UNC-10在致密核心囊泡分泌过程中的作用

Rim是囊泡分泌活性区中的重要组成蛋白,它与细胞分泌和突触可塑性相关．在秀丽隐感线虫中只存在一种编码Rim的基因即unc-10．我们的研究发现,在线虫中Rim的基因突变unc-10(md1117)会导致致密核心囊泡的分泌缺陷．在活体中,unc-10突变虫系的神经多肽分泌显著下降．此外,在主要分泌致密核心囊泡的ALA神经元内,钙光解释放促发的快相分泌也比野生型减少．运用全内反射荧光显微成像技术,我们观察在unc-10缺失的情况下ALA 神经元中致密核心囊泡的锚定过程,结果显示在细胞膜附近停留的囊泡数目减少,表明囊泡锚定受到阻碍．上述试验结果表明,UNC-10能够影响致密核心囊泡的分泌过程,其机制可能是影响了囊泡的锚定过程．     

PKA activation bypasses the requirement for UNC-31 in the docking of dense core vesicles from C. elegans neurons

The nematode C. elegans provides a powerful model system for exploring the molecular basis of synaptogenesis and neurotransmission. However, the lack of direct functional assays of release processes has largely prevented an in depth understanding of the mechanism of vesicular exocytosis and endocytosis in C. elegans. We address this technical limitation by developing direct electrophysiological assays, including membrane capacitance and amperometry measurements, in primary cultured C. elegans neurons. In addition, we have succeeded in monitoring the docking and fusion of single dense core vesicles (DCVs) employing total internal reflection fluorescence microscopy. With these approaches and mutant perturbation analysis, we provide direct evidence that UNC-31 is required for the docking of DCVs at the plasma membrane. Interestingly, the defect in DCV docking caused by UNC-31 mutation can be fully rescued by PKA activation. We also demonstrate that UNC-31 is required for UNC-13-mediated augmentation of DCV exocytosis.     

The non-neuronal syntaxin SYN-1 regulates defecation behavior and neural activity in C. elegans through interaction with the Munc13-like protein AEX-1

We have previously shown that the AEX-1 protein, which is expressed in postsynaptic muscles, retrogradely regulates presynaptic neural activity at the Caenorhabditis elegans neuromuscular junctions. AEX-1 is similar to vertebrate Munc13-4 protein, suggesting a function for vesicle exocytosis from a kind of cells. Compared to emerging evidences of the role of Munc13 proteins in synaptic vesicle release, however, the precise mechanism for vesicle exocytosis by AEX-1 and Munc13-4 is little understood. Here we have identified SYN-1 as a candidate molecule of AEX-1-dependent vesicle exocytosis from non-neuronal cells. The syn-1 gene encodes a C. elegans syntaxin, which is distantly related to the neuronal syntaxin UNC-64. The syn-1 gene is predominantly expressed in non-neuronal tissues and genetically interacts with aex-1 for presynaptic activity. However, the two proteins did not interact physically in our yeast two-hybrid system and mutational SYN-1 did not bypass the requirement of AEX-1 for the behavioral defects in aex-1 mutants, whereas mutant UNC-64 does in unc-13 mutants. These results suggest that a novel molecular interaction between the AEX-1 and syntaxin may regulate vesicle exocytosis for retrograde signal release.     

Extracellular Caper se inhibits quantal size of catecholamine release in adrenal slice chromaffin cells

Classic calcium hypothesis states that depolarization-induced increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) triggers vesicle exocytosis by increasing vesicle release probability in neurons and neuroendocrine cells. The extracellular Ca²⁺, in this calcium hypothesis, serves as a reservoir of Ca²⁺ source. Recently we find that extracellular Ca²⁺per se inhibits the [Ca²⁺]_i dependent vesicle exocytosis, but it remains unclear whether quantal size is regulated by extracellular, or intracellular Ca²⁺ or both [1]. In this work we showed that, in physiological condition, extracellular Ca²⁺per se specifically inhibited the quantal size of single vesicle release in rat adrenal slice chromaffin cells. The extracellular Ca²⁺ in physiological concentration (2.5 mM) directly regulated fusion pore kinetics of spontaneous quantal release of catecholamine. In addition, removal of extracellular Ca²⁺ directly triggered vesicle exocytosis without eliciting intracellular Ca²⁺. We propose that intracellular Ca²⁺ and extracellular Ca²⁺per se cooperately regulate single vesicle exocytosis. The vesicle release probability was jointly modulated by both intracellular and extracellular Ca²⁺, while the vesicle quantal size was mainly determined by extracellular Ca²⁺ in chromaffin cells physiologically.     

