Dysregulation of Norepinephrine Release in the Absence of Functional Synaptotagmin 7.

Synaptotagmin 7 (Syt7) is expressed in cardiac sympathetic nerve terminals where norepinephrine (NE) is released in both Ca(2+)-dependent exocytosis and Ca(2+)-independent norepinephrine transporter (NET)-mediated overflow. The role of Syt7 in the regulation of NE release from cardiac sympathetic nerve terminals is tested by employing a Syt7 knock-in mouse line that expresses a non-functional mutant form of Syt7. In cardiac sympathetic nerve terminals prepared from these Syt7 knock-in mice, the Ca(2+)-dependent component of NE release was diminished. However, these terminals displayed upregulated function of NET (∼130% of controls) and a significant increase in Ca(2+)-independent NE overflow (∼140% of controls), which is greater than the Ca(2+)-dependent component of NE exocytosis occurring in wild-type controls. Consistent with a significant increase in NE overflow, the Syt7 knock-in mice showed significantly higher blood pressures compared to those of littermate wild-type and heterozygous mice. Our results indicate that the lack of functional Syt7 dysregulates NE release from cardiac sympathetic nerve terminals.

Inositol hexakisphosphate suppresses excitatory neurotransmission via synaptotagmin-1 C2B domain in the hippocampal neuron.

Inositol hexakisphosphate (InsP(6)) levels rise and fall with neuronal excitation and silence, respectively, in the hippocampus, suggesting potential signaling functions of this inositol polyphosphate in hippocampal neurons. We now demonstrate that intracellular application of InsP(6) caused a concentration-dependent inhibition of autaptic excitatory postsynaptic currents (EPSCs) in cultured hippocampal neurons. The treatment did not alter the size and replenishment rate of the readily releasable pool in autaptic neurons. Intracellular exposure to InsP(6) did not affect spontaneous EPSCs or excitatory amino acid-activated currents in neurons lacking autapses. The InsP(6)-induced inhibition of autaptic EPSCs was effectively abolished by coapplication of an antibody to synaptotagmin-1 C2B domain. Importantly, preabsorption of the antibody with a GST-WT synaptotagmin-1 C2B domain fragment but not with a GST-mutant synaptotagmin-1 C2B domain fragment that poorly reacted with the antibody impaired the activity of the antibody on the InsP(6)-induced inhibition of autaptic EPSCs. Furthermore, K(+) depolarization significantly elevated endogenous levels of InsP(6) and occluded the inhibition of autaptic EPSCs by exogenous InsP(6). These data reveal that InsP(6) suppresses excitatory neurotransmission via inhibition of the presynaptic synaptotagmin-1 C2B domain-mediated fusion via an interaction with the synaptotagmin Ca(2+)-binding sites rather than via interference with presynaptic Ca(2+) levels, synaptic vesicle trafficking, or inactivation of postsynaptic ionotropic glutamate receptors. Therefore, elevated InsP(6) in activated neurons serves as a unique negative feedback signal to control hippocampal excitatory neurotransmission.

Synaptotagmin-12, a synaptic vesicle phosphoprotein that modulates spontaneous neurotransmitter release.

Central synapses exhibit spontaneous neurotransmitter release that is selectively regulated by cAMP-dependent protein kinase A (PKA). We now show that synaptic vesicles contain synaptotagmin-12, a synaptotagmin isoform that differs from classical synaptotagmins in that it does not bind Ca(2+). In synaptic vesicles, synaptotagmin-12 forms a complex with synaptotagmin-1 that prevents synaptotagmin-1 from interacting with SNARE complexes. We demonstrate that synaptotagmin-12 is phosphorylated by cAMP-dependent PKA on serine(97), and show that expression of synaptotagmin-12 in neurons increases spontaneous neurotransmitter release by approximately threefold, but has no effect on evoked release. Replacing serine(97) by alanine abolishes synaptotagmin-12 phosphorylation and blocks its effect on spontaneous release. Our data suggest that spontaneous synaptic-vesicle exocytosis is selectively modulated by a Ca(2+)-independent synaptotagmin isoform, synaptotagmin-12, which is controlled by cAMP-dependent phosphorylation.

