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Cold Spring Harb Perspect Biol. 2012 January; 4(1): a011353.
PMCID: PMC3249630

Calcium Control of Neurotransmitter Release


Upon entering a presynaptic terminal, an action potential opens Ca2+ channels, and transiently increases the local Ca2+ concentration at the presynaptic active zone. Ca2+ then triggers neurotransmitter release within a few hundred microseconds by activating synaptotagmins Ca2+. Synaptotagmins bind Ca2+ via two C2-domains, and transduce the Ca2+ signal into a nanomechanical activation of the membrane fusion machinery; this activation is mediated by the Ca2+-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, Ca2+-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.

Synaptic transmission is initiated when an action potential invades a nerve terminal, opening Ca2+ channels, which gate a highly localized, transient increase in intracellular Ca2+ at the active zone (Fig. 1A). Ca2+ triggers synaptic vesicle exocytosis, thereby releasing the neurotransmitters contained in the vesicles and initiating synaptic transmission. This fundamental mechanism was discovered in pioneering work on the neuromuscular junction by Katz and Miledi (1967). Its precise time course was studied in many “model” synapses, including giant squid axon synapses (Augustine et al. 1985) and rat cerebellar parallel fiber synapses (Sabatini and Regehr 1996), but characterized in greatest detail in the calyx of Held synapses in the brainstem (Fig. 1B; reviewed in Meinrenken et al. 2003). Overall, these high-resolution electrophysiological studies on neurotransmitter release revealed that a presynaptic action potential is tightly coupled to Ca2+ influx and synaptic vesicle fusion, such that under physiological conditions, Ca2+ triggers fusion in a few hundred microseconds, or possibly even in less than 100 microseconds (Sabatini and Regehr 1996).

Figure 1.
Principle and time course of Ca2+-triggered synaptic transmission. (A) Schematic diagram of a synapse illustrating the localized influx of Ca2+ at the active zone (red = secreted neurotransmitters). (B) Schematic illustration of the sequence and time ...

The amazing speed and precision of Ca2+-triggered neurotransmitter release raised the question of how such speed might be possible—how can Ca2+ induce exocytosis in a few hundred microseconds, on the same timescale as the gating of an ion channel? As we will describe in this review, the speed and precision of release are mediated, at least in part, by the properties of the Ca2+ sensor synaptotagmin (Syt) and its cofactor complexin. Moreover, increasing evidence indicates that the principal mechanism of Ca2+-triggered exocytosis, although not the organization of this mechanism, is conserved in other, slower forms of Ca2+-induced exocytosis. Thus, the Syt-based fusion apparatus emerges as a general paradigm that accounts for most Ca2+-regulated exocytosis in eukaryotic cells.

In addition to normal action potential-evoked neurotransmitter release, synapses exhibit two additional forms of Ca2+-dependent synaptic exocytosis: spontaneous “mini” release and asynchronous release (Pang and Südhof 2010). Spontaneous mini release was discovered by Katz (Fatt and Katz 1952), and is thought to represent the background activity of a nerve terminal that is triggered by spontaneous Ca2+ fluctuations. Although the biological significance of spontaneous release remains debated, its presence can be observed in all neurons. When calculated on a per-synapse basis, spontaneous release occurs only once every 2–3 hours at an individual excitatory synapse of a pyramidal neuron in the CA1 region of the hippocampus, and approximately once every 3 min at individual inhibitory synapses. Asynchronous release is undetectable in most synapses under physiological conditions, but becomes apparent when the principal Syt Ca2+ sensor for regular release is ablated (Geppert et al. 1994). Some inhibitory synapses exhibit a slow form of release on repetitive stimulation (Hefft and Jonas 2005), which may represent either genuine physiological asynchronous release or simply synaptotagmin-dependent delayed release.


Syts are defined as proteins containing a short amino-terminal noncytoplasmic sequence followed by a transmembrane region, a central linker sequence of variable length, and two carboxy-terminal C2-domains that account for the majority of the protein (Fig. 2A; Perin et al. 1990). Sixteen genes encoding canonical Syts are expressed in mammals; in addition, mammals contain a Syt-related gene encoding a similar protein (the so-called B/K protein) that lacks a transmembrane region, and is occasionally called Syt17. Synaptotagmins are evolutionarily conserved, and invertebrates also express multiple Syt isoforms. However, synaptotagmins are absent from plants and unicellular eukaryotes, suggesting that they evolved at the same time as animals. Moreover, a large family of Syt-like proteins is expressed in animals; these proteins also contain two C2-domains, but will not be discussed here further (see Pang and Südhof 2010). Syt1, Syt2, Syt9, and Syt12 are present on synaptic vesicles and on secretory granules in neuroendocrine cells; the latter also contain Syt7. In addition, Syt10 was recently found on secretory vesicles containing IGF-1 in olfactory mitral neurons (Cao et al. 2011); Syt4 was localized to the trans-Golgi complex, synaptic vesicles, and postsynaptic organelles, and may be widely distributed in neuronal organelles (reviewed in Südhof 2004); and Syt7 was additionally localized to “secretory lysosomes” in nonneuronal and nonendocrine cells (Andrews and Chakrabarti 2005). Other Syts appear to be primarily localized to transport vesicles, although some Syt isoforms are enriched on the plasma membrane (Butz et al. 1999), and the identity of many of the Syt-containing vesicles remains unknown.