Signaling role of the voltage-gated calcium channel as the molecular on/off-switch of secretion

Voltage-gated calcium channels (VGCC) are involved in a large variety of cellular Ca²⁺ signaling processes, including exocytosis, a Ca²⁺ dependent release of neurotransmitters and hormones.Great progress has been made in understanding the mode of action of VGCC in exocytosis, a process distinguished by two sequential yet independent Ca²⁺ binding reactions. First, Ca²⁺ binds at the selectivity filter, the EEEE motif of the VGCC, and second, subsequent to a brief and intense Ca²⁺ inflow to synaptotagmin, a vesicular protein. Inquiry into the functional and physical interactions of the channels with synaptic proteins has demonstrated that exocytosis is triggered during the initial Ca²⁺ binding at the channel pore, prior to Ca²⁺ entry. Accordingly, a cycle of secretion begins by an incoming stimulus that releases vesicles from a releasable pool upon Ca²⁺ binding at the pore, and at the same time, the transient increase in [Ca²⁺]_i primes a fresh set of non-releasable vesicles, to be fused by the next incoming stimulus.We propose a model, in which the Ca²⁺ binding at the EEEE motif and the consequent conformational changes in the channel are the primary event in triggering secretion, while synaptotagmin acts as a vesicle docking protein. Thus, the channel serves as the molecular On/Off signaling switch, where the predominance of a conformational change in Ca²⁺-bound channel provides for the fast secretory process.     

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.     

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.     

Open syntaxin docks synaptic vesicles

Synaptic vesicles dock to the plasma membrane at synapses to facilitate rapid exocytosis. Docking was originally proposed to require the soluble N-ethylmaleimide–sensitive fusion attachment protein receptor (SNARE) proteins; however, perturbation studies suggested that docking was independent of the SNARE proteins. We now find that the SNARE protein syntaxin is required for docking of all vesicles at synapses in the nematode Caenorhabditis elegans. The active zone protein UNC-13, which interacts with syntaxin, is also required for docking in the active zone. The docking defects in unc-13 mutants can be fully rescued by overexpressing a constitutively open form of syntaxin, but not by wild-type syntaxin. These experiments support a model for docking in which UNC-13 converts syntaxin from the closed to the open state, and open syntaxin acts directly in docking vesicles to the plasma membrane. These data provide a molecular basis for synaptic vesicle docking.     

UNC-108/RAB-2 and its effector RIC-19 are involved in dense core vesicle maturation in Caenorhabditis elegans

Small guanosine triphosphatases of the Rab family regulate intracellular vesicular trafficking. Rab2 is highly expressed in the nervous system, yet its function in neurons is unknown. In Caenorhabditis elegans, unc-108/rab-2 mutants have been isolated based on their locomotory defects. We show that the locomotion defects of rab-2 mutants are not caused by defects in synaptic vesicle release but by defects in dense core vesicle (DCV) signaling. DCVs in rab-2 mutants are often enlarged and heterogeneous in size; however, their number and distribution are not affected. This implicates Rab2 in the biogenesis of DCVs at the Golgi complex. We demonstrate that Rab2 is required to prevent DCV cargo from inappropriately entering late endosomal compartments during DCV maturation. Finally, we show that RIC-19, the C. elegans orthologue of the human diabetes autoantigen ICA69, is also involved in DCV maturation and is recruited to Golgi membranes by activated RAB-2. Thus, we propose that RAB-2 and its effector RIC-19 are required for neuronal DCV maturation.     

The role of C. elegans Ena/VASP homolog UNC-34 in neuronal polarity and motility

Ena/VASP proteins mediate the effects of guidance cues on the actin cytoskeleton. The single C. elegans homolog of the Ena/VASP family of proteins, UNC-34, is required for the migrations of cells and growth cones. Here we show that unc-34 mutant alleles also interact genetically with Wnt mutants to reveal a role for unc-34 in the establishment of neuronal polarity along the C. elegans anterior-posterior axis. Our mutant analysis shows that eliminating UNC-34 function results in neuronal migration and polarity phenotypes that are enhanced at higher temperatures, revealing a heat-sensitive process that is normally masked by the presence of UNC-34. Finally, we show that the UNC-34 protein is expressed broadly and accumulates in axons and at the apical junctions of epithelial cells. While most mutants lacked detectable UNC-34, three unc-34 mutants that contained missense mutations in the EVH1 domain produced full-length UNC-34 that failed to localize to apical junctions and axons, supporting the role for the EVH1 domain in localizing Ena/VASP family members.     

