SNARE complex alters the interactions of the Ca2+ sensor synaptotagmin 1 with lipid bilayers

Release of neuronal transmitters from nerve terminals is triggered by the molecular Ca²⁺ sensor synaptotagmin 1 (Syt1). Syt1 is a transmembrane protein attached to the synaptic vesicle (SV), and its cytosolic region comprises two domains, C2A and C2B, which are thought to penetrate into lipid bilayers upon Ca²⁺ binding. Before fusion, SVs become attached to the presynaptic membrane (PM) by the four-helical SNARE complex, which is thought to bind the C2B domain in vivo. To understand how the interactions of Syt1 with lipid bilayers and the SNARE complex trigger fusion, we performed molecular dynamics (MD) simulations at a microsecond scale. We investigated how the isolated C2 modules and the C2AB tandem of Syt1 interact with membranes mimicking either SV or PM. The simulations showed that the C2AB tandem can either bridge SV and PM or insert into PM with its Ca²⁺-bound tips and that the latter configuration is more favorable. Surprisingly, C2 domains did not cooperate in penetrating into PM but instead mutually hindered their insertion into the bilayer. To test whether the interaction of Syt1 with lipid bilayers could be affected by the C2B-SNARE attachment, we performed systematic conformational analysis of the C2AB-SNARE complex. Notably, we found that the C2B-SNARE interface precludes the coupling of C2 domains and promotes their insertion into PM. We performed the MD simulations of the prefusion protein complex positioned between the lipid bilayers mimicking PM and SV, and our results demonstrated in silico that the presence of the Ca²⁺ bound C2AB tandem promotes lipid merging. Altogether, our MD simulations elucidated the role of the Syt1-SNARE interactions in the fusion process and produced the dynamic all-atom model of the prefusion protein-lipid complex.     

The Juxtamembrane Linker of Full-length Synaptotagmin 1 Controls Oligomerization and Calcium-dependent Membrane Binding

Synaptotagmin 1 (Syt1) is the calcium sensor for synchronous neurotransmitter release. The two C2 domains of Syt1, which may mediate fusion by bridging the vesicle and plasma membranes, are connected to the vesicle membrane by a 60-residue linker. Here, we use site-directed spin labeling and a novel total internal reflection fluorescence vesicle binding assay to characterize the juxtamembrane linker and to test the ability of reconstituted full-length Syt1 to interact with opposing membrane surfaces. EPR spectroscopy demonstrates that the majority of the linker interacts with the membrane interface, thereby limiting the extension of the C2A and C2B domains into the cytoplasm. Pulse dipolar EPR spectroscopy provides evidence that purified full-length Syt1 is oligomerized in the membrane, and mutagenesis indicates that a glycine zipper/GXXXG motif within the linker helps mediate oligomerization. The total internal reflection fluorescence-based vesicle binding assay demonstrates that full-length Syt1 that is reconstituted into supported lipid bilayers will capture vesicles containing negatively charged lipid in a Ca²⁺-dependent manner. Moreover, the rate of vesicle capture increases with Syt1 density, and mutations in the GXXXG motif that inhibit oligomerization of Syt1 reduce the rate of vesicle capture. This work demonstrates that modifications within the 60-residue linker modulate both the oligomerization of Syt1 and its ability to interact with opposing bilayers. In addition to controlling its activity, the oligomerization of Syt1 may play a role in organizing proteins within the active zone of membrane fusion.     

Postsynaptic regulation of synaptic plasticity by synaptotagmin 4 requires both C2 domains

Ca²⁺ influx into synaptic compartments during activity is a key mediator of neuronal plasticity. Although the role of presynaptic Ca²⁺ in triggering vesicle fusion though the Ca²⁺ sensor synaptotagmin 1 (Syt 1) is established, molecular mechanisms that underlie responses to postsynaptic Ca²⁺ influx remain unclear. In this study, we demonstrate that fusion-competent Syt 4 vesicles localize postsynaptically at both neuromuscular junctions (NMJs) and central nervous system synapses in Drosophila melanogaster. Syt 4 messenger RNA and protein expression are strongly regulated by neuronal activity, whereas altered levels of postsynaptic Syt 4 modify synaptic growth and presynaptic release properties. Syt 4 is required for known forms of activity-dependent structural plasticity at NMJs. Synaptic proliferation and retrograde signaling mediated by Syt 4 requires functional C2A and C2B Ca²⁺–binding sites, as well as serine 284, an evolutionarily conserved substitution for a key Ca²⁺-binding aspartic acid found in other synaptotagmins. These data suggest that Syt 4 regulates activity-dependent release of postsynaptic retrograde signals that promote synaptic plasticity, similar to the role of Syt 1 as a Ca²⁺ sensor for presynaptic vesicle fusion.     