Figure 2.
Structures and Ca2+-binding properties of synaptotagmins. (A) Canonical domain structuresand classification of synaptotagmins. Mammals express 16 synaptotagmins composed of an amino-terminal transmembrane region preceded by a short noncytoplasmic sequence ...

C2-domains were named as sequence motifs in protein kinase C isoforms (Coussens et al. 1986), but their functional activity as autonomously folded Ca2+-binding domains was first identified in Syt1 (Davletov and Südhof 1993). The atomic structures of the C2A-domain of Syt1, the first C2-domain structure determined (Sutton et al. 1995), revealed that C2-domains are composed of a stable β-sandwich with flexible loops emerging from the top and bottom (Fig. 2B); all subsequently determined C2-domain structures described a similar fold. Ca2+ binds exclusively to the top loops of the C2-domains, which form binding sites for two to three Ca2+ ions in close vicinity (Shao et al. 1996). Although not all C2-domains bind Ca2+ (see below), all of those that do and have been studied in detail contain the same Ca2+-binding site architecture, with the Ca2+ ions ligated primarily by aspartate side chains (Fig. 2C). C2-domains are now recognized as general Ca2+-binding modules which are found in a large number of signal transduction and membrane trafficking proteins.

Approximately half of all C2-domains lack the canonical aspartate residues required for Ca2+ binding, including the C2A-domains of Syt4 and Syt11 (von Poser et al. 1997), and all C2-domains of Syt8 and Syt12–Syt16, which are thus thought to be Ca2+ independent. Even the C2B-domains of Syt4 and Syt11 which include all of the requisite Ca2+-binding sequences were found to be unable to bind Ca2+ (Dai et al. 2004). This initially surprising result was explained by the atomic structure of the Syt4 C2B-domain, which showed that the canonical Ca2+-binding residues in the top loops of the domain are too far apart to be able to ligate Ca2+ (Dai et al. 2004). Thus, the 16 mammalian Syts can be broadly classified into Ca2+-dependent and Ca2+-independent groups (Fig. 2A). The first group can be further subclassified based on whether or not their amino-terminal noncytoplasmic sequences contain disulfide-bonded cysteine residues (Fukuda et al. 1999). Similarly, the second group can be subclassified based on evolutionary conservation and sequence homologies, which reveal two sets of evolutionarily conserved synaptotagmins formed by Syt4 and Syt11 and by Syt14–16, and into a heterogeneous set of nonconserved synaptotagmins (Syt8, Syt12, and Syt13).

Initial studies on Syt1 revealed that both of its C2-domains bind to phospholipids in a Ca2+-dependent manner (Davletov and Südhof 1992; Fernandez et al. 2001), and that they bind to SNARE proteins (Bennett et al. 1992; Chapman et al. 1995; Li et al. 1995). Syt1 binding to phospholipids requires Ca2+ and negatively charged phospholipids, with phosphatidyl-inositolphosphates being most effective (Pang et al. 2006). Interestingly, the intrinsic Ca2+ affinity of the Syt1 C2-domains is relatively low (millimolar), but the presence of negatively charged phospholipids increases their apparent Ca2+ affinity by two to three orders of magnitude. This probably occurs because the Ca2+-coordination spheres of the Syt1 C2-domains are incomplete, but are completed by the negatively charged phospholipid headgroups during Ca2+-dependent phospholipid binding (Fernández-Chacón et al. 2001; Fernandez et al. 2002). In contrast to the Ca2+-dependent interaction of Syt1 with phospholipids, its binding to SNARE proteins and SNARE complexes is less well understood. There seems to be a direct interaction of the Syt1 C2-domains with syntaxin-1 that is greatly enhanced by Ca2+ (Li et al. 1995); moreover, Syt1 also binds to assembled SNARE complexes in a Ca2+-dependent manner (Pang et al. 2006). However, the interaction of Syt1 with SNARE complexes is highly sensitive to ionic strength in that it becomes Ca2+ independent at lower ionic strength, and at least some Ca2+-independent SNARE-complex binding by Syt1 may occur physiologically (Pang et al. 2006).

All Syt isoforms that bind Ca2+ appear to bind to phospholipids and to SNARE proteins in a Ca2+-dependent manner (Li et al. 1995; Sutton et al. 1999; Sugita et al. 2002; Hui et al. 2005). Interestingly, at least during Ca2+-dependent phospholipid binding, different Syts exhibit quite different apparent Ca2+ affinities, suggesting that they normally differentially respond to Ca2+ signals (Sugita et al. 2002). The biochemical properties of most Ca2+-independent Syts remain unknown, apart from Syt12, which has been shown to bind to Syt1 on the synaptic vesicle membrane (Maximov et al. 2007).


From the very beginning, Syt1 was hypothesized to function as a Ca2+ sensor for neurotransmitter release (Perin et al. 1990; Brose et al. 1992; Davletov and Südhof 1993). Knockout (KO) of Syt1 confirmed this hypothesis, showing that the Syt1 deletion abolished fast synchronous neurotransmitter release (Fig. 3A; Geppert et al. 1994). Strikingly, not all Ca2+-stimulated release was abolished in Syt1 KO neurons, which retained a delayed asynchronous form of release that is increased on repetitive stimulation (Maximov and Südhof 2005). Moreover, release induced by hypertonic sucrose, which is thought to cause the Ca2+-independent exocytosis of all vesicles in the readily releasable pool of a synapse was unchanged, indicating that the Syt1 KO did not interfere with vesicle fusion as such, but only with the Ca2+ triggering of fusion (Geppert et al. 1994).

