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.     

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.     

Choice of reconstitution protocol modulates the aggregation state of full-length membrane-reconstituted synaptotagmin-1

Synaptotagmin-1 (Syt1) functions as the Ca²⁺ sensor in neuronal exocytosis, and it is routinely incorporated into lipid bilayers along with other components of the fusion machinery in order to reconstruct the in vivo fusion process. Here, we demonstrate that the detergent used to reconstitute full-length Syt1 has a significant effect on the state of the protein in bilayers. When octyl-β-d -glucopyranoside is used to reconstitute the protein, Syt1 is present in an aggregated state that is mediated by the long juxta-membrane linker. EPR spectra from spin labels in the two C2 domains of Syt1 no longer resemble those obtained from a soluble construct containing these domains, and the C2B domain no longer exhibits a Ca²⁺-dependent membrane insertion. In contrast, when reconstituted using 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, Syt1 is largely monomeric and the EPR spectra from C2A and C2B resemble those of the soluble construct. This result demonstrates that the choice of detergent used to reconstitute Syt1 can modulate the state of the neuronal Ca²⁺-sensor.     

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: 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.     

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.     

Extracellular Ca2+ ions reduce NMDA receptor conductance and gating

Brief intracellular Ca²⁺ transients initiate signaling routines that direct cellular activities. Consequently, activation of Ca²⁺-permeable neurotransmitter-gated channels can both depolarize and initiate remodeling of the postsynaptic cell. In particular, the Ca²⁺ transient produced by NMDA receptors is essential to normal synaptic physiology, drives the development and plasticity of excitatory central synapses, and also mediates glutamate excitotoxicity. The amplitude and time course of the Ca²⁺ signal depends on the receptor’s conductance and gating kinetics; these properties are themselves influenced both directly and indirectly by fluctuations in the extracellular Ca²⁺ concentration. Here, we used electrophysiology and kinetic modeling to delineate the direct effects of extracellular Ca²⁺ on recombinant GluN1/GluN2A receptor conductance and gating. We report that, in addition to decreasing unitary conductance, Ca²⁺ also decreased channel open probability primarily by lengthening closed-channel periods. Using one-channel current recordings, we derive a kinetic model for GluN1/GluN2A receptors in physiological Ca²⁺ concentrations that accurately describes macroscopic channel behaviors. This model represents a practical instrument to probe the mechanisms that control the Ca²⁺ transients produced by NMDA receptors during both normal and aberrant synaptic signaling.     

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.     

Synaptotagmin IV regulates dense core vesicle (DCV) release in LbetaT2 cells

Synaptotagmins (Syts) are calcium-binding proteins which are conserved from nematodes to humans. Fifteen Syts have been identified in mammalian species. Syt I is recognized as a Ca²⁺ sensor for the synchronized release of synaptic vesicles in some types of neurons, but its role in the secretion of dense core vesicles (DCVs) remains unclear. The function of Syt IV is of particular interest because it is rapidly up-regulated by chronic depolarization and seizures. Using RNAi-mediated gene silencing, we have explored the role of Syt I and IV on secretion in a pituitary gonadotrope cell line. Downregulation of Syt IV clearly reduced Ca²⁺-triggered exocytosis of dense core vesicles (DCVs) in LβT2 cells. Syt I silencing, however, had no effect on vesicular release.     

Synaptotagmin I: A major Ca2+ sensor for transmitter release at a central synapse

Mice carrying a mutation in the synaptotagmin I gene were generated by homologous recombination. Mutant mice are phenotypically normal as heterozygotes, but die within 48 hr after birth as homozygotes. Studies of hippocampal neurons cultured from homozygous mutant mice reveal that synaptic transmission is severely impaired. The synchronous, fast component of Ca²⁺-dependent neurotransmitter release is decreased, whereas asynchronous release processes, including spontaneous synaptic activity (miniature excitatory postsynaptic current frequency) and release triggered by hypertonic solution or α-latrotoxin, are unaffected. Our findings demonstrate that synaptotagmin I function is required for Ca²⁺-triggering of synchronous neurotransmitter release, but is not essential for asynchronous or Ca²⁺-independent release. We propose that synaptotagmin I is the major low affinity Ca²⁺ sensor mediating Ca²⁺ regulation of synchronous neurotransmitter release in hippocampal neurons.     

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 Å 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.     

