cells, thus establishing an essential role for syntaxin in Ca2+-triggered exocytosis. These mutants can generate syntaxin-free cells as a useful experimental system for research, and may lead to pharmaceuticals that target syntaxin selectively.

Synaptotagmin (syt) 7 is one of three syt isoforms found in all metazoans; it is ubiquitously expressed, yet its function in neurons remains obscure. Here, we resolved Ca2+-dependent and Ca2+-independent synaptic vesicle (SV) replenishment pathways, and found that syt 7 plays a selective and critical role in the Ca2+-dependent pathway. Mutations that disrupt Ca2+-binding to syt 7 abolish this function, suggesting that syt 7 functions as a Ca2+-sensor for replenishment. The Ca2+-binding protein calmodulin (CaM) has also been implicated in SV replenishment, and we found that loss of syt 7 was phenocopied by a CaM antagonist. Moreover, we discovered that syt 7 binds to CaM in a highly specific and Ca2+-dependent manner; this interaction requires intact Ca2+-binding sites within syt 7. Together, these data indicate that a complex of two conserved Ca2+-binding proteins, syt 7 and CaM, serve as a key regulator of SV replenishment in presynaptic nerve terminals. DOI: http://dx.doi.org/10.7554/eLife.01524.001, eLife digest Neurons communicate with one another at junctions called synapses. The arrival of an electrical signal called an action potential at the first neuron triggers the release of chemicals called neurotransmitters into the synapse. These chemicals then diffuse across the gap between the neurons and bind to receptors on the second cell. The neurotransmitter molecules are stored in the first cell in packages known as vesicles, which release their contents by fusing with the cell membrane. Following a fusion event, neurons must replenish their vesicle stocks to ensure that they are ready for the arrival of the next action potential. This replenishment process is known to involve a calcium-dependent pathway and a calcium-independent pathway. A protein called calmodulin, that binds calcium ions, has an important role in the first of these pathways. Now, Liu et al. have shown that another protein, synaptotagmin 7, also has a key role in the replenishment of synaptic vesicles, possibly as a sensor for calcium ions. Moreover, Liu et al. found that synaptotagmin 7 and calmodulin bind to each other to form a complex, which suggests that the calcium-dependent replenishment pathway is regulated by this complex. The synaptotagmins are a family of 17 proteins, three of which are present in all animals. Two of these were known to have roles in synapses, but the role of the third—synaptotagmin 7—had been unclear. In addition to providing a more complete understanding of the replenishment of synaptic vesicles, the work of Liu et al. also supplies the final piece of the jigsaw regarding the role of the synaptotagmins that are present in all animals. DOI: http://dx.doi.org/10.7554/eLife.01524.002

Real-time voltammetry measurements from cracked PC12 cells were used to analyze the role of synaptotagmin–SNARE interactions during Ca2+-triggered exocytosis. The isolated C2A domain of synaptotagmin I neither binds SNAREs nor inhibits norepinephrine secretion. In contrast, two C2 domains in tandem (either C2A-C2B or C2A-C2A) bind strongly to SNAREs, displace native synaptotagmin from SNARE complexes, and rapidly inhibit exocytosis. The tandem C2 domains of synaptotagmin cooperate via a novel mechanism in which the disruptive effects of Ca2+ ligand mutations in one C2 domain can be partially alleviated by the presence of an adjacent C2 domain. Complete disruption of Ca2+-triggered membrane and target membrane SNARE interactions required simultaneous neutralization of Ca2+ ligands in both C2 domains of the protein. We conclude that synaptotagmin–SNARE interactions regulate membrane fusion and that cooperation between synaptotagmin's C2 domains is crucial to its function.

Synaptotagmin has been proposed to function as a Ca(2+) sensor that regulates synaptic vesicle exocytosis, whereas the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is thought to form the core of a conserved membrane fusion machine. Little is known concerning the functional relationships between synaptotagmin and SNAREs. Here we report that synaptotagmin can facilitate SNARE complex formation in vitro and that synaptotagmin mutations disrupt SNARE complex formation in vivo. Synaptotagmin oligomers efficiently bind SNARE complexes, whereas Ca(2+) acting via synaptotagmin triggers cross-linking of SNARE complexes into dimers. Mutations in Drosophila that delete the C2B domain of synaptotagmin disrupt clathrin AP-2 binding and endocytosis. In contrast, a mutation that blocks Ca(2+)-triggered conformational changes in C2B and diminishes Ca(2+)-triggered synaptotagmin oligomerization results in a postdocking defect in neurotransmitter release and a decrease in SNARE assembly in vivo. These data suggest that Ca(2+)-driven oligomerization via the C2B domain of synaptotagmin may trigger synaptic vesicle fusion via the assembly and clustering of SNARE complexes.