Figure 3.
Syt1, Syt2, and Syt9 function as synaptic Ca2+ sensors for neurotransmitter release. (A) Deletion of Syt1 in cortical neurons blocks fast synchronous neurotransmitter release. Panels depict representative inhibitory postsynaptic currents (IPSCs) monitored ...

Given that eight Ca2+-binding Syts are expressed in neurons, which of these Syts function in neurotransmitter release? A systematic screen of Ca2+-binding Syts for rescue of the Syt1 KO phenotype revealed that only Syt1, Syt2, and Syt9 were capable of rescue (Fig. 3B; Xu et al. 2007). This result is surprising given the close similarity of Ca2+-binding Syts with each other, but is consistent with the differential expression patterns of Syt1, Syt2, and Syt9, and also explains why Syt1 KO neurons lose all release despite expressing high levels of Syt7, Syt3, and Syt5. Strikingly, Syt1, Syt2, and Syt9 were found to trigger release with quite distinct kinetics, with Syt2 exhibiting the fastest onset and the fastest decline in release, and Syt9 the slowest onset and the slowest decline (Fig. (Fig.3C,D;3C,D; Xu et al. 2007). The distinct kinetic properties of Syt1, Syt2, and Syt9 fit well with the expression of Syt9 primarily in the limbic system, whereas Syt2 is primarily expressed in synapses requiring very fast synaptic transmission, such as the auditory system or the neuromuscular junction.

The demonstration that Syt1 is essential for fast Ca2+-triggered neurotransmitter release was consistent with the notion that Syt1 functions as a Ca2+ sensor for release, but did not represent proof that Ca2+ binding to Syt1 triggers release, nor did it tell us which of the principal two biochemical activities of Syt1—Ca2+-dependent phospholipid- and SNARE-binding—was involved in its function. Definitive evidence that Syt1, and by analogy other Syts, is indeed a Ca2+ sensor for release came from knockin experiments in which point mutations that selectively alter the Ca2+ affinity and specificity of Ca2+-dependent interactions were introduced into the endogenous mouse Syt1 (Fernández-Chacón et al. 2001; Pang et al. 2006). Biochemical studies revealed that the D232N mutation in the C2A-domain of Syt1 increases the strength of the Ca2+-dependent binding of the double C2-domain fragment to SNARE complexes without altering phospholipid binding, whereas both the R233Q mutation and, to a lesser degree, the D238N mutation decrease the apparent Ca2+ affinity of the double C2-domain fragment of Syt1 during phospholipid binding without major effects on SNARE-complex binding (Fernández-Chacón et al. 2001; Pang et al. 2006). Strikingly, the D232N mutation increased the apparent Ca2+ affinity of evoked neurotransmitter release (i.e., decreased the Kd for Ca2+), whereas the R233Q and the D232N decreased the apparent Ca2+ affinity of evoked release without changing the apparent Ca2+ cooperativity of release (Fig. 4A). Thus, changes in the apparent Ca2+ affinity of Syt1 lead to corresponding changes in the apparent Ca2+ affinity of release, proving that Syt1 is a true Ca2+ sensor. Moreover, these experiments show that both SNARE- and phospholipid-binding by Syt1 are involved in release, suggesting that Ca2+ binding to Syt1 triggers release by inducing a fast simultaneous interaction of Syt1 with both SNARE complexes and with phospholipids.

Figure 4.
Syt1 Ca2+-binding site point mutations illustrate Syt1 function as a Ca2+ sensor for evoked and spontaneous neurotransmitter release. (A) Evoked IPSCs measured in neurons cultured from knockin mice carrying single amino-acid substitutions in the Ca2+ ...

Ca2+ binding to both C2-domains of Syt1 contributes to triggering release, although the two C2-domains are not equivalent. Mutations in the C2B-domain Ca2+-binding sites block release similarly to the Syt1 KO, whereas mutations in the C2A-domain Ca2+-binding sites have a much less severe effect (Mackler and Reist 2001; Stevens and Sullivan 2003; Shin et al. 2009). Initially it was hypothesized that the C2A-domain Ca2+-binding sites are of little importance, but careful Ca2+ titrations revealed that blocking Ca2+ binding to the C2A-domain not only decreases the magnitude of release, but also decreases the apparent Ca2+ cooperativity of release (Shin et al. 2009). Among others, these experiments confirmed that the two C2-domains of Syt1 function cooperatively, and that Ca2+ binding to both C2-domains triggers release to achieve the high apparent cooperativity of Ca2+ triggering of release (Meinrenken et al. 2003).


In a typical central neuron, spontaneous release events add up over time to a sizable signal, and may have an important physiological role (McKinney et al. 1999; Carter and Regehr 2002; Sutton et al. 2006). Most spontaneous release is Ca2+ dependent, suggesting it is regulated (Xu et al. 2009). Although spontaneous release was traditionally considered to be determined primarily if not exclusively by the number of synapses and the release probability of these synapses, increasing evidence indicates that spontaneous release may be derived from a vesicle pool that is different from that used for evoked release (Sara et al. 2005), and that it does not always, and possibly not even in most cases, correlate with the number of synapses and their release probability.