The C2 Domains of Human Synaptotagmin 1 Have Distinct Mechanical Properties

Synaptotagmin 1 (Syt1) is the Ca⁺² receptor for fast, synchronous vesicle fusion in neurons. Because membrane fusion is an inherently mechanical, force-driven event, Syt1 must be able to adapt to the energetics of the fusion apparatus. Syt1 contains two C2 domains (C2A and C2B) that are homologous in sequence and three-dimensional in structure; yet, a number of observations have suggested that they have distinct biochemical and biological properties. In this study, we analyzed the mechanical stability of the C2A and C2B domains of human Syt1 using single-molecule atomic force microscopy. We found that stretching the C2AB domains of Syt1 resulted in two distinct unfolding force peaks. The larger force peak of ∼100 pN was identified as C2B and the second peak of ∼50 pN as C2A. Furthermore, a significant fraction of C2A domains unfolded through a low force intermediate that was not observed in C2B. We conclude that these domains have different mechanical properties. We hypothesize that a relatively small stretching force may be sufficient to deform the effector-binding regions of the C2A domain and modulate the affinity for soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs), phospholipids, and Ca⁺².     

Synaptotagmin 1 and SNAREs form a complex that is structurally heterogeneous

Synaptotagmin 1 (syt1) functions as a Ca²⁺-sensor for neuronal exocytosis. Here, site-directed spin labeling was used to examine the complex formed between a soluble fragment of syt1, which contains its two C2 domains, and the neuronal core soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. Changes in electron paramagnetic resonance lineshape and accessibility for spin-labeled syt1 mutants indicate that in solution, the assembled core SNARE complex contacts syt1 in several regions. For the C2B domain, contact occurs in the polybasic face and sites opposite the Ca²⁺-binding loops. For the C2A domain, contact is seen with the SNARE complex in a region near loop 2. Double electron-electron resonance was used to estimate distances between the two C2 domains of syt1. These distances have broad distributions in solution, which do not significantly change when syt1 is fully associated with the core SNARE complex. The broad distance distributions indicate that syt1 is structurally heterogeneous when bound to the SNAREs and does not assume a well-defined structure. Simulated annealing using electron paramagnetic resonance-derived distance restraints produces a family of syt1 structures where the Ca²⁺-binding regions of each domain face in roughly opposite directions. The results suggest that when associated with the SNAREs, syt1 is configured to bind opposing bilayers, but that the syt1/SNARE complex samples multiple conformational states.     

Ca2+ releases E‐Syt1 autoinhibition to couple ER‐plasma membrane tethering with lipid transport

The extended synaptotagmins (E‐Syts) are endoplasmic reticulum (ER) proteins that bind the plasma membrane (PM) via C2 domains and transport lipids between them via SMP domains. E‐Syt1 tethers and transports lipids in a Ca²⁺‐dependent manner, but the role of Ca²⁺ in this regulation is unclear. Of the five C2 domains of E‐Syt1, only C2A and C2C contain Ca²⁺‐binding sites. Using liposome‐based assays, we show that Ca²⁺ binding to C2C promotes E‐Syt1‐mediated membrane tethering by releasing an inhibition that prevents C2E from interacting with PI(4,5)P₂‐rich membranes, as previously suggested by studies in semi‐permeabilized cells. Importantly, Ca²⁺ binding to C2A enables lipid transport by releasing a charge‐based autoinhibitory interaction between this domain and the SMP domain. Supporting these results, E‐Syt1 constructs defective in Ca²⁺ binding in either C2A or C2C failed to rescue two defects in PM lipid homeostasis observed in E‐Syts KO cells, delayed diacylglycerol clearance from the PM and impaired Ca²⁺‐triggered phosphatidylserine scrambling. Thus, a main effect of Ca²⁺ on E‐Syt1 is to reverse an autoinhibited state and to couple membrane tethering with lipid transport.     

The cytoplasmic C2A domain of synaptotagmin shows sequence specific interaction with its own mRNA

Synaptotagmin-1 (Syt1) is essential in Ca²⁺-dependent neurotransmitter release, but its expression regulation is unknown. Here we report that the cytoplasmic Syt1 fragment forms ribonucleoprotein complex by interacting with the 3′ untranslated region (3^′UTR) of its own mRNA. Two protein-binding domains, GU₁₅ repeat and GUCAAUG, within the Syt 3′UTR and the C2 domains in Syt1, especially C2A, are essential in this ribonucleoprotein complex formation. Furthermore, in in vitro assay the translation efficiency of Syt1 mRNA was downregulated in presence of 3′UTR. These results demonstrate for the fist time that the soluble fraction of Syt1 can interact with its own mRNA in a highly sequence specific manner.     