The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca2+ sensor for rapid exocytosis. Synaptotagmin spans the vesicle membrane once and possesses a large cytoplasmic domain that contains two C2 domains, C2A and C2B. Multiple Ca2+ ions bind to the membrane proximal C2A domain. However, it is not known whether the C2B domain also functions as a Ca2+-sensing module. Here, we report that Ca2+ drives conformational changes in the C2B domain of synaptotagmin and triggers the homo- and hetero-oligomerization of multiple isoforms of the protein. These effects of Ca2+ are mediated by a set of conserved acidic Ca2+ ligands within C2B; neutralization of these residues results in constitutive clustering activity. We addressed the function of oligomerization using a dominant negative approach. Two distinct reagents that block synaptotagmin clustering potently inhibited secretion from semi-intact PC12 cells. Together, these data indicate that the Ca2+-driven clustering of the C2B domain of synaptotagmin is an essential step in excitation-secretion coupling. We propose that clustering may regulate the opening or dilation of the exocytotic fusion pore.

The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca(2+) sensor for rapid exocytosis. Synaptotagmin spans the vesicle membrane once and possesses a cytoplasmic domain largely comprised of two C2 domains designated C2A and C2B. We have determined how deep the Ca(2+)-binding loops of Ca(2+).C2A penetrate into the lipid bilayer and report mutations in synaptotagmin that can uncouple membrane penetration from Ca(2+)-triggered interactions with the SNARE complex. To determine whether C2A penetrates into the vesicle ("cis") or plasma ("trans") membrane, we reconstituted a fragment of synaptotagmin that includes the membrane-spanning and C2A domain (C2A-TMR) into proteoliposomes. Kinetics experiments revealed that cis interactions are rapid (< or =500 micros). Binding in the trans mode was distinguished by the slow diffusion of trans target vesicles. Both modes of binding were observed, indicating that the linker between the membrane anchor and C2A domain functions as a flexible tether. C2A-TMR assembled into oligomers via a novel N-terminal oligomerization domain suggesting that synaptotagmin may form clusters on the surface of synaptic vesicles. This novel mode of clustering may allow for rapid Ca(2+)-triggered oligomerization of the protein via the membrane distal C2B domain.

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.

Communication within the nervous system is mediated by Ca2+-triggered fusion of synaptic vesicles with the presynaptic plasma membrane. Genetic and biochemical evidence indicates that synaptotagmin I may function as a Ca2+ sensor in neuronal exocytosis because it can bind Ca2+ and penetrate into lipid bilayers1,2,3,4. Chronic depolarization or seizure activity results in the upregulation of a distinct and unusual isoform of the synaptotagmin family, synaptotagmin IV (ref. 5). We have identified a Drosophila homologue of synaptotagmin IV that is enriched on synaptic vesicles and contains an evolutionarily conserved substitution of aspartate to serine that abolishes its ability to bind membranes in response to Ca2+ influx. Synaptotagmin IV forms hetero-oligomers with synaptotagmin I, resulting in synaptotagmin clusters that cannot effectively penetrate lipid bilayers and are less efficient at coupling Ca2+ to secretion in vivo : upregulation of synaptotagmin IV, but not synaptotagmin I, decreases evoked neurotransmission. These findings indicate that modulating theexpression of synaptotagmins with different Ca2+-binding affinities can lead to heteromultimers that can regulate the efficiency of excitation–secretion coupling in vivo and represent a new molecular mechanism for synaptic plasticity.

The C2A and C2B domains of Doc2 have markedly distinct Ca2+-sensing and lipid-binding properties. When expressed in wild-type neurons, a Ca2+-ligand mutant form of Doc2, that lacks apparent Ca2+-binding activity and is constitutively bound to the plasma membrane, results in an anomalous enhancement of evoked and spontaneous synaptic transmission., Double C2-domain protein (Doc2) is a Ca2+-binding protein implicated in asynchronous and spontaneous neurotransmitter release. Here we demonstrate that each of its C2 domains senses Ca2+; moreover, the tethered tandem C2 domains display properties distinct from the isolated domains. We confirm that overexpression of a mutant form of Doc2β, in which two acidic Ca2+ ligands in the C2A domain and two in the C2B domain have been neutralized, results in markedly enhanced asynchronous release in synaptotagmin 1–knockout neurons. Unlike wild-type (wt) Doc2β, which translocates to the plasma membrane in response to increases in [Ca2+]i, the quadruple Ca2+-ligand mutant does not bind Ca2+ but is constitutively associated with the plasma membrane; this effect is due to substitution of Ca2+ ligands in the C2A domain. When overexpressed in wt neurons, Doc2β affects only asynchronous release; in contrast, Doc2β Ca2+-ligand mutants that constitutively localize to the plasma membrane enhance both the fast and slow components of synaptic transmission by increasing the readily releasable vesicle pool size; these mutants also increase the frequency of spontaneous release events. Thus, mutations in the C2A domain of Doc2β that were intended to disrupt Ca2+ binding result in an anomalous enhancement of constitutive membrane-binding activity and endow Doc2β with novel functional properties.

