Conformational Dynamics of Calcium-Triggered Activation of Fusion by Synaptotagmin

Synaptotagmin triggers rapid exocytosis of neurotransmitters from synaptic vesicles in response to Calcium (Ca²⁺) ions. Here, we use a novel Nanodisc-based system, designed to be a soluble mimetic of the clamped synaptic vesicle-bilayer junction, combined with fluorescence resonance energy transfer (FRET) spectroscopy to monitor the structural relationships among SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptor), Synaptotagmin C2 domains, and the lipid bilayer in real time during the Ca²⁺-activation process. We report that Synaptotagmin remains rigidly fixed on the partially assembled SNARE complex with no detectable internal rearrangement of its C2 domains, even as it rapidly inserts into the bilayer. We hypothesize that this straightforward, one-step physical mechanism could explain how this Ca²⁺- sensor rapidly activates neurotransmitter release from the clamped state.     

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.     

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.     

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.     

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.     

UNC-31/CAPS docks and primes dense core vesicles in C. elegans neurons

UNC-31 or its mammalian homologue, Ca²⁺-dependent activator protein for secretion (CAPS), is indispensable for exocytosis of dense core vesicle (DCV) and synaptic vesicle (SV). From N- to the C-terminus, UNC-31 contains putative functional domains, including dynactin 1 binding domain (DBD), C2, PH, (M)UNC-13 homology domain (MHD) and DCV binding domain (DCVBD), the last four we examined in this study. We employed UNC-31 null mutant C. elegans worms to examine whether UNC-31 functions could be rescued by ectopic expression of full length UNC-31 vs each of these four domain-deleted mutants. Full length UNC-31 cDNA rescued the phenotypes of C. elegans null mutants in response to Ca²⁺-elevation in ALA neurons. Surprisingly, MHD deletion also rescued UNC-31 exocytotic function in part because the relatively high Ca²⁺ level (pre-flash Ca²⁺ was 450 nM) used in the capacitance study could bypass the MHD defect. Nonetheless, the three other domain-truncation cDNAs had almost no rescue on Ca²⁺ evoked secretion. Importantly, this genetic null mutant rescue strategy enabled physiological studies at levels of whole organism to single cells, such as locomotion assay, pharmacological study of neurotransmission at neuromuscular junction, in vivo neuropeptide release measurement and analysis of vesicular docking. Our results suggest that each of these UNC-31 domains support distinct sequential molecular actions of UNC-31 in vesicular exocytosis, including steps in vesicle tethering and docking that bridge vesicle with plasma membrane, and subsequently priming vesicle by initiating the formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) core complex.     

A novel dual Ca2+ sensor system regulates Ca2+-dependent neurotransmitter release

Ca²⁺-dependent neurotransmitter release requires synaptotagmins as Ca²⁺ sensors to trigger synaptic vesicle (SV) exocytosis via binding of their tandem C2 domains—C2A and C2B—to Ca²⁺. We have previously demonstrated that SNT-1, a mouse synaptotagmin-1 (Syt1) homologue, functions as the fast Ca²⁺ sensor in Caenorhabditis elegans. Here, we report a new Ca²⁺ sensor, SNT-3, which triggers delayed Ca²⁺-dependent neurotransmitter release. snt-1;snt-3 double mutants abolish evoked synaptic transmission, demonstrating that C. elegans NMJs use a dual Ca²⁺ sensor system. SNT-3 possesses canonical aspartate residues in both C2 domains, but lacks an N-terminal transmembrane (TM) domain. Biochemical evidence demonstrates that SNT-3 binds both Ca²⁺ and the plasma membrane. Functional analysis shows that SNT-3 is activated when SNT-1 function is impaired, triggering SV release that is loosely coupled to Ca²⁺ entry. Compared with SNT-1, which is tethered to SVs, SNT-3 is not associated with SV. Eliminating the SV tethering of SNT-1 by removing the TM domain or the whole N terminus rescues fast release kinetics, demonstrating that cytoplasmic SNT-1 is still functional and triggers fast neurotransmitter release, but also exhibits decreased evoked amplitude and release probability. These results suggest that the fast and slow properties of SV release are determined by the intrinsically different C2 domains in SNT-1 and SNT-3, rather than their N-termini–mediated membrane tethering. Our findings therefore reveal a novel dual Ca²⁺ sensor system in C. elegans and provide significant insights into Ca²⁺-regulated exocytosis.     