Partial Metal Ion Saturation of C2 Domains Primes Synaptotagmin 1-Membrane Interactions

Synaptotagmin 1 (Syt1) is an integral membrane protein whose phospholipid-binding tandem C2 domains, C2A and C2B, act as Ca²⁺ sensors of neurotransmitter release. Our objective was to understand the role of individual metal-ion binding sites of these domains in the membrane association process. We used Pb²⁺, a structural and functional surrogate of Ca²⁺, to generate the protein states with well-defined protein-metal ion stoichiometry. NMR experiments revealed that binding of one divalent metal ion per C2 domain results in loss of conformational plasticity of the loop regions, potentially pre-organizing them for additional metal-ion and membrane-binding events. In C2A, a divalent metal ion in site 1 is sufficient to drive its weak association with phosphatidylserine-containing membranes, whereas in C2B, it enhances the interactions with the signaling lipid phosphatidylinositol-4,5-bisphosphate. In full-length Syt1, both Pb²⁺-complexed C2 domains associate with phosphatidylserine-containing membranes. Electron paramagnetic resonance experiments show that the extent of membrane insertion correlates with the occupancy of the C2 metal ion sites. Together, our results indicate that upon partial metal ion saturation of the intra-loop region, Syt1 adopts a dynamic, partially membrane-bound state. The properties of this state, such as conformationally restricted loop regions and positioning of C2 domains in close proximity to anionic lipid headgroups, “prime” Syt1 for cooperative binding of a full complement of metal ions and deeper membrane insertion.     

Regulation of Exocytosis and Fusion Pores by Synaptotagmin-Effector Interactions

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.     

Solution and Membrane-Bound Conformations of the Tandem C2A and C2B Domains of Synaptotagmin 1: Evidence for Bilayer Bridging

Synaptotagmin 1 (syt1) is a synaptic vesicle membrane protein that functions as the Ca²⁺ sensor in neuronal exocytosis. Here, site-directed spin labeling was used to generate models for the solution and membrane-bound structures of a soluble fragment of syt1 containing its two C2 domains, C2A and C2B. In solution, distance restraints between the two C2 domains of syt1 were measured using double electron-electron resonance and used in a simulated annealing routine to generate models for the structure of the tandem C2A-C2B fragment. The data indicate that the two C2 domains are flexibly linked and do not interact with each other in solution, with or without Ca²⁺. However, the favored orientation is one where the Ca²⁺-binding loops are oriented in opposite directions. A similar approach was taken for membrane-associated C2A-C2B, combining both distances and bilayer depth restraints with simulated annealing. The restraints can only be satisfied if the Ca²⁺ and membrane-binding surfaces of the domains are oriented in opposite directions so that C2A and C2B are docked to opposing bilayers. The result suggests that syt1 functions to bridge across the vesicle and plasma membrane surfaces in a Ca²⁺-dependent manner.     

Synaptotagmin I and IX function redundantly in controlling fusion pore of large dense core vesicles

Synaptotagmins (Syts) constitute a large family of at least 16 members and individual Syt isoforms exhibit distinct Ca²⁺-binding properties and subcellular localization. It remains to be demonstrated whether multiple Syt isoforms can function independently or cooperatively on certain type of vesicle. In the current study, we have developed NPY-pHluorin to specifically assess exocytosis of large dense core vesicles (LDCVs) and studied the requirement of Syt I and Syt IX for LDCV exocytosis in PC12 cells. We found that down-regulation of both Syt I and Syt IX resulted in a significant loss of Ca²⁺-dependent LDCV exocytosis. Moreover, our results suggest Syt I and Syt IX play redundant role in controlling the choice of fusion modes. Down-regulation of both Syt I and Syt IX renders more fusion in the kiss-and-run mode. We conclude that Syt I and Syt IX function redundantly in Ca²⁺-sensing and fusion pore dilation on LDCVs in PC12 cells.     