The Ca2+ dependence of spontaneous release suggested that a Ca2+ sensor should trigger it. The logical choice for such a Ca2+ sensor would be Syt1 and its homologs. Indeed, analysis of the Ca2+ dependence of spontaneous release in knockin mutant mice in which the affinity and binding-specificity of the Syt1 Ca2+-binding sites were altered revealed that increases in the Ca2+-dependent interaction of Syt1 with SNARE complexes induced by the D232N mutation caused an increase in the apparent Ca2+ affinity of spontaneous release (Fig. 4B; Xu et al. 2009). Conversely, decreases in the apparent Ca2+ affinity of Syt1 during phospholipid binding induced by the R233Q or the D238N mutation caused a decrease in the apparent Ca2+ affinity of spontaneous release (Fig. 4B). These results indicated that Syt1 functions as a Ca2+ sensor for spontaneous release, consistent with the previous finding that other elements of the canonical release machinery (e.g., SNARE proteins, Munc18-1, Munc13, and RIMs) are similarly involved.

However, deletion of Syt1 produces a large increase in spontaneous release frequency instead of blocking spontaneous release (Littleton et al. 1994; Maximov and Südhof 2005), suggesting that Syt1 is a clamp for spontaneous release instead of a Ca2+ sensor. This apparently contradictory observation was explained when it was found that the increased spontaneous release in Syt1 KO neurons was also completely Ca2+ dependent, but exhibited a higher apparent Ca2+ affinity and a lower apparent Ca2+ cooperativity than spontaneous release triggered by Ca2+ binding to Syt1 (Xu et al. 2009). Thus, deletion of Syt1 unmasked a secondary Ca2+ sensor with different properties that was normally clamped by Syt1. The nature of this Ca2+ sensor remains unclear, but it may be the same as the Ca2+ sensor that triggers asynchronous release and that has similar properties (Sun et al. 2007).

In addition to Syts, Doc2 proteins were implicated in regulating spontaneous release. KO or shRNA-mediated knockdown of Doc2 proteins, which are Ca2+-binding proteins related to Syts that also bind to SNAREs and to phospholipids, decreases spontaneous release significantly (Groffen et al. 2010; Pang et al. 2011). Interestingly, the Doc2 KO and knockdown had no effect on evoked release, confirming the differential nature of spontaneous and evoked release. Although the Ca2+-binding properties of Doc2 proteins suggested that they may function as Ca2+ sensors for spontaneous exocytosis (Groffen et al. 2010), the decrease in mini frequency induced by Doc2 knockdown was fully rescued by expression of Doc2 proteins lacking Ca2+-binding sites, indicating that Doc2 proteins do not function as Ca2+ sensors for spontaneous exocytosis (Pang et al. 2011). Moreover, the Doc2 knockdown had no effect on the increased spontaneous release observed in Syt1 KO neurons, and also did not affect asynchronous release, suggesting that Doc2 proteins do not function in these types of release.


Complexins were identified as SNARE-complex binding proteins, giving rise to their name (McMahon et al. 1995). Complexins are small soluble proteins of approximately 120 residues that are evolutionarily conserved in all animals similar to synaptotagmins. The crystal structure of complexin bound to the assembled SNARE complex revealed that complexin contains a central short ~20-residue α-helix that nestles into the groove formed by the synaptobrevin and syntaxin SNARE motifs in the SNARE complex (Chen et al. 2002). In addition, complexin contains a similarly short accessory amino-terminal α-helix that does not contact the SNARE complex in the crystal structure, with amino- and carboxy-terminal unstructured sequences flanking the two helices. Interestingly, complexin binds to the SNARE complex in an antiparallel orientation at a membrane-proximal site of the complex, suggesting that complexin can only bind to the SNARE complex after it has assembled to more than 50%.

Mammals express four complexin isoforms, two that are widely distributed in the body and abundant in brain (complexin-1 and -2; McMahon et al. 1995), and two additional isoforms that are enriched in retina (complexin-3 and -4; Reim et al. 2005). Complexin-3 and -4 include a carboxy-terminal isoprenylation sequence that is absent from complexin-1 and -2 and likely attaches complexin-3 and -4 to the plasma membrane. Deletion of complexin-1 and -2 in mice led to a striking phenotype that represented a milder phenocopy of the Syt1 knockout phenotype, suggesting that complexins act in the same pathway as synaptotagmins (Reim et al. 2001). Specifically, these mice exhibited a selective but only partial loss of synchronous neurotransmitter release, without a major decrease in asynchronous release. Subsequent knockdown experiments using shRNAs confirmed this phenotype, but extended it to reveal an increase in spontaneous release analogous to that observed in Syt1 knockout neurons, with the same shift to a higher apparent Ca2+ affinity and lower apparent Ca2+ cooperativity (Maximov et al. 2009; Yang et al. 2010). Thus, complexins act both as activators and as clamps of neurotransmitter release. Moreover, unlike the Syt1 KO, which had no effect on the size of the readily-releasable pool, the complexin knockdown produced a ~40% decrease in the readily-releasable pool (Yang et al. 2010). Rescue experiments revealed that SNARE-complex binding by complexin is essential for its function (Maximov et al. 2009). Moreover, such experiments showed that the complexin amino terminus is crucial for its activating role (Xue et al. 2007), whereas its accessory α-helix mediates its clamping function (Maximov et al. 2009; Yang et al. 2010). Considerable controversy developed at one point about whether or not complexins have a clamping function, in that such function was not obvious in autapses (Reim et al. 2001), but was the only function observed in in vitro fusion assays (Giraudo et al. 2009). Although the activation function of complexin is likely more important overall than its clamping function, the fact that the two functions can be localized to distinct autonomously acting sequences in complexin (Maximov et al. 2009; Yang et al. 2010) and that both phenocopy a function observed for Syts strongly supports their physiological importance.