Synaptotagmin I increases the probability of vesicle fusion at low [Ca2+] in pituitary cells

Synaptotagmin I (Syt I),a low-affinity Ca²⁺-binding protein, is thought to serve asthe Ca²⁺ sensor in the release of neurotransmitter.However, functional studies on the calyx of Held synapse revealed thatthe rapid release of neurotransmitter requires only approximatelymicromolar [Ca²⁺], suggesting that Syt I may play a morecomplex role in determining the high-affinity Ca²⁺dependence of exocytosis. Here we tested this hypothesis by studying pituitary cells, which possess high- and low-affinityCa²⁺-dependent exocytic pathways and express Syt I. Usingpatch-clamp capacitance measurements to monitor secretion and the acuteantisense deletion of Syt I from differentiated cells, we have shownthat the rapid and the most Ca²⁺-sensitive pathway ofexocytosis in rat melanotrophs requires Syt I. Furthermore, stimulationof the Ca²⁺-dependent exocytosis by cytosol dialysis withsolutions containing 1 µM [Ca²⁺] was completelyabolished in the absence of Syt I. Similar results were obtained by thepreinjection of antibodies against the CAPS (Ca²⁺-dependentactivator protein for secretion) protein. These results indicate thatsynaptotagmin I and CAPS proteins increase the probability of vesiclefusion at low cytosolic [Ca²⁺].

     

Ca2+-Dependent Inactivation of GluN2A and GluN2B NMDA Receptors Occurs by a Common Kinetic Mechanism

N-Methyl-d-aspartate (NMDA) receptors are Ca²⁺-permeable channels gated by glutamate and glycine that are essential for central excitatory transmission. Ca²⁺-dependent inactivation (CDI) is a regulatory feedback mechanism that reduces GluN2A-type NMDA receptor responses in an activity-dependent manner. Although CDI is mediated by calmodulin binding to the constitutive GluN1 subunit, prior studies suggest that GluN2B-type receptors are insensitive to CDI. We examined the mechanism of CDI subtype dependence using electrophysiological recordings of recombinant NMDA receptors expressed in HEK-293 cells. In physiological external Ca²⁺, we observed robust CDI of whole-cell GluN2A currents (0.42 ± 0.05) but no CDI in GluN2B currents (0.08 ± 0.07). In contrast, when Ca²⁺ was supplied intracellularly, robust CDI occurred for both GluN2A and GluN2B currents (0.75 ± 0.03 and 0.67 ± 0.02, respectively). To examine how the source of Ca²⁺ affects CDI, we recorded one-channel Na⁺ currents to quantify the receptor gating mechanism while simultaneously monitoring ionomycin-induced intracellular Ca²⁺ elevations with fluorometry. We found that CDI of both GluN2A and GluN2B receptors reflects receptor accumulation in long-lived closed (desensitized) states, suggesting that the observed subtype-dependent differences in macroscopic CDI reflect intrinsic differences in equilibrium open probabilities (P_o). We tested this hypothesis by measuring substantial macroscopic CDI, in physiologic conditions, for high P_o GluN2B receptors (GluN1^A652Y/GluN2B). Together, these results show that Ca²⁺ flux produces activity-dependent inactivation for both GluN2A and GluN2B receptors and that the extent of CDI varies with channel P_o. These results are consistent with CDI as an autoinhibitory feedback mechanism against excessive Ca²⁺ load during high P_o activation.     

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.     

Glutamate triggers intracellular Ca2+ oscillations and nitric oxide release by inducing NAADP- and InsP3-dependent Ca2+ release in mouse brain endothelial cells

The neurotransmitter glutamate increases cerebral blood flow by activating postsynaptic neurons and presynaptic glial cells within the neurovascular unit. Glutamate does so by causing an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in the target cells, which activates the Ca²⁺/Calmodulin-dependent nitric oxide (NO) synthase to release NO. It is unclear whether brain endothelial cells also sense glutamate through an elevation in [Ca²⁺]_i and NO production. The current study assessed whether and how glutamate drives Ca²⁺-dependent NO release in bEND5 cells, an established model of brain endothelial cells. We found that glutamate induced a dose-dependent oscillatory increase in [Ca²⁺]_i, which was maximally activated at 200 μM and inhibited by α-methyl-4-carboxyphenylglycine, a selective blocker of Group 1 metabotropic glutamate receptors. Glutamate-induced intracellular Ca²⁺ oscillations were triggered by rhythmic endogenous Ca²⁺ mobilization and maintained over time by extracellular Ca²⁺ entry. Pharmacological manipulation revealed that glutamate-induced endogenous Ca²⁺ release was mediated by InsP₃-sensitive receptors and nicotinic acid adenine dinucleotide phosphate (NAADP) gated two-pore channel 1. Constitutive store-operated Ca²⁺ entry mediated Ca²⁺ entry during ongoing Ca²⁺ oscillations. Finally, glutamate evoked a robust, although delayed increase in NO levels, which was blocked by pharmacologically inhibition of the accompanying intracellular Ca²⁺ signals. Of note, glutamate induced Ca²⁺-dependent NO release also in hCMEC/D3 cells, an established model of human brain microvascular endothelial cells. This investigation demonstrates for the first time that metabotropic glutamate-induced intracellular Ca²⁺ oscillations and NO release have the potential to impact on neurovascular coupling in the brain.     