The Calcium-Dependent and Calcium-Independent Membrane Binding of Synaptotagmin 1: Two Modes of C2B Binding

The Ca²⁺-independent membrane interactions of the soluble C2 domains from synaptotagmin 1 (syt1) were characterized using a combination of site-directed spin labeling and vesicle sedimentation. The second C2 domain of syt1, C2B, binds to membranes containing phosphatidylserine and phosphatidylcholine in a Ca²⁺-independent manner with a lipid partition coefficient of approximately 3.0 × 10² M^− 1. A soluble fragment containing the first and second C2 domains of syt1, C2A and C2B, has a similar affinity, but C2A alone has no detectable affinity to phosphatidylcholine/phosphatidylserine bilayers in the absence of Ca²⁺. Although the Ca²⁺-independent membrane affinity of C2B is modest, it indicates that this domain will never be free in solution within the cell. Site-directed spin labeling was used to obtain bilayer depth restraints, and a simulated annealing routine was used to generate a model for the membrane docking of C2B in the absence of Ca²⁺. In this model, the polybasic strand of C2B forms the membrane binding surface for the domain; however, this face of C2B does not penetrate the bilayer but is localized within the aqueous double layer when C2B is bound. This double-layer location indicates that C2B interacts in a purely electrostatic manner with the bilayer interface. In the presence of Ca²⁺, the membrane affinity of C2B is increased approximately 20-fold, and the domain rotates so that the Ca²⁺-binding loops of C2B insert into the bilayer. This Ca²⁺-triggered conformational change may act as a switch to modulate the accessibility of the polybasic face of C2B and control interactions of syt1 with other components of the fusion machinery.     

Phosphatidylserine Regulation of Ca2+-triggered Exocytosis and Fusion Pores in PC12 Cells

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.     

Synaptotagmin-1 and Doc2b Exhibit Distinct Membrane-Remodeling Mechanisms

Synaptotagmin-1 (Syt1) is a calcium sensor protein that is critical for neurotransmission and is therefore extensively studied. Here, we use pairs of optically trapped beads coated with SNARE-free synthetic membranes to investigate Syt1-induced membrane remodeling. This activity is compared with that of Doc2b, which contains a conserved C₂AB domain and induces membrane tethering and hemifusion in this cell-free model. We find that the soluble C₂AB domain of Syt1 strongly affects the probability and strength of membrane-membrane interactions in a strictly Ca²⁺- and protein-dependent manner. Single-membrane loading of Syt1 yielded the highest probability and force of membrane interactions, whereas in contrast, Doc2b was more effective after loading both membranes. A lipid-mixing assay with confocal imaging reveals that both Syt1 and Doc2b are able to induce hemifusion; however, significantly higher Syt1 concentrations are required. Consistently, both C₂AB fragments cause a reduction in the membrane-bending modulus, as measured by a method based on atomic force microscopy. This lowering of the energy required for membrane deformation may contribute to Ca²⁺-induced fusion.     

CaV2.1 (P/Q channel) interaction with synaptic proteins is essential for depolarization-evoked release

It is well established that syntaxin 1A (Sx1A), SNAP-25 and synaptotagmin (Syt1) either alone or in combination, modify the kinetic properties of voltage-gated Ca²⁺ channels (VGCCs). The interaction interface resides mainly at the cytosolic II-III domain of the alpha1 subunit of the channels, while Sx1A interacts with the channel also via two highly conserved cysteine residues at the transmembrane domain. In the present study, we characterized Ca²⁺-independent coupling of the human neuronal P/Q-type calcium channel (Ca_V2.1) with Sx1A, SNAP-25, Syt1 and synaptobrevin (VAMP) in BAPTA-injected Xenopus oocytes. The co-expression of Ca_V2.1 with Sx1A, SNAP-25 and Syt1, produced a multiprotein complex with distinctive kinetic properties analogous to the excitosome complexes generated by Ca_V1.2, Ca_V2.2, and Ca_V2.3. The distinct kinetic properties of Ca_V2.1 acquired by its close association with Syt1 and t-SNAREs suggest that the vesicle is tethered to the neuronal channel and to the exocytotic machinery independently of intracellular Ca²⁺. To explore the relevance of these interactions to secretion we exploited a BotC1-and a BotA-sensitive secretion system developed for Xenopus oocytes not buffered by BAPTA, in which depolarization-evoked secretion is monitored by a change in membrane capacitance. The reconstituted Ca_V2.1 release is consistent with the model in which the VGCC acts from within the exocytotic complex playing a signaling role in triggering release. The relevance of these results to secretion posits the role of possible rearrangements within the excitosome subsequent to Ca²⁺ entry, setting the stage for the fusion of channel-tethered-vesicles upon the arrival of an action potential.     