How do complexin and Syts act in promoting Ca2+ triggering of synaptic vesicle fusion? Biochemical data indicate that the central α-helix of complexin and Syt1 bind to SNARE complexes at overlapping sites, and that Ca2+ binding to Syt1 triggers the displacement of the central α-helix of complexin from its binding site on SNARE complexes (Tang et al. 2006). Based on these electrophysiological and biochemical studies, a plausible current model is that complexin binding to primed synaptic vesicles containing partially assembled SNARE complexes “superprimes” these vesicles into an activated state, and simultaneously clamps them (Fig. 5). Ca2+ binding to synaptotagmin then triggers synaptotagmin binding to the SNARE complex and the phospholipid bilayer, dislodging the complexin clamp and pulling on the SNARE complex, thereby opening the fusion pore. This model accounts for most of the existing data, although it offers no explanation for the clamping function of synaptotagmin, and thus is unlikely to be definitive.

Figure 5.
Schematic diagram of the mechanism of action of synaptotagmin and its complexin cofactor in Ca2+-triggered exocytosis. The top diagram displays the sequential priming of synaptic vesicles by partial SNARE/SM protein complex assembly, superpriming by binding ...


Many forms of exocytosis are induced by Ca2+, including hormone exocytosis in endocrine cells such as chromaffin cells, neuropeptide secretion in neurons, mast cell degranulation, and the acrosome reaction in sperm. Strikingly, for most of these forms of exocytosis the same Syts that mediate synaptic vesicle fusion (Syt1, Syt2, and Syt9) appear to be involved; in addition, Syt7 was also shown to play a central role. Specifically, for chromaffin granule exocytosis in chromaffin cells, nearly all Ca2+ triggering is mediated in equal parts by Syt1 and Syt7, such that only the double Syt1/7 KO has a major phenotype in exocytosis (Schonn et al. 2008). In contrast, in pancreatic insulin- and glucagon-secreting cells, Syt7 appears to be the dominant Ca2+ sensor (Gustavsson et al. 2008, 2009), whereas in mast cell degranulation, Syt2 is the most important Ca2+ sensor (Melicoff et al. 2009). In addition, Syt7 has been linked to exocytosis in non-neuronal cells, although it is not quite clear whether this is a physiological event (Martinez et al. 2000; Jaiswal et al. 2004; Wang and Hilgemann 2008). At least in chromaffin cells, complexins are obligatory cofactors for Syt-triggered exocytosis (Cai et al. 2008). Thus, despite the different kinetics of various types of exocytosis and the different localizations of various Syts, the same Syt paradigm appears to widely operate for all of these forms of exocytosis.

Until recently, no definitive function was identified for the second class of Ca2+-binding Syts that contain amino-terminal disulfide bonds, which lead to constitutive dimerization (Fig. 2A). These Syts exhibit a higher apparent Ca2+ affinity than Syt1, and also interact with phospholipids and SNAREs in a Ca2+-dependent manner (Li et al. 1995; Sugita et al. 2002), suggesting that they may also function as Ca2+ sensors in exocytosis. This hypothesis was recently confirmed for Syt10, which was identified as the Ca2+ sensor for exocytosis of IGF-1 containing secretory vesicles in mitral neurons from the olfactory bulb (Cao et al. 2011). Interestingly, in the same neurons Syt1 functions as the Ca2+ sensor for synaptic vesicle exocytosis, and the two Syts act completely independently of each other (Fig. 6). Thus, different synaptotagmin isoforms can operate in the same cell to mediate the Ca2+-regulated fusion of distinct types of secretory vesicles.

Figure 6.
Illustration of distinct but similar functions of different synaptotagmins: Syt10 acts as a Ca2+ sensor for IGF-1 containing vesicles in mitral neurons from olfactory bulb. (A) IGF-1 secretion from cultured olfactory bulb neurons is stimulated by depolarization ...


Over the last two decades, a universal mechanism by which Ca2+ binding to Syts triggers fusion has emerged. This process involves a pas de deux of Syts and complexin, which act simultaneously on SNARE complexes and phospholipid membranes. The mechanism ensures the millisecond control of neurotransmitter release from presynaptic terminals on arrival of an action potential, but goes far beyond the synapse. The mechanism similarly applies to many other Ca2+-induced secretory events, as exemplified by the role of Syts and complexins in neuroendocrine secretion including IGF-1 exocytosis, and might further extend to all Ca2+-regulated membrane trafficking events in general.


This work was supported by the Howard Hughes Medical Institute and by grants from the NIMH.