The Ca2+ Affinity of Synaptotagmin 1 Is Markedly Increased by a Specific Interaction of Its C2B Domain with Phosphatidylinositol 4,5-Bisphosphate

The synaptic vesicle protein synaptotagmin 1 is thought to convey the calcium signal onto the core secretory machinery. Its cytosolic portion mainly consists of two C2 domains, which upon calcium binding are enabled to bind to acidic lipid bilayers. Despite major advances in recent years, it is still debated how synaptotagmin controls the process of neurotransmitter release. In particular, there is disagreement with respect to its calcium binding properties and lipid preferences. To investigate how the presence of membranes influences the calcium affinity of synaptotagmin, we have now measured these properties under equilibrium conditions using isothermal titration calorimetry and fluorescence resonance energy transfer. Our data demonstrate that the acidic phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂), but not phosphatidylserine, markedly increases the calcium sensitivity of synaptotagmin. PI(4,5)P₂ binding is confined to the C2B domain but is not affected significantly by mutations of a lysine-rich patch. Together, our findings lend support to the view that synaptotagmin functions by binding in a trans configuration whereby the C2A domain binds to the synaptic vesicle and the C2B binds to the PI(4,5)P₂-enriched plasma membrane.Calcium-dependent secretion of neurotransmitter-loaded synaptic vesicles is at the heart of synaptic transmission. The underlying membrane fusion reaction between vesicle and plasma membrane has been intensively studied and found to be promoted by both protein-protein as well as protein-lipid interactions. From the multitude of proteins involved in this membrane fusion event, the Ca²⁺-binding protein synaptotagmin 1 is one of its central regulating factors (for review, see Refs. 1–6). Synaptotagmin 1 is anchored in the membrane of synaptic vesicles via a single transmembrane region. Its N-terminal region comprises a short luminal domain, whereas the larger cytoplasmic C-terminal region consists of tandem C2 domains, termed C2A and C2B, tethered to each other via a short linker (7) (a schematic outline of the structural features of synaptotagmin 1 is given in Fig. 1A). Several isoforms with similar domain structure have been identified (8).Open in a separate windowFIGURE 1.Structure of synaptotagmin 1. Synaptotagmin 1 protein consists of two C2 domains, C2A and C2B, that coordinate three and two calcium ions, respectively (16). The acidic residues that coordinate calcium binding is shown schematically, with the residues mutated in the calcium binding mutants (i.e. C2Ab*, C2a*B, and C2a*b*) shown in red. The Lys-rich patch is represented as a ball-and-stick model colored blue with the single cysteine site for the FRET assay (S342C) colored in green (A). The different mutants and constructs used in the study are schematically depicted (B).C2 domains are Ca²⁺ binding modules of ∼130 amino acids, first described as the second conserved region of protein kinase C (PKC)² (9). The C2A domain of synaptotagmin 1 was the first C2 domain structure to be determined (10). In subsequent studies other C2 domains, including the C2B domain of synaptotagmin, were shown to exhibit very similar three-dimensional structures. They have a conserved eight-stranded anti-parallel β-sandwich connected by surface loops. C2 modules are most commonly found in enzymes involved in lipid modifications and signal transduction (PKC, phospholipases, phosphatidylinositol 3-kinases, etc.) and proteins involved in membrane trafficking (synaptotagmins, rabphilin, DOC2, etc.) (11).Calcium ions bind in a cup-shaped depression formed by the N- and C-terminal loops of the C2 key motifs of C2 domains. Notably, the coordination spheres for the Ca²⁺ ions are incomplete (12, 13). In canonical C2 domains, this incomplete coordination sphere can be occupied by anionic and neutral (14, 15) phospholipids, enabling the C2 domain to be attached to the membrane. Hence, it is thought that the general function of C2 domains is to mediate Ca²⁺-triggered binding of the protein to a membrane. In fact, upon rise of the intracellular