Interaction of synaptotagmin with lipid bilayers, analyzed by single-molecule force spectroscopy

Synaptotagmin I is the major Ca²⁺ sensor for membrane fusion during neurotransmitter release. The cytoplasmic domain of synaptotagmin consists of two C2 domains, C2A and C2B. On binding Ca²⁺, the tips of the two C2 domains rapidly and synchronously penetrate lipid bilayers. We investigated the forces of interaction between synaptotagmin and lipid bilayers using single-molecule force spectroscopy. Glutathione-S-transferase-tagged proteins were attached to an atomic force microscope cantilever via a glutathione-derivatized polyethylene glycol linker. With wild-type C2AB, the force profile for a bilayer containing phosphatidylserine had both Ca²⁺-dependent and Ca²⁺-independent components. No force was detected when the bilayer lacked phosphatidylserine, even in the presence of Ca²⁺. The binding characteristics of C2A and C2B indicated that the two C2 domains cooperate in binding synaptotagmin to the bilayer, and that the relatively weak Ca²⁺-independent force depends only on C2A. When the lysine residues K189-192 and K326, 327 were mutated to alanine, the strong Ca²⁺-dependent binding interaction was either absent or greatly reduced. We conclude that synaptotagmin binds to the bilayer via C2A even in absence of Ca²⁺, and also that positively charged regions of both C2A and C2B are essential for the strong Ca²⁺-dependent binding of synaptotagmin to the bilayer.     

Rat and Drosophila Synaptotagmin 4 Have Opposite Effects during SNARE-catalyzed Membrane Fusion

Synaptotagmins (Syt) are a large family of proteins that regulate membrane traffic in neurons and other cell types. One isoform that has received considerable attention is SYT4, with apparently contradictory reports concerning the function of this isoform in fruit flies and mice. SYT4 was reported to function as a negative regulator of neurotrophin secretion in mouse neurons and as a positive regulator of secretion of a yet to be identified growth factor from muscle cells in flies. Here, we have directly compared the biochemical and functional properties of rat and fly SYT4. We report that rat SYT4 inhibited SNARE-catalyzed membrane fusion in both the absence and presence of Ca²⁺. In marked contrast, fly SYT4 stimulated SNARE-mediated membrane fusion in response to Ca²⁺. Analysis of chimeric molecules, isolated C2 domains, and point mutants revealed that the C2B domain of the fly protein senses Ca²⁺ and is sufficient to stimulate fusion. Rat SYT4 was able to stimulate fusion in response to Ca²⁺ when the conserved Asp-to-Ser Ca²⁺ ligand substitution in its C2A domain was reversed. In summary, rat SYT4 serves as an inhibitory isoform, whereas fly SYT4 is a bona fide Ca²⁺ sensor capable of coupling Ca²⁺ to membrane fusion.     

Silence of Synaptotagmin VII inhibits release of dense core vesicles in PC12 cells

Synaptotagmin VII (Syt VII), which has a higher Ca²⁺ affinity and slower disassembly kinetics with lipid than Syt I and Syt IX, was regarded as being uninvolved in synaptic vesicle (SV) exocytosis but instead possibly as a calcium sensor for the slower kinetic phase of dense core vesicles (DCVs) release. By using high temporal resolution capacitance and amperometry measurements, it was demonstrated that the knockdown of endogenous Syt VII attenuated the fusion of DCV with the plasma membrane, reduced the amplitude of the exocytotic burst of the Ca²⁺-triggered DCV release without affecting the slope of the sustained component, and blocked the fusion pore expansion. This suggests that Syt VII is the Ca²⁺ sensor of DCV fusion machinery and is an essential factor for the establishment and maintenance of the pool size of releasable DCVs in PC12 cells.     

Synaptotagmin IV Acts as a Multi-Functional Regulator of Ca<Superscript>2+</Superscript>-Dependent Exocytosis

In response to stimuli, secretary cells secrete a variety of signaling molecules packed in vesicles (e.g., neurotransmitters and peptide hormones) into the extracellular space by exocytosis. The vesicle secretion is often triggered by calcium ion (Ca²⁺) entered into secretary cells and achieved by the fusion of secretory vesicles with the plasma membrane. Recent accumulating evidence has indicated that members of the synaptotagmin (Syt) family play a major role in Ca²⁺-dependent exocytosis, and Syt I, in particular, is now widely accepted as the major Ca²⁺-sensor for synchronous neurotransmitter release. Involvement of other Syt isoforms in Ca²⁺-dependent exocytotic events other than neurotransmitter release has also been reported, and the Syt IV isoform is of particular interest, because Syt IV has several unique features not found in Syt I (e.g., immediate early gene product induced by deporalization and postsynaptic localization). In this article, we summarize the literature on the multi-functional role of Syt IV in Ca²⁺-dependent exocytosis.     

A Highlights from MBoC Selection: Functional analysis of the interface between the tandem C2 domains of synaptotagmin-1

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.