Editors: Morgan Sheng, Bernardo Sabatini, and Thomas Südhof

Additional Perspectives on The Synapse available at


  • Andrews NW, Chakrabarti S 2005. There’s more to life than neurotransmission: The regulation of exocytosis by synaptotagmin VII. Trends Cell Biol 15: 626–631 [PubMed]
  • Augustine GJ, Charlton MP, Smith SJ 1985. Calcium entry and transmitter release at voltage-clamped nerve terminals of squid. J Physiol 367: 163–181 [PubMed]
  • Barber CF, Jorquera RA, Melom JE, Littleton JT 2009. Postsynaptic regulation of synaptic plasticity by synaptotagmin 4 requires both C2 domains. J Cell Biol 187: 295–310 [PMC free article] [PubMed]
  • Bennett MK, Calakos N, Scheller RH 1992. Syntaxin: A synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones. Science 257: 255–259 [PubMed]
  • Brose N, Petrenko AG, Südhof TC, Jahn R 1992. Synaptotagmin: A Ca2+ sensor on the synaptic vesicle surface. Science 256: 1021–1025 [PubMed]
  • Butz S, Fernández-Chacón R, Schmitz F, Jahn R, Südhof TC 1999. The subcellular localizations of atypical synaptotagmins: Synaptotagmin III is enriched in synapses and synaptic plasma membranes but not in synaptic vesicles. J Biol Chem 274: 18290–18296 [PubMed]
  • Cai H, Reim K, Varoqueaux F, Tapechum S, Hill K, Sørensen JB, Brose N, Chow RH 2008. Complexin II plays a positive role in Ca2+-triggered exocytosis by facilitating vesicle priming. Proc Natl Acad Sci 105: 19538–19543 [PubMed]
  • Cao P, Maximov A, Südhof TC 2011. Activity-dependent IGF-1 exocytosis is controlled by the Ca2+-sensor synaptotagmin-10. Cell 145: 300–311 [PMC free article] [PubMed]
  • Carter AG, Regehr WG 2002. Quantal events shape cerebellar interneuron firing. Nat Neurosci 5: 1309–1318 [PubMed]
  • Chapman ER, Hanson PI, An S, Jahn R 1995. Ca2+ regulates the interaction between synaptotagmin and syntaxin 1. J Biol Chem 270: 23667–23671 [PubMed]
  • Chen X, Tomchick DR, Kovrigin E, Arac D, Machius M, Südhof TC, Rizo J 2002. Three-dimensional structure of the complexin/SNARE complex. Neuron 33: 397–409 [PubMed]
  • Coussens L, Parker PJ, Rhee L, Yang-Feng TL, Chen E, Waterfield MD, Francke U, Ullrich A 1986. Multiple, distinct forms of bovine and human protein kinase C suggest diversity in cellular signaling pathways. Science 233: 859–866 [PubMed]
  • Dai H, Shin O-K, Machius M, Tomchick DR, Südhof TC, Rizo J 2004. Structural basis for the evolutionary inactivation of Ca2+-binding to synaptotagmin 4. Nature Struct Mol Biol 11: 844–849 [PubMed]
  • Davletov BA, Südhof TC 1993. A single C2-domain from synaptotagmin I is sufficient for high affinity Ca2+/−phospholipid-binding. J Biol Chem 268: 26386–26390 [PubMed]
  • Dean C, Liu H, Dunning FM, Chang PY, Jackson MB, Chapman ER 2009. Synaptotagmin-IV modulates synaptic function and long-term potentiation by regulating BDNF release. Nat Neurosci 12: 767–776 [PMC free article] [PubMed]
  • Fatt P, Katz B 1952. Spontaneous subthreshold activity at motor nerve endings. J Physiol 117: 109–128 [PubMed]
  • Fernandez I, Arac D, Ubach J, Gerber SH, Shin O, Gao Y, Anderson RGW, Südhof TC, Rizo J 2001. Three-dimensional structure of the synaptotagmin 1 C2B-domain: Synaptotagmin 1 as a phospholipid binding machine. Neuron 32: 1057–1069 [PubMed]
  • Fernández-Chacón R, Königstorffer A, Gerber SH, Garcia J, Matos MF, Stevens CF, Brose N, Rizo J Rosenmund C, Südhof TC 2001. Synaptotagmin I functions as a Ca2+-regulator of release probability. Nature 410: 41–49 [PubMed]
  • Fukuda M, Kanno E, Mikoshiba K 1999. Conserved N-terminal cysteine motif is essential for homo- and heterodimer formation of synaptotagmins III, V, VI, and X. J Biol Chem 274: 31421–31427 [PubMed]
  • Geppert M, Goda Y, Hammer RE, Li C, Rosahl TW, Stevens CF, Südhof TC 1994. Synaptotagmin I: A major Ca2+ sensor for transmitter release at a central synapse. Cell 79: 717–727 [PubMed]
  • Giraudo CG, Garcia-Diaz A, Eng WS, Chen Y, Hendrickson WA, Melia TJ, Rothman JE 2009. Alternative zippering as an on-off switch for SNARE-mediated fusion. Science 323: 512–516 [PMC free article] [PubMed]
  • Groffen AJ, Martens S, Diez Arazola R, Cornelisse LN, Lozovaya N, de Jong AP, Goriounova NA, Habets RL, Takai Y, Borst JG, et al. 2010. Doc2b is a high-affinity Ca2+ sensor for spontaneous neurotransmitter release. Science 327: 1614–1618 [PMC free article] [PubMed]