While there is compelling evidence that the synaptic vesicle protein synaptotagmin serves as the major Ca2+ sensor for regulated exocytosis, it is not known how Ca2+ binding initiates membrane fusion. Here we report that Ca2+ increases the affinity, by approximately 2 orders of magnitude, between synaptotagmin and syntaxin 1, a component of the synaptic fusion apparatus. This effect is specific for divalent cations which can stimulate exocytosis of synaptic vesicles (Ca2+Ba2+, Sr2+Mg2+). The Ca(2+)-dependence of the interaction was composed of two components with EC50 values of 0.7 and 180 microM Ca2+. The interaction is mediated by the carboxyl-terminal region of syntaxin 1 (residues 194-288) and is regulated by a novel Ca(2+)-binding site(s) which does not require phospholipids and is not disrupted by mutations that abolish Ca(2+)-dependent phospholipid binding to synaptotagmin. We propose that this interaction constitutes an essential step in excitation-secretion coupling.

Synaptotagmin is the major calcium sensor for fast synaptic transmission that requires the synchronous fusion of synaptic vesicles. Synaptotagmin contains two calcium-binding domains: C2A and C2B. Mutation of a positively charged residue (R233Q in rat) showed that Ca2+-dependent interactions between the C2A domain and membranes play a role in the electrostatic switch that initiates fusion. Surprisingly, aspartate-to-asparagine mutations in C2A that inhibit Ca2+binding support efficient synaptic transmission, suggesting that Ca2+binding by C2A is not required for triggering synchronous fusion. Based on a structural analysis, we generated a novel mutation of a single Ca2+-binding residue in C2A (D229E inDrosophila) that inhibited Ca2+binding but maintained the negative charge of the pocket. This C2A aspartate-to-glutamate mutation resulted in ∼80% decrease in synchronous transmitter release and a decrease in the apparent Ca2+affinity of release. Previous aspartate-to-asparagine mutations in C2A partially mimicked Ca2+binding by decreasing the negative charge of the pocket. We now show that the major function of Ca2+binding to C2A is to neutralize the negative charge of the pocket, thereby unleashing the fusion-stimulating activity of synaptotagmin. Our results demonstrate that Ca2+binding by C2A is a critical component of the electrostatic switch that triggers synchronous fusion. Thus, Ca2+binding by C2B is necessary and sufficient to regulate the precise timing required for coupling vesicle fusion to Ca2+influx, but Ca2+binding by both C2domains is required to flip the electrostatic switch that triggers efficient synchronous synaptic transmission.

Synaptotagmin I (syt1) is required for normal rates of synaptic vesicle endo- and exocytosis. However, whether the kinetic defects observed during endocytosis in Syt1 knockout neurons are secondary to defective exocytosis or whether syt1 directly regulates the rate of vesicle retrieval remains unknown. To address this question, we sought to dissociate these two activities. We uncoupled the function of syt1 in exo- and endocytosis in mouse neurons either by re-targeting the protein or via mutagenesis of its tandem C2 domains. The effect of these manipulations on exo- and endocytosis were analyzed using electrophysiology, in conjunction with optical imaging of the vesicle cycle. Our results indicate that syt1 is directly involved in endocytosis. Notably, either of the C2 domains of syt1, C2A or C2B, was able to function as a Ca(2+) sensor for endocytosis. Thus, syt1 functions as a dual Ca(2+) sensor for both endo- and exocytosis, potentially coupling these two components of the vesicle cycle.

Different synaptotagmin isoforms (syt I, VII, and IX) sort to populations of dense-core vesicles with different sizes. These isoforms differ in their sensitivities to divalent cations and trigger different modes of exocytosis. Exocytosis triggered by these isoforms also differs in its sensitivity to inhibition by another isoform, syt IV., Many cells release multiple substances in different proportions according to the specific character of a stimulus. PC12 cells, a model neuroendocrine cell line, express multiple isoforms of the exocytotic Ca2+ sensor synaptotagmin. We show that these isoforms sort to populations of dense-core vesicles that differ in size. These synaptotagmins differ in their Ca2+ sensitivities, their preference for full fusion or kiss-and-run, and their sensitivity to inhibition by synaptotagmin IV. In PC12 cells, vesicles that harbor these different synaptotagmin isoforms can be preferentially triggered to fuse by different forms of stimulation. The mode of fusion is specified by the synaptotagmin isoform activated, and because kiss-and-run exocytosis can filter small molecules through a size-limiting fusion pore, the activation of isoforms that favor kiss-and-run will select smaller molecules over larger molecules packaged in the same vesicle. Thus synaptotagmin isoforms can provide multiple levels of control in the release of different molecules from the same cell.