calcium level, C2 domain-containing enzymes are translocated to the membrane so that the catalytic domains can interact with lipids or membrane-anchored protein substrates (11). Yet synaptotagmin 1 does not contain such a catalytic domain, suggesting that the properties of its tandem C2 domains are the sole key to understanding its molecular function. In neurotransmission, synaptotagmin is thought to transmit the Ca²⁺ signal onto the core membrane fusion machinery, composed of the three SNARE (soluble N-ethylmaleimide sensitive factor attachment receptor) proteins syntaxin 1, SNAP-25 (Q-SNAREs, residing on the plasma membrane), and synaptobrevin 2 (also referred to as VAMP2 (vesicle-associated membrane protein) (R-SNARE, residing on the synaptic vesicle)). So far the multifarious interplay between the SNARE machinery, the two fusing membranes, and synaptotagmin 1 is not well understood. The crystal structure of the entire cytosolic domain of synaptotagmin in the absence of Ca²⁺ has revealed an interesting domain arrangement with the two C2 domains facing in opposite directions (16), hinting at the possibility that the molecule might interact with two opposing membranes upon rise of intracellular Ca²⁺.Although the underlying processes of Ca²⁺ binding and Ca²⁺-dependent membrane binding of synaptotagmin 1 have been studied by a multitude of structural and biochemical investigations, they have not revealed features of synaptotagmin C2 domains that are different from those of other C2 domain-containing proteins. Calcium binding to synaptotagmin in the absence of membranes has been studied by NMR. These studies showed that the isolated C2A domain of synaptotagmin 1 binds three calcium ions with an apparent affinity of ∼60–75 μm, ∼400–500 μm, and more than 1 mm (17). The isolated C2B domain binds two calcium ions with similar calcium affinities in the range of ∼300–600 μm (18). The relatively low intrinsic Ca²⁺ affinities of both C2 domains are difficult to reconcile with the role of synaptotagmin 1 as the Ca²⁺ sensor for fast and synchronous neurotransmitter release, suggesting that interaction with phospholipids contributes to its Ca²⁺ sensitivity. Indeed, Ca²⁺-triggered binding of isolated C2 domains to lipid membranes was first shown in an in vitro study of synaptotagmin 1 using a fluorescence-based approach (19). Subsequent equilibrium fluorescence studies have shed more light on the molecular process underlying membrane binding of synaptotagmin 1, for example by demonstrating that the isolated C2A domain dips into the membrane bilayer upon Ca²⁺ binding (20). This penetration was corroborated by electro-paramagnetic resonance (EPR) spectroscopy studies, which also showed that the penetration depth increased when both C2 domains of synaptotagmin 1 were attached to each other (21) as compared with the single domains (22, 23). However, a variety of different Ca²⁺ and lipid preferences for the individual C2 domains of synaptotagmin has been reported (3, 5, 6).To resolve these discrepancies and to shed more light on the molecular interactions of synaptotagmin 1, we have now used quantitative approaches to study the Ca²⁺ concentration and the lipid composition needed for synaptotagmin to bind to membranes. We employed isothermal titration calorimetry (ITC) to measure the intrinsic calcium binding affinities of synaptotagmin 1 C2 domains both as isolated domains as well as in the context of the tandem C2AB protein. Then, we investigated whether the intrinsic calcium affinity is modulated in the presence of lipids using a newly developed fluorescence resonance energy transfer (FRET) approach. In addition, we investigated how Ca²⁺ and phospholipid binding of synaptotagmin is affected when the Ca²⁺ binding sites in both C2 domains and the putative phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂)-interacting site in the C2 domain are inactivated. We found that the two C2 domains bind calcium largely independently but cooperate in membrane binding. Furthermore, we confirmed that the C2B domain interacts specifically with PI(4,5)P₂. Remarkably, in the presence of PI(4,5)P₂, drastically lower amounts of calcium were needed for membrane binding.     