Differential but convergent functions of Ca2+ binding to synaptotagmin-1 C2 domains mediate neurotransmitter release.

Neurotransmitter release is triggered by cooperative Ca2+-binding to the Ca2+-sensor protein synaptotagmin-1. Synaptotagmin-1 contains two C2 domains, referred to as the C2A and C2B domains, that bind Ca2+ with similar properties and affinities. However, Ca2+ binding to the C2A domain is not required for release, whereas Ca2+ binding to the C2B domain is essential for release. We now demonstrate that despite its expendability, Ca2+-binding to the C2A domain significantly contributes to the overall triggering of neurotransmitter release, and determines its Ca2+ cooperativity. Biochemically, Ca2+ induces more tight binding of the isolated C2A domain than of the isolated C2B domain to standard liposomes composed of phosphatidylcholine and phosphatidylserine. However, here we show that surprisingly, the opposite holds true when the double C2A/B-domain fragment of synaptotagmin-1 is used instead of isolated C2 domains, and when liposomes containing a physiological lipid composition are used. Under these conditions, Ca2+ binding to the C2B domain but not the C2A domain becomes the primary determinant of phospholipid binding. Thus, the unique requirement for Ca2+ binding to the C2B domain for synaptotagmin-1 in Ca2+-triggered neurotransmitter release may be accounted for, at least in part, by the unusual phospholipid-binding properties of its double C2A/B-domain fragment.

Interactions among the SNARE proteins and complexin analyzed by a yeast four-hybrid assay.

Exocytosis is one of the most crucial and ubiquitous processes in all of biology. This event is mediated by the formation of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes, ternary assemblies of syntaxin, SNAP23/SNAP25 (synaptosomal-associated protein of 23 or 25 kDa), and synaptobrevin. The exocytotic process can be further regulated by complexin, which interacts with the SNARE complex. Complexin is involved in a Ca(2+)-triggered exocytotic process. In eukaryotic cells, multiple isoforms of SNARE proteins are expressed and are involved in distinct types of exocytosis. To understand the underlying biochemical mechanism of various exocytotic processes mediated by different SNARE protein isoforms, we systematically analyzed the interactions among syntaxin, SNAP23/SNAP25, synaptobrevin, and complexin by employing a newly developed yeast four-hybrid interaction assay. The efficiency of SNARE complex formation and the specificity of complexin binding are regulated by the different SNARE protein isoforms. Therefore, various types of exocytosis, occurring on different time scales with different efficiencies, can be explained by the involved SNARE complexes composed of different combinations of SNARE protein isoforms.

Evolutionary diversity of the dual Ca2+ sensor system for neurotransmitter release.

Several proteins containing C2 domains have been identified as Ca2+ sensors for neurotransmitter release. In several cases, multiple C2 domain containing proteins function together to sustain evoked synchronous and asynchronous release as well as Ca2+-dependent forms of spontaneous release. Most recent publication by Li and colleagues have identified a novel Ca2+ sensor at the C. elegans neuromuscular junction [8] that complements the fast Ca2+ sensor synaptotagmin-1 in mediating a slower form of evoked release. Here, we discuss these results as well as earlier work suggesting an evolutionarily conserved diversity of Ca2+ sensors mediating distinct forms of neurotransmitter release.

Presynaptic store-operated Ca2+ entry drives excitatory spontaneous neurotransmission and augments endoplasmic reticulum stress.

Store-operated calcium entry (SOCE) is activated by depletion of Ca2+ from the endoplasmic reticulum (ER) and mediated by stromal interaction molecule (STIM) proteins. Here, we show that in rat and mouse hippocampal neurons, acute ER Ca2+ depletion increases presynaptic Ca2+ levels and glutamate release through a pathway dependent on STIM2 and the synaptic Ca2+ sensor synaptotagmin-7 (syt7). In contrast, synaptotagmin-1 (syt1) can suppress SOCE-mediated spontaneous release, and STIM2 is required for the increase in spontaneous release seen during syt1 loss of function. We also demonstrate that chronic ER stress activates the same pathway leading to syt7-dependent potentiation of spontaneous glutamate release. During ER stress, inhibition of SOCE or syt7-driven fusion partially restored basal neurotransmission and decreased expression of pro-apoptotic markers, indicating that these processes participate in the amplification of ER-stress-related damage. Taken together, we propose that presynaptic SOCE links ER stress and augmented spontaneous neurotransmission, which may, in turn, facilitate neurodegeneration.