Calcium Promotes the Formation of Syntaxin 1 Mesoscale Domains through Phosphatidylinositol 4,5-Bisphosphate

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) is a minor component of total plasma membrane lipids, but it has a substantial role in the regulation of many cellular functions, including exo- and endocytosis. Recently, it was shown that PI(4,5)P₂ and syntaxin 1, a SNARE protein that catalyzes regulated exocytosis, form domains in the plasma membrane that constitute recognition sites for vesicle docking. Also, calcium was shown to promote syntaxin 1 clustering in the plasma membrane, but the molecular mechanism was unknown. Here, using a combination of superresolution stimulated emission depletion microscopy, FRET, and atomic force microscopy, we show that Ca²⁺ acts as a charge bridge that specifically and reversibly connects multiple syntaxin 1/PI(4,5)P₂ complexes into larger mesoscale domains. This transient reorganization of the plasma membrane by physiological Ca²⁺ concentrations is likely to be important for Ca²⁺-regulated secretion.     

UNC-13 interaction with syntaxin is required for synaptic transmission

Neurotransmitter secretion at synapses is controlled by several processes-morphological docking of vesicles at release sites, priming of docked vesicles to make them fusion competent, and calcium-dependent fusion of vesicles with the plasma membrane . In worms, flies, and mice, mutants lacking UNC-13 have defects in vesicle priming . Current models propose that UNC-13 primes vesicles by stabilizing Syntaxin's "open" conformation by directly interacting with its amino-terminal regulatory domain . However, the functional significance of the UNC-13/Syntaxin interaction has not been tested directly. A truncated protein containing the Munc homology domains (MHD1 and MHD2) and the carboxy-terminal C2 domain partially rescued both the behavioral and secretion defects of unc-13 mutants in C. elegans. A double mutation in MHD2 (F1000A/K1002A) disrupts the UNC-13/Syntaxin interaction. The rate of endogenous synaptic events and the amplitude of nerve-evoked excitatory post-synaptic currents (EPSCs) were both significantly reduced in UNC-13S(F1000A/K1002A). However, the pool of primed (i.e., fusion-competent) vesicles was normal. These results suggest that the UNC-13/Syntaxin interaction is conserved in C. elegans and that, contrary to current models, the UNC-13/Syntaxin interaction is required for nerve-evoked vesicle fusion rather than synaptic-vesicle priming. Thus, UNC-13 may regulate multiple steps of the synaptic-vesicle cycle.     

SLO-2 isoforms with unique Ca2+- and voltage-dependence characteristics confer sensitivity to hypoxia in C. elegans

Slo channels are large conductance K⁺ channels that display marked differences in their gating by intracellular ions. Among them, the Slo1 and C. elegans SLO-2 channels are gated by calcium (Ca²⁺), while mammalian Slo2 channels are activated by both sodium (Na⁺) and chloride (Cl⁻). Here, we report that SLO-2 channels, SLO-2a and a novel N-terminal variant isoform, SLO-2b, are activated by Ca²⁺ and voltage, but in contrast to previous reports they do not exhibit Cl⁻ sensitivity. Most importantly, SLO-2 provides a unique case in the Slo family for sensing Ca²⁺ with the high-affinity Ca²⁺ regulatory site in the RCK1 but not the RCK2 domain, formed through interactions with residues E319 and E487 (that correspond to D362 and E535 of Slo1, respectively). The SLO-2 RCK2 domain lacks the Ca²⁺ bowl structure and shows minimal Ca²⁺ dependence. In addition, in contrast to SLO-1, SLO-2 loss-of-function mutants confer resistance to hypoxia in C. elegans. Thus, the C. elegans SLO-2 channels possess unique biophysical and functional properties.     