  • Gustavsson N, Lao Y, Maximov A, Chuang JC, Kostromina E, Repa JJ, Li C, Radda GK, Südhof TC, Han W 2008. Impaired insulin secretion and glucose intolerance in synaptotagmin-7 null mutant mice. Proc Natl Acad Sci 105: 3992–3997 [PubMed]
  • Gustavsson N, Wei SH, Hoang DN, Lao Y, Zhang Q, Radda GK, Rorsman P, Südhof TC, Han W 2009. Synaptotagmin-7 is a principal Ca2+ sensor for Ca2+-induced glucagon exocytosis in pancreas. J Physiol 587: 1169–1178 [PubMed]
  • Han W, Rhee JS, Maximov A, Lao Y, Mashimo T, Rosenmund C, Südhof TC 2004. N-glycosylation is essential for vesicular targeting of synaptotagmin 1. Neuron 41: 85–99 [PubMed]
  • Hefft S, Jonas P 2005. Asynchronous GABA release generates long-lasting inhibition at a hippocampal interneuron-principal neuron synapse. Nat Neurosci 8: 1319–1328 [PubMed]
  • Hui E, Bai J, Wang P, Sugimori M, Llinas RR, Chapman ER 2005. Three distinct kinetic groupings of the synaptotagmin family: Candidate sensors for rapid and delayed exocytosis. Proc Natl Acad Sci 102: 5210–5214 [PubMed]
  • Jaiswal JK, Chakrabarti S, Andrews NW, Simon SM 2004. Synaptotagmin VII restricts fusion pore expansion during lysosomal exocytosis. PLoS Biol 2: E233. [PMC free article] [PubMed]
  • Katz B, Miledi R 1967. Ionic requirements of synaptic transmitter release. Nature 215: 651. [PubMed]
  • Li C, Ullrich B, Zhang ZZ, Anderson RGW, Brose N, Südhof TC 1995. Ca2+-dependent and Ca2+-independent activities of neural and nonneural synaptotagmins. Nature 375: 594–599 [PubMed]
  • Littleton JT, Stern M, Perin M, Bellen HJ 1994. Calcium dependence of neurotransmitter release and rate of spontaneous vesicle fusions are altered in Drosophila synaptotagmin mutants. Proc Natl Acad Sci 91: 10888–10892 [PubMed]
  • Littleton JT, Serano TL, Rubin GM, Ganetzky B, Chapman ER 1999. Synaptic function modulated by changes in the ratio of synaptotagmin I and IV. Nature 400: 757–760 [PubMed]
  • Mackler JM, Reist NE 2001. Mutations in the second C2 domain of synaptotagmin disrupt synaptic transmission at Drosophila neuromuscular junctions. J Comp Neurol 436: 4–16 [PubMed]
  • Martinez I, Chakrabarti S, Hellevik T, Morehead J, Fowler K, Andrews NW 2000. Synaptotagmin VII regulates Ca2+-dependent exocytosis of lysosomes in fibroblasts. J Cell Biol 148: 1141–1149 [PMC free article] [PubMed]
  • Maximov A, Südhof TC 2005. Autonomous function of synaptotagmin 1 in triggering synchronous release independent of asynchronous release. Neuron 48: 547–554 [PubMed]
  • Maximov A, Shin O-H, Südhof TC 2007. Synaptotagmin-12, a synaptic vesicle phosphoprotein that modulates spontaneous neurotransmitter release. J Cell Biol 176: 113–124 [PMC free article] [PubMed]
  • Maximov A, Tang J, Yang X, Pang Z, Südhof TC 2009. Complexin controls the force transfer from SNARE complexes to membranes in fusion. Science 323: 516–521 [PMC free article] [PubMed]
  • McKinney RA, Capogna M, Dürr R, Gähwiler BH, Thompson SM 1999. Miniature synaptic events maintain dendritic spines via AMPA receptor activation. Nat Neurosci 2: 44–49 [PubMed]
  • McMahon HT, Missler M, Li C, Südhof TC 1995. Complexins: Cytosolic proteins that regulate SNAP-receptor function. Cell 83: 111–119 [PubMed]
  • Meinrenken CJ, Borst JG, Sakmann B 2003. Local routes revisited: The space and time dependence of the Ca2+ signal for phasic transmitter release at the rat calyx of Held. J Physiol 547: 665–689 [PubMed]
  • Melicoff E, Sansores-Garcia L, Gomez A, Moreira DC, Datta P, Thakur P, Petrova Y, Siddiqi T, Murthy JN, Dickey BF, et al. 2009. Synaptotagmin-2 controls regulated exocytosis but not other secretory responses of mast cells. J Biol Chem 284: 19445–19451 [PMC free article] [PubMed]
  • Pang ZP, Südhof TC 2010. Cell biology of Ca2+-triggered exocytosis. Curr Opinion Cell Biol 22: 496–505 [PMC free article] [PubMed]
  • Pang ZP, Shin O-H, Meyer AC, Rosenmund C, Südhof TC 2006. A gain-of-function mutation in synaptotagmin-1 reveals a critical role of Ca2+-dependent SNARE-complex binding in synaptic exocytosis. J Neurosci 26: 12556–12565 [PubMed]
  • Pang ZP, Bacaj T, Yang X, Zhou P, Xu W, Südhof TC 2011. Doc2 supports spontaneous synaptic transmission by a calcium-independent mechanism. Neuron 70: 244–251 [PMC free article] [PubMed]
  • Perin MS, Fried VA, Mignery GA, Jahn R, Südhof TC 1990. Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C. Nature 345: 260–263 [PubMed]