Role of Aberrant Spontaneous Neurotransmission in SNAP25-Associated Encephalopathies.

SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) complex, composed of synaptobrevin, syntaxin, and SNAP25, forms the essential fusion machinery for neurotransmitter release. Recent studies have reported several mutations in the gene encoding SNAP25 as a causative factor for developmental and epileptic encephalopathies of infancy and childhood with diverse clinical manifestations. However, it remains unclear how SNAP25 mutations give rise to these disorders. Here, we show that although structurally clustered mutations in SNAP25 give rise to related synaptic transmission phenotypes, specific alterations in spontaneous neurotransmitter release are a key factor to account for disease heterogeneity. Importantly, we identified a single mutation that augments spontaneous release without altering evoked release, suggesting that aberrant spontaneous release is sufficient to cause disease in humans.

Structural basis for the evolutionary inactivation of Ca2+ binding to synaptotagmin 4.

The neuronal protein synaptotagmin 1 functions as a Ca(2+) sensor in exocytosis via two Ca(2+)-binding C(2) domains. The very similar synaptotagmin 4, which includes all the predicted Ca(2+)-binding residues in the C(2)B domain but not in the C(2)A domain, is also thought to function as a neuronal Ca(2+) sensor. Here we show that, unexpectedly, both C(2) domains of fly synaptotagmin 4 exhibit Ca(2+)-dependent phospholipid binding, whereas neither C(2) domain of rat synaptotagmin 4 binds Ca(2+) or phospholipids efficiently. Crystallography reveals that changes in the orientations of critical Ca(2+) ligands, and perhaps their flexibility, render the rat synaptotagmin 4 C(2)B domain unable to form full Ca(2+)-binding sites. These results indicate that synaptotagmin 4 is a Ca(2+) sensor in the fly but not in the rat, that the Ca(2+)-binding properties of C(2) domains cannot be reliably predicted from sequence analyses, and that proteins clearly identified as orthologs may nevertheless have markedly different functional properties.

Sr2+ binding to the Ca2+ binding site of the synaptotagmin 1 C2B domain triggers fast exocytosis without stimulating SNARE interactions.

Sr(2+) triggers neurotransmitter release similar to Ca(2+), but less efficiently. We now show that in synaptotagmin 1 knockout mice, the fast component of both Ca(2+)- and Sr(2+)-induced release is selectively impaired, suggesting that both cations partly act by binding to synaptotagmin 1. Both the C(2)A and the C(2)B domain of synaptotagmin 1 bind Ca(2+) in phospholipid complexes, but only the C(2)B domain forms Sr(2+)/phospholipid complexes; therefore, Sr(2+) binding to the C(2)B domain is sufficient to trigger fast release, although with decreased efficacy. Ca(2+) induces binding of the synaptotagmin C(2) domains to SNARE proteins, whereas Sr(2+) even at high concentrations does not. Thus, triggering of the fast component of release by Sr(2+) as a Ca(2+) agonist involves the formation of synaptotagmin/phospholipid complexes, but does not require stimulated SNARE binding.

Synaptotagmin function in dense core vesicle exocytosis studied in cracked PC12 cells.