Ca-regulated secretory granule exocytosis in pancreatic and parotid acinar cells

Protein secretion from acinar cells of the pancreas and parotid glands is controlled by G-protein coupled receptor activation and generation of the cellular messengers Ca²⁺, diacylglycerol and cAMP. Secretory granule (SG) exocytosis shares some common characteristics with nerve, neuroendocrine and endocrine cells which are regulated mainly by elevated cell Ca²⁺. However, in addition to diverse signaling pathways, acinar cells have large ∼1 μm diameter SGs (∼30 fold larger diameter than synaptic vesicles), respond to stimulation at slower rates (seconds versus milliseconds), demonstrate significant constitutive secretion, and in isolated acini, undergo sequential compound SG–SG exocytosis at the apical membrane. Exocytosis proceeds as an initial rapid phase that peaks and declines over 3 min followed by a prolonged phase that decays to near basal levels over 20–30 min. Studies indicate the early phase is triggered by Ca²⁺ and involves the SG proteins VAMP2 (vesicle associated membrane protein2), Ca²⁺-sensing protein synatotagmin 1 (syt1) and the accessory protein complexin 2. The molecular details for regulation of VAMP8-mediated SG exocytosis and the prolonged phase of secretion are still emerging. Here we review the known regulatory molecules that impact the sequential exocytic process of SG tethering, docking, priming and fusion in acinar cells.     

Effect of Calcium ion on synaptotagmin-like protein during pre-fusion of vesicle for exocytosis in blood-brain barrier

BackgroundCalcium signaling and membrane fusion play key roles in exocytosis of drug-containing vesicles through the blood-brain barrier (BBB). Identifying the role of synaptotagmin-like protein4-a (Slp4-a) in the presence of Ca²⁺ ions, at the pre-fusion stage of a vesicle with the basolateral membrane of endothelial cell, can reveal brain drug transportation across BBB.MethodsWe utilized molecular dynamics (MD) simulations with a coarse-grained PACE force field to investigate the behaviors of Slp4-a with vesicular and endothelial membranes at the pre-fusion stage of exocytosis since all-atom MD simulation or experiments are more time-consuming and expensive to capture these behaviors.ResultsThe Slp4-a pulls lipid membranes (vesicular and endothelial) into close proximity and disorganizes lipid arrangement at contact points, which are predictors for initiation of fusion. Our MD results also indicate that Slp4-a needs Ca²⁺ to bind with weakly-charged POPE lipids (phosphatidylethanolamine).ConclusionsSlp4-a is an important trigger for membrane fusion in BBB exocytosis. It binds to lipid membranes at multiple binding sites and triggers membrane disruption for fusion in calcium-dependent case.General significanceUnderstanding the prefusion process of the vesicle will help to design better drug delivery mechanisms to the brain through formidable BBB.     

CAPS Activity in Priming Vesicle Exocytosis Requires CK2 Phosphorylation

CAPS (Ca²⁺-dependent activator protein for secretion) functions in priming Ca²⁺-dependent vesicle exocytosis, but the regulation of CAPS activity has not been characterized. Here we show that phosphorylation by protein kinase CK2 is required for CAPS activity. Dephosphorylation eliminated CAPS activity in reconstituting Ca²⁺-dependent vesicle exocytosis in permeable and intact PC12 cells. Ser-5, -6, and -7 and Ser-1281 were identified by mass spectrometry as the major phosphorylation sites in the 1289 residue protein. Ser-5, -6, and -7 but not Ser-1281 to Ala substitutions abolished CAPS activity. Protein kinase CK2 phosphorylated CAPS in vitro at these sites and restored the activity of dephosphorylated CAPS. CK2 is the likely in vivo CAPS protein kinase based on inhibition of phosphorylation by tetrabromo-2-benzotriazole in PC12 cells and by the identity of in vivo and in vitro phosphorylation sites. CAPS phosphorylation by CK2 was constitutive, but the elevation of Ca²⁺ in synaptosomes increased CAPS Ser-5 and -6 dephosphorylation, which terminates CAPS activity. These results identify a functionally important N-terminal phosphorylation site that regulates CAPS activity in priming vesicle exocytosis.Regulated neurotransmitter secretion is central to intercellular communication in the nervous system. Two types of secretory vesicles mediate neurotransmitter release; that is, synaptic vesicles that release transmitters such as glutamate at synapses and dense-core vesicles that release modulatory transmitters and neuropeptides at non-synaptic sites. Both types of secretory vesicles are recruited to docking sites on the plasma membrane where they are primed to a ready release state to undergo fusion in response to Ca²⁺ elevations. Many of the proteins that mediate the targeting, docking, priming, and Ca²⁺-dependent fusion of vesicles with the plasma membrane function in both synaptic vesicle and dense-core vesicle pathways (1). CAPS-1² (also known as Cadps1) is a 1289-residue protein that reconstitutes Ca²⁺-triggered dense-core vesicle exocytosis in permeable neuroendocrine cells at a priming step (2–4). CAPS is required for secretion of a subset of transmitters in Caenorhabditis elegans (5) and Drosophila melanogaster (6) and for priming dense-core vesicle exocytosis in neuroendocrine cells (7) and synaptic vesicle exocytosis in neurons (8). Vesicle priming reactions are extensively modulated during physiological demand (9), but mechanisms that regulate CAPS function remain to be identified.Reversible protein phosphorylation is a major mechanism for the regulation of cellular processes including vesicle exocytosis. Many proteins that function in evoked vesicle exocytosis are phosphoproteins (10, 11). The neuronal SNARE proteins syntaxin 1A, VAMP-2, and SNAP-25 are phosphorylated by several protein kinases in vitro (12–14). Protein kinase C and protein kinase A sites on SNAP-25 affect refilling rates and size, respectively, of the primed pool of vesicles in chromaffin cells (15, 16). Several SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-binding proteins such as munc18, RIM1, and rabphilin undergo regulated phosphorylation, but it is not known whether phosphorylation affects function (10, 11, 17).Because the function of CAPS at a priming step in vesicle exocytosis may be regulated, we determined whether CAPS is phosphorylated. We show that CAPS is a phosphoprotein with functionally essential N-terminal phosphorylated Ser residues. Ser-5, -6, and -7 in CAPS were substrates for protein kinase CK2 in vitro and in vivo as well as for a Ca²⁺-dependent dephosphorylation mechanism. The results indicate that phosphorylation by protein kinase CK2 is necessary for CAPS activity in priming vesicle exocytosis and that regulated dephosphorylation may constitute a mechanism for terminating CAPS activity.     