Calmodulin Suppresses Synaptotagmin-2 Transcription in Cortical Neurons

Calmodulin (CaM) is a ubiquitous Ca²⁺ sensor protein that plays a pivotal role in regulating innumerable neuronal functions, including synaptic transmission. In cortical neurons, most neurotransmitter release is triggered by Ca²⁺ binding to synaptotagmin-1; however, a second delayed phase of release, referred to as asynchronous release, is triggered by Ca²⁺ binding to an unidentified secondary Ca²⁺ sensor. To test whether CaM could be the enigmatic Ca²⁺ sensor for asynchronous release, we now use in cultured neurons short hairpin RNAs that suppress expression of ∼70% of all neuronal CaM isoforms. Surprisingly, we found that in synaptotagmin-1 knock-out neurons, the CaM knockdown caused a paradoxical rescue of synchronous release, instead of a block of asynchronous release. Gene and protein expression studies revealed that both in wild-type and in synaptotagmin-1 knock-out neurons, the CaM knockdown altered expression of >200 genes, including that encoding synaptotagmin-2. Synaptotagmin-2 expression was increased several-fold by the CaM knockdown, which accounted for the paradoxical rescue of synchronous release in synaptotagmin-1 knock-out neurons by the CaM knockdown. Interestingly, the CaM knockdown primarily activated genes that are preferentially expressed in caudal brain regions, whereas it repressed genes in rostral brain regions. Consistent with this correlation, quantifications of protein levels in adult mice uncovered an inverse relationship of CaM and synaptotagmin-2 levels in mouse forebrain, brain stem, and spinal cord. Finally, we employed molecular replacement experiments using a knockdown rescue approach to show that Ca²⁺ binding to the C-lobe but not the N-lobe of CaM is required for suppression of synaptotagmin-2 expression in cortical neurons. Our data describe a previously unknown, Ca²⁺/CaM-dependent regulatory pathway that controls the expression of synaptic proteins in the rostral-caudal neuraxis.     

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.     

Simultaneous Release of Multiple Vesicles from Rods Involves Synaptic Ribbons and Syntaxin 3B

First proposed as a specialized mode of release at sensory neurons possessing ribbon synapses, multivesicular release has since been described throughout the central nervous system. Many aspects of multivesicular release remain poorly understood. We explored mechanisms underlying simultaneous multivesicular release at ribbon synapses in salamander retinal rod photoreceptors. We assessed spontaneous release presynaptically by recording glutamate transporter anion currents (I_A(glu)) in rods. Spontaneous I_A(glu) events were correlated in amplitude and kinetics with simultaneously measured miniature excitatory postsynaptic currents in horizontal cells. Both measures indicated that a significant fraction of events is multiquantal, with an analysis of I_A(glu) revealing that multivesicular release constitutes ∼30% of spontaneous release events. I_A(glu) charge transfer increased linearly with event amplitude showing that larger events involve greater glutamate release. The kinetics of large and small I_A(glu) events were identical as were rise times of large and small miniature excitatory postsynaptic currents, indicating that the release of multiple vesicles during large events is highly synchronized. Effects of exogenous Ca²⁺ buffers suggested that multiquantal, but not uniquantal, release occurs preferentially near Ca²⁺ channels clustered beneath synaptic ribbons. Photoinactivation of ribbons reduced the frequency of spontaneous multiquantal events without affecting uniquantal release frequency, showing that spontaneous multiquantal release requires functional ribbons. Although both occur at ribbon-style active zones, the absence of cross-depletion indicates that evoked and spontaneous multiquantal release from ribbons involve different vesicle pools. Introducing an inhibitory peptide into rods to interfere with the SNARE protein, syntaxin 3B, selectively reduced multiquantal event frequency. These results support the hypothesis that simultaneous multiquantal release from rods arises from homotypic fusion among neighboring vesicles on ribbons and involves syntaxin 3B.     

高锶离子亲和力传感蛋白介导CALYX突触囊泡异步和自发释放

突触囊泡在钙离子(Ca2+)触发下释放神经递质普遍存在着同步和异步两种形式.突触囊泡膜蛋白(synaptotagmin 2,Syt-2)已被证实是Calyx of Held突触囊泡同步释放的Ca2+传感蛋白,而相关的异步释放Ca2+传感蛋白还有待于探索.虽然锶离子(Sr2+)因其物理和化学性质都接近Ca2+,且能触发更多的囊泡异步释放成分而成为研究异步释放机制的常用工具,但有关Sr2+触发异步释放的机制存在着争议.本文在胞外以Sr2+替换Ca2+的条件下,通过对野生型(WT)和Syt-2敲除型(Z2B-/-)小鼠Calyx突触囊泡自发和诱发释放的电生理特性分析,发现Syt-2是介导Sr2+诱发的突触囊泡快速释放的传感蛋白,但不是介导Sr2+相关神经递质异步释放和自发释放的传感蛋白;而未知的触发囊泡异步释放的传感蛋白相比Syt-2对Sr2+具有更高的亲和力,同时也介导突触囊泡的自发释放.这一研究为探索并最终发现触发囊泡异步释放的未知传感蛋白提供了新的线索.