Ca(2+)-triggered dense-core vesicle exocytosis in PC12 cells does not require vesicular synaptotagmins 1 and 2, but may use plasma membrane synaptotagmins 3 and 7 as Ca(2+) sensors. In support of this hypothesis, C(2) domains from the plasma membrane but not vesicular synaptotagmins inhibit PC12 cell exocytosis. Ca(2+) induces binding of both plasma membrane and vesicular synaptotagmins to phospholipids and SNAREs (soluble N-ethylmaleimide-sensitive attachment protein receptors), although with distinct apparent Ca(2+) affinities. Here we used gain-of-function C(2)-domain mutants of synaptotagmin 1 and loss-of-function C(2)-domain mutants of synaptotagmin 7 to examine how synaptotagmins function in dense-core vesicle exocytosis. Our data indicate that phospholipid- but not SNARE-binding by plasma membrane synaptotagmins is the primary determinant of Ca(2+)-triggered dense-core vesicle exocytosis. These results support a general lipid-based mechanism of action of synaptotagmins in exocytosis, with the specificity of various synaptotagmins for different types of fusion governed by their differential localizations and Ca(2+) affinities.

Structural determinants of synaptobrevin 2 function in synaptic vesicle fusion.

Deletion of synaptobrevin/vesicle-associated membrane protein, the major synaptic vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor (R-SNARE), severely decreases but does not abolish spontaneous and evoked synaptic vesicle exocytosis. We now show that the closely related R-SNARE protein cellubrevin rescues synaptic transmission in synaptobrevin-deficient neurons but that deletion of both cellubrevin and synaptobrevin does not cause a more severe decrease in exocytosis than deletion of synaptobrevin alone. We then examined the structural requirements for synaptobrevin to function in exocytosis. We found that substituting glutamine for arginine in the zero-layer of the SNARE motif did not significantly impair synaptobrevin-dependent exocytosis, whereas insertion of 12 or 24 residues between the SNARE motif and transmembrane region abolished the ability of synaptobrevin to mediate Ca2+-evoked exocytosis. Surprisingly, however, synaptobrevin with the 12-residue but not the 24-residue insertion restored spontaneous release in synaptobrevin-deficient neurons. Our data suggest that synaptobrevin mediates Ca2+-triggered exocytosis by tight coupling of the SNARE motif to the transmembrane region and hence forcing the membranes into close proximity for fusion. Furthermore, the fusion reactions underlying evoked and spontaneous release differ mechanistically.

A gain-of-function mutation in synaptotagmin-1 reveals a critical role of Ca2+-dependent soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex binding in synaptic exocytosis.

Synaptotagmin-1, the Ca2+ sensor for fast neurotransmitter release, was proposed to function by Ca2+-dependent phospholipid binding and/or by Ca2+-dependent soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex binding. Extensive in vivo data support the first hypothesis, but testing the second hypothesis has been difficult because no synaptotagmin-1 mutation is known that selectively interferes with SNARE complex binding. Using knock-in mice that carry aspartate-to-asparagine substitutions in a Ca2+-binding site of synaptotagmin-1 (the D232N or D238N substitutions), we now show that the D232N mutation dramatically increases Ca2+-dependent SNARE complex binding by native synaptotagmin-1, but leaves phospholipid binding unchanged. In contrast, the adjacent D238N mutation does not significantly affect SNARE complex binding, but decreases phospholipid binding. Electrophysiological recordings revealed that the D232N mutation increased Ca2+-triggered release, whereas the D238N mutation decreased release. These data establish that fast vesicle exocytosis is driven by a dual Ca2+-dependent activity of synaptotagmin-1, namely Ca2+-dependent binding both to SNARE complexes and to phospholipids.

Structure/function analysis of Ca2+ binding to the C2A domain of synaptotagmin 1.