Vesicular Calcium Regulates Coat Retention,Fusogenicity, and Size of Pre-Golgi Intermediates

The significance and extent of Ca²⁺ regulation of the biosynthetic secretory pathway have been difficult to establish, and our knowledge of regulatory relationships integrating Ca²⁺ with vesicle coats and function is rudimentary. Here, we investigated potential roles and mechanisms of luminal Ca²⁺ in the early secretory pathway. Specific depletion of luminal Ca²⁺ in living normal rat kidney cells using cyclopiazonic acid (CPA) resulted in the extreme expansion of vesicular tubular cluster (VTC) elements. Consistent with this, a suppressive role for vesicle-associated Ca²⁺ in COPII vesicle homotypic fusion was demonstrated in vitro using Ca²⁺ chelators. The EF-hand–containing protein apoptosis-linked gene 2 (ALG-2), previously implicated in the stabilization of sec31 at endoplasmic reticulum exit sites, inhibited COPII vesicle fusion in a Ca²⁺-requiring manner, suggesting that ALG-2 may be a sensor for the effects of vesicular Ca²⁺ on homotypic fusion. Immunoisolation established that Ca²⁺ chelation inhibits and ALG-2 specifically favors residual retention of the COPII outer shell protein sec31 on pre-Golgi fusion intermediates. We conclude that vesicle-associated Ca²⁺, acting through ALG-2, favors the retention of residual coat molecules that seem to suppress membrane fusion. We propose that in cells, these Ca²⁺-dependent mechanisms temporally regulate COPII vesicle interactions, VTC biogenesis, cargo sorting, and VTC maturation.     

CPNA-1, a copine domain protein,is located at integrin adhesion sites and is required for myofilament stability in Caenorhabditis elegans

We identify cpna-1 (F31D5.3) as a novel essential muscle gene in the nematode Caenorhabditis elegans. Antibodies specific to copine domain protein atypical-1 (CPNA-1), as well as a yellow fluorescent protein translational fusion, are localized to integrin attachment sites (M-lines and dense bodies) in the body-wall muscle of C. elegans. CPNA-1 contains an N-terminal predicted transmembrane domain and a C-terminal copine domain and binds to the M-line/dense body protein PAT-6 (actopaxin) and the M-line proteins UNC-89 (obscurin), LIM-9 (FHL), SCPL-1 (SCP), and UNC-96. Proper CPNA-1 localization is dependent upon PAT-6 in embryonic and adult muscle. Nematodes lacking cpna-1 arrest elongation at the twofold stage of embryogenesis and display disruption of the myofilament lattice. The thick-filament component myosin heavy chain MYO-3 and the M-line component UNC-89 are initially localized properly in cpna-1–null embryos. However, in these embryos, when contraction begins, MYO-3 and UNC-89 become mislocalized into large foci and animals die. We propose that CPNA-1 acts as a linker between an integrin-associated protein, PAT-6, and membrane-distal components of integrin adhesion complexes in the muscle of C. elegans.     

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.     

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.