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.     

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.     

A quaternary SNARE-synaptotagmin-Ca2+-phospholipid complex in neurotransmitter release

The function of synaptotagmin as a Ca²⁺ sensor in neurotransmitter release involves Ca²⁺-dependent phospholipid binding to its two C₂ domains, but this activity alone does not explain why Ca²⁺ binding to the C₂B domain is more critical for release than Ca²⁺ binding to the C₂A domain. Synaptotagmin also binds to SNARE complexes, which are central components of the membrane fusion machinery, and displaces complexins from the SNAREs. However, it is unclear how phospholipid binding to synaptotagmin is coupled to SNARE binding and complexin displacement. Using supported lipid bilayers deposited within microfluidic channels, we now show that Ca²⁺ induces simultaneous binding of synaptotagmin to phospholipid membranes and SNARE complexes, resulting in an intimate quaternary complex that we name SSCAP complex. Mutagenesis experiments show that Ca²⁺ binding to the C₂B domain is critical for SSCAP complex formation and displacement of complexin, providing a clear rationale for the preponderant role of the C₂B domain in release. This and other correlations between the effects of mutations on SSCAP complex formation and their functional effects in vivo suggest a key role for this complex in release. We propose a model whereby the highly positive electrostatic potential at the tip of the SSCAP complex helps to induce membrane fusion during release.     

A role for synaptotagmin (p65) in regulated exocytosis

Proteins that are specifically localized to synaptic vesicles in the nervous system have been proposed to mediate aspects of synaptic transmission. Antibodies raised against the cytoplasmic domains of five of these proteins, vamp, rab3A, synaptophysin, synaptotagmin, and SV2, were used to investigate their function. Microinjection of monoclonal and polyclonal antibodies raised against synaptotagmin (p65), but not the other vesicle proteins, decreases K⁺/Ca²⁺-mediated dopamine β-hydroxylase surface staining, a measure of regulated secretion in PC12 cells. Microinjection of a soluble fragment of synaptotagmin encompassing one of the domains homologous to the C2 regulatory region of protein kinase C, but lacking the membrane anchor, also inhibits evoked dopamine β-hydroxylase surface staining. These results provide support for the hypothesis that synaptotagmin, a Ca²⁺- and phospholipid-binding protein, is important for regulated exocytosis in neurons.     

A Highlights from MBoC Selection: Mutations that disrupt Ca2+-binding activity endow Doc2β with novel functional properties during synaptic transmission

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

Ca2+–Calmodulin regulates SNARE assembly and spontaneous neurotransmitter release via v-ATPase subunit V0a1

Most chemical neurotransmission occurs through Ca²⁺-dependent evoked or spontaneous vesicle exocytosis. In both cases, Ca²⁺ sensing is thought to occur shortly before exocytosis. In this paper, we provide evidence that the Ca²⁺ dependence of spontaneous vesicle release may partly result from an earlier requirement of Ca²⁺ for the assembly of soluble N-ethylmaleimide–sensitive fusion attachment protein receptor (SNARE) complexes. We show that the neuronal vacuolar-type H⁺-adenosine triphosphatase V0 subunit a1 (V100) can regulate the formation of SNARE complexes in a Ca²⁺–Calmodulin (CaM)-dependent manner. Ca²⁺–CaM regulation of V100 is not required for vesicle acidification. Specific disruption of the Ca²⁺-dependent regulation of V100 by CaM led to a >90% loss of spontaneous release but only had a mild effect on evoked release at Drosophila melanogaster embryo neuromuscular junctions. Our data suggest that Ca²⁺–CaM regulation of V100 may control SNARE complex assembly for a subset of synaptic vesicles that sustain spontaneous release.     

Synaptotagmin perturbs the structure of phospholipid bilayers

Synaptotagmin I (syt), an integral protein of the synaptic vesicle membrane, is believed to act as a Ca2+ sensor for neuronal exocytosis. Syt's cytoplasmic domain consists largely of two C2 domains, C2A and C2B. In response to Ca2+ binding, the C2 domains interact with membranes, becoming partially embedded in the lipid bilayer. We have imaged syt C2AB in association with lipid bilayers under fluid, using AFM. As expected, binding of C2AB to bilayers required both an anionic phospholipid [phosphatidylserine (PS)] and Ca2+. C2AB associated with bilayers in the form of aggregates of varying stoichiometries, and aggregate size increased with an increase in PS content. Repeated scanning of bilayers revealed that as C2AB dissociated it left behind residual indentations in the bilayer. The mean depth of these identations was 1.81 nm, indicating that they did not span the bilayer. Individual C2 domains (C2A and C2B) also formed aggregates and produced bilayer indentations. Binding of C2AB to bilayers and the formation of indentations were significantly compromised by mutations that interfere with binding of Ca2+ to syt or reduce the positive charge on the surface of C2B. We propose that bilayer perturbation by syt might be significant with respect to its ability to promote membrane fusion.     