Synaptotagmin 1, a Ca2+ sensor for fast synaptic vesicle exocytosis, contains two C2 domains that form Ca2+-dependent complexes with phospholipids. To examine the functional importance of Ca2+ binding to the C2A domain of synaptotagmin 1, we studied two C2A domain mutations, D232N and D238N, using recombinant proteins and knock-in mice. Both mutations severely decreased intrinsic Ca2+ binding and Ca2+-dependent phospholipid binding by the isolated C2A domain. Both mutations, however, did not alter the apparent Ca2+ affinity of the double C2 domain fragment, although both decreased the tightness of the Ca2+/phospholipid/double C2 domain complex. When introduced into the endogenous synaptotagmin 1 gene in mice, the D232N and D238N mutations had no apparent effect on morbidity and mortality and caused no detectable alteration in the Ca2+-dependent properties of synaptotagmin 1. Electrophysiological recordings of cultured hippocampal neurons from knock-in mice revealed that neither mutation induced major changes in synaptic transmission. The D232N mutation, however, caused increased synaptic depression during repetitive stimulation, whereas the D238N mutation did not exhibit this phenotype. Our data indicate that Ca2+ binding to the C2A domain of synaptotagmin 1 may be important but not essential, consistent with the finding that the two C2 domains cooperate and may be partially redundant in Ca2+-dependent phospholipid binding. Moreover, although the apparent Ca2+ affinity of the synaptotagmin 1/phospholipid complex is critical, the tightness of the Ca2+/phospholipid complex is not. Our data also demonstrate that subtle changes in the biochemical properties of synaptotagmin 1 can result in significant alterations in synaptic responses.

Assays and properties of arfaptin 2 binding to Rac1 and ADP-ribosylation factors (Arfs).

Arfaptin 1 and 2 were identified as targets for GTP bound ADP-ribosylation factors (Arfs). Arfaptin 1 had no significant effects on guanine nucleotide binding to Arfs, nor enzymatic activities of guanine nucleotide exchange factor (GEF) and GTPase activating protein (GAP) acting on Arfs. However, arfaptin 1 inhibited Arf activation of cholera toxin and phospholipase D (PLD) in a dose-dependent manner. Only GTP-bound forms of Arf1, 5, and 6 interacted with arfaptin 1 and 2, but GTP-Arf1 showed the strongest binding to the arfaptins. In contrast to the binding of Arfs to arfaptins, GDP-Rac1 or dominant negative Rac1-N17N bound to arfaptin 2, whereas GTP-Rac1 or dominant active Rac1-Q61L did not bind to arfaptin 2. Neither GTP-Rac1 nor GDP-Rac1 bound to arfaptin 1. Based on our observation, we propose that arfaptin 2 is a target for GDP-Rac1 and for GTP-Arf1, and is involved in interactions between the Rac1 and Arfs signaling pathways. This chapter describes methods for investigating the interactions of arfaptins 1 and 2 with GTP- or GDP-liganded Arfs and Rac1.

Exocytosis and synaptic vesicle function.

Synaptic vesicles release their vesicular contents to the extracellular space by Ca(2+)-triggered exocytosis. The Ca(2+)-triggered exocytotic process is regulated by synaptotagmin (Syt), a vesicular Ca(2+)-binding C2 domain protein. Synaptotagmin 1 (Syt1), the most studied major isoform among 16 Syt isoforms, mediates Ca(2+)-triggered synaptic vesicle exocytosis by interacting with the target membranes and SNARE/complexin complex. In synapses of the central nervous system, synaptobrevin 2, a major vesicular SNARE protein, forms a ternary SNARE complex with the plasma membrane SNARE proteins, syntaxin 1 and SNAP25. The affinities of Ca(2+)-dependent interactions between Syt1 and its targets (i.e., SNARE complexes and membranes) are well correlated with the efficacies of the corresponding exocytotic processes. Therefore, different SNARE protein isoforms and membrane lipids, which interact with Syt1 with various affinities, are capable of regulating the efficacy of Syt1-mediated exocytosis. Otoferlin, another type of vesicular C2 domain protein that binds to the membrane in a Ca(2+)-dependent manner, is also involved in the Ca(2+)-triggered synaptic vesicle exocytosis in auditory hair cells. However, the functions of otoferlin in the exocytotic process are not well understood. In addition, at least five different types of synaptic vesicle proteins such as synaptic vesicle protein 2, cysteine string protein α, rab3, synapsin, and a group of proteins containing four transmembrane regions, which includes synaptophysin, synaptogyrin, and secretory carrier membrane protein, are involved in modulating the exocytotic process by regulating the formation and trafficking of synaptic vesicles.

Structural and mutational analysis of functional differentiation between synaptotagmins-1 and -7.