  • Reim K, Mansour M, Varoqueaux F, McMahon HT, Südhof TC, Brose N, Rosenmund C 2001. Complexins regulate the Ca2+ sensitivity of the synaptic neurotransmitter release machinery. Cell 104: 71–81 [PubMed]
  • Reim K, Wegmeyer H, Brandstätter JH, Xue M, Rosenmund C, Dresbach T, Hofmann K, Brose N 2005. Structurally and functionally unique complexins at retinal ribbon synapses. J Cell Biol 169: 669–680 [PMC free article] [PubMed]
  • Sabatini BL, Regehr WG 1996. Timing of neurotransmission at fast synapses in the mammalian brain. Nature 384: 170–172 [PubMed]
  • Sara Y, Virmani T, Deák F, Liu X, Kavalali ET 2005. An isolated pool of vesicles recycles at rest and drives spontaneous neurotransmission. Neuron 45: 563–573 [PubMed]
  • Schonn JS, Maximov A, Lao Y, Südhof TC, Sorensen JB 2008. Synaptotagmin-1 and -7 are functionally overlapping Ca2+ sensors for exocytosis in adrenal chromaffin cells. Proc Natl Acad Sci 105: 3998–4003 [PubMed]
  • Shao X, Davletov BA, Sutton RB, Südhof TC, Rizo J 1996. Bipartite Ca2+-binding motif in C2 domains of synaptotagmin and protein kinase C. Science 273: 248–251 [PubMed]
  • Shin O-H, Xu J, Rizo J, Südhof TC 2009. Differential but convergent functions of Ca2+-binding to synaptotagmin-1 C2-domains mediate neurotransmitter release. Proc Natl Acad Sci 106: 16469–16474 [PubMed]
  • Stevens CF, Sullivan JM 2003. The synaptotagmin C2A domain is part of the calcium sensor controlling fast synaptic transmission. Neuron 39: 299–308 [PubMed]
  • Sun J, Pang ZP, Qin D, Fahim AT, Adachi R, Südhof TC 2007. A dual Ca2+-sensor model for neuro-transmitter release in a central synapse. Nature 450: 676–682 [PMC free article] [PubMed]
  • Südhof TC 2002. Synaptotagmins: Why so many? J Biol Chem 277: 7629–7632 [PubMed]
  • Südhof TC 2004. The synaptic vesicle cycle. Annu Rev Neurosci 27: 509–547 [PubMed]
  • Sugita S, Shin O-H, Han W, Lao Y, Südhof TC 2002. Synaptotagmins form hierarchy of exocytotic Ca2+-sensors with distinct Ca2+-affinities. EMBO J 21: 270–280 [PubMed]
  • Sutton AB, Davletov BA, Berghuis AM, Südhof TC, Sprang SR 1995. Structure of the first C2-domain of synaptotagmin I: A novel Ca2+/phospholipid binding fold. Cell 80: 929–938 [PubMed]
  • Sutton RB, Ernst JA, Brunger AT 1999. Crystal structure of the cytosolic C2A-C2B domains of synaptotagmin III. Implications for Ca2+-independent snare complex interaction. J Cell Biol 147: 589–598 [PMC free article] [PubMed]
  • Sutton MA, Ito HT, Cressy P, Kempf C, Woo JC, Schuman EM 2006. Miniature neurotransmission stabilizes synaptic function via tonic suppression of local dendritic protein synthesis. Cell 125: 785–799 [PubMed]
  • Tang J, Maximov A, Shin O-H, Dai H, Rizo J, Südhof TC 2006. A complexin/synaptotagmin-1 switch controls fast synaptic vesicle exocytosis. Cell 126: 1175–1187 [PubMed]
  • Ubach J, Zhang X, Shao X, Südhof TC, Rizo J 1998. Ca2+ binding to synaptotagmin: How many Ca2+ ions bind to the tip of a C2-domain? EMBO J 17: 3921–3930 [PubMed]
  • Von Poser C, Ichtchenko K, Shao X, Rizo J, Südhof TC 1997. The evolutionary pressure to inactivate: A subclass of synaptotagmins with an amino acid substitution that abolishes Ca2+ binding. J. Biol Chem 272: 14314–14319 [PubMed]
  • Wang TM, Hilgemann DW 2008. Ca-dependent nonsecretory vesicle fusion in a secretory cell. J Gen Physiol 132: 51–65 [PMC free article] [PubMed]
  • Wang CT, Lu JC, Bai J, Chang PY, Martin TF, Chapman ER, Jackson MB 2003. Different domains of synaptotagmin control the choice between kiss-and-run and full fusion. Nature 424: 943–947 [PubMed]
  • Xu J, Mashimo T, Südhof TC 2007. Synaptotagmin-1, -2, and -9: Ca2+-sensors for fast release that specify distinct presynaptic properties in subsets of neurons. Neuron 54: 801–812 [PubMed]
  • Xu J, Pang ZP, Shin OH, Südhof TC 2009. Synaptotagmin-1 functions as a Ca2+ sensor for spontaneous release. Nat Neurosci 12: 759–766 [PMC free article] [PubMed]
  • Xue M, Reim K, Chen X, Chao HT, Deng H, Rizo J, Brose N, Rosenmund C 2007. Distinct domains of complexin I differentially regulate neurotransmitter release. Nat Struct Mol Biol 14: 949–958 [PubMed]
  • Yang X, Kaeser Woo YJ, Pang ZP, Xu W, Südhof TC 2010. Complexin clamps asynchronous release by blocking a secondary Ca2+-sensor via its accessory-helix. Neuron 68: 907–920 [PMC free article] [PubMed]

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