Synaptotagmin’s Role in Neurotransmitter Release Likely Involves Ca2+-induced Conformational Transition

Neuronal exocytosis is mediated by a Ca²⁺-triggered membrane fusion event that joins synaptic vesicles and presynaptic membrane. In this event, synaptotagmin I plays a key role as a Ca²⁺ sensor protein that binds to and bends the presynaptic membrane with its C2B domain, and thereby initiates membrane fusion. We report free energy calculations according to which C2B-induced membrane bending is preceded by a Ca²⁺- and membrane-dependent conformational transition. In this transition C2B attaches to the membrane, moves its C-terminal helix from the orientation seen in the available (but membrane-free) crystal/NMR structures as pointing away from the membrane (helix-up), to an orientation pointing toward the membrane (helix-down). In the C2B helix-down state, lipid tails in the proximal membrane bilayer leaflet interact with the moved helix and become disordered, whereas tails in the distal leaflet, to keep in contact with the proximal leaflet, become stretched and ordered. The difference in lipid tail packing between the two leaflets results in an imbalance of pressure across the membrane, and thereby causes membrane bending. The lipid-disordering monitored in the simulations is well suited to facilitate Ca²⁺-triggered membrane fusion.     

Partitioning of synaptotagmin I C2 domains between liquid-ordered and liquid-disordered inner leaflet lipid phases

Synaptotagmin I is the calcium sensor in synchronous neurotransmitter release caused by fusion of synaptic vesicles with the presynaptic membrane in neurons. Synaptotagmin I interacts with acidic phospholipids, but also with soluble N-ethylmaleimide-sensitive factor attachment receptors (SNAREs), at various stages in presynaptic membrane fusion. Because SNAREs can be organized into small cholesterol-dependent clusters in membranes, it is important to determine whether the C2 domains of synaptotagmin target membrane domains with different cholesterol contents. To address this question, we used a previously developed asymmetric two-phase lipid bilayer system to investigate the membrane binding and lipid phase targeting of soluble C2A and C2AB domains of synaptotagmin. We found that both domains target more disordered cholesterol-poor domains better than highly ordered cholesterol-rich domains. The selectivity is greatest (～3-fold) for C2A binding to disordered domains that are formed in the presence of 5 mol % PIP(2) and 15 mol % PS. It is smallest (～1.4-fold) for C2AB binding to disordered domains that are formed in the presence of 40 mol % PS. In the course of these experiments, we also found that C2A domains in the presence of Ca(2+) and C2AB domains in the absence of Ca(2+) are quite reliable reporters of the acidic lipid distribution between ordered and disordered lipid phases. Accordingly, PS prefers the liquid-disordered phase over the liquid-ordered phase by ～2-fold, but PIP(2) has an up to 3-fold preference for the liquid-disordered phase.     

Synaptotagmin’s Role in Neurotransmitter Release Likely Involves Ca-induced Conformational Transition

Neuronal exocytosis is mediated by a Ca²⁺-triggered membrane fusion event that joins synaptic vesicles and presynaptic membrane. In this event, synaptotagmin I plays a key role as a Ca²⁺ sensor protein that binds to and bends the presynaptic membrane with its C2B domain, and thereby initiates membrane fusion. We report free energy calculations according to which C2B-induced membrane bending is preceded by a Ca²⁺- and membrane-dependent conformational transition. In this transition C2B attaches to the membrane, moves its C-terminal helix from the orientation seen in the available (but membrane-free) crystal/NMR structures as pointing away from the membrane (helix-up), to an orientation pointing toward the membrane (helix-down). In the C2B helix-down state, lipid tails in the proximal membrane bilayer leaflet interact with the moved helix and become disordered, whereas tails in the distal leaflet, to keep in contact with the proximal leaflet, become stretched and ordered. The difference in lipid tail packing between the two leaflets results in an imbalance of pressure across the membrane, and thereby causes membrane bending. The lipid-disordering monitored in the simulations is well suited to facilitate Ca²⁺-triggered membrane fusion.     

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.