Synaptotagmins are known to mediate diverse forms of Ca2+-triggered exocytosis through their C2 domains, but the principles underlying functional differentiation among them are unclear. Synaptotagmin-1 functions as a Ca2+ sensor in neurotransmitter release at central nervous system synapses, but synaptotagmin-7 does not, and yet both isoforms act as Ca2+ sensors in chromaffin cells. To shed light into this apparent paradox, we have performed rescue experiments in neurons from synaptotagmin-1 knockout mice using a chimera that contains the synaptotagmin-1 sequence with its C2B domain replaced by the synaptotagmin-7 C2B domain (Syt1/7). Rescue was not achieved either with the WT Syt1/7 chimera or with nine mutants where residues that are distinct in synaptotagmin-7 were restored to those present in synaptotagmin-1. To investigate whether these results arise because of unique conformational features of the synaptotagmin-7 C2B domain, we determined its crystal structure at 1.44 A resolution. The synaptotagmin-7 C2B domain structure is very similar to that of the synaptotagmin-1 C2B domain and contains three Ca2+-binding sites. Two of the Ca2+-binding sites of the synaptotagmin-7 C2B domain are also present in the synaptotagmin-1 C2B domain and have analogous ligands to those determined for the latter by NMR spectroscopy, suggesting that a discrepancy observed in a crystal structure of the synaptotagmin-1 C2B domain arose from crystal contacts. Overall, our results suggest that functional differentiation in synaptotagmins arises in part from subtle sequence changes that yield dramatic functional differences.

Munc13 C2B domain is an activity-dependent Ca2+ regulator of synaptic exocytosis.

Munc13 is a multidomain protein present in presynaptic active zones that mediates the priming and plasticity of synaptic vesicle exocytosis, but the mechanisms involved remain unclear. Here we use biophysical, biochemical and electrophysiological approaches to show that the central C(2)B domain of Munc13 functions as a Ca(2+) regulator of short-term synaptic plasticity. The crystal structure of the C(2)B domain revealed an unusual Ca(2+)-binding site with an amphipathic alpha-helix. This configuration confers onto the C(2)B domain unique Ca(2+)-dependent phospholipid-binding properties that favor phosphatidylinositolphosphates. A mutation that inactivated Ca(2+)-dependent phospholipid binding to the C(2)B domain did not alter neurotransmitter release evoked by isolated action potentials, but it did depress release evoked by action-potential trains. In contrast, a mutation that increased Ca(2+)-dependent phosphatidylinositolbisphosphate binding to the C(2)B domain enhanced release evoked by isolated action potentials and by action-potential trains. Our data suggest that, during repeated action potentials, Ca(2+) and phosphatidylinositolphosphate binding to the Munc13 C(2)B domain potentiate synaptic vesicle exocytosis, thereby offsetting synaptic depression induced by vesicle depletion.

Synaptotagmin-1 functions as a Ca2+ sensor for spontaneous release.

Spontaneous 'mini' release occurs at all synapses, but its nature remains enigmatic. We found that >95% of spontaneous release in murine cortical neurons was induced by Ca2+-binding to synaptotagmin-1 (Syt1), the Ca2+ sensor for fast synchronous neurotransmitter release. Thus, spontaneous and evoked release used the same Ca2+-dependent release mechanism. As a consequence, Syt1 mutations that altered its Ca2+ affinity altered spontaneous and evoked release correspondingly. Paradoxically, Syt1 deletions (as opposed to point mutations) massively increased spontaneous release. This increased spontaneous release remained Ca2+ dependent but was activated at lower Ca2+ concentrations and with a lower Ca2+ cooperativity than synaptotagmin-driven spontaneous release. Thus, in addition to serving as a Ca2+ sensor for spontaneous and evoked release, Syt1 clamped a second, more sensitive Ca2+ sensor for spontaneous release that resembles the Ca2+ sensor for evoked asynchronous release. These data suggest that Syt1 controls both evoked and spontaneous release at a synapse as a simultaneous Ca2+-dependent activator and clamp of exocytosis.

A complexin/synaptotagmin 1 switch controls fast synaptic vesicle exocytosis.

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