Neuronal Exocytosis Research Articles

Conformational states of syntaxin-1 govern the necessity of N-peptide binding in exocytosis of PC12 cells and Caenorhabditis elegans.

Syntaxin-1 is the central SNARE protein for neuronal exocytosis. It interacts with Munc18-1 through its cytoplasmic domains, including the N-terminal peptide (N-peptide). Here we examine the role of the N-peptide binding in two conformational states ("closed" vs. "open") of syntaxin-1 using PC12 cells and Caenorhabditis elegans. We show that expression of "closed" syntaxin-1A carrying N-terminal single point mutations (D3R, L8A) that perturb interaction with the hydrophobic pocket of Munc18-1 rescues impaired secretion in syntaxin-1-depleted PC12 cells and the lethality and lethargy of unc-64 (C. elegans orthologue of syntaxin-1)-null mutants. Conversely, expression of the "open" syntaxin-1A harboring the same mutations fails to rescue the impairments. Biochemically, the L8A mutation alone slightly weakens the binding between "closed" syntaxin-1A and Munc18-1, whereas the same mutation in the "open" syntaxin-1A disrupts it. Our results reveal a striking interplay between the syntaxin-1 N-peptide and the conformational state of the protein. We propose that the N-peptide plays a critical role in intracellular trafficking of syntaxin-1, which is dependent on the conformational state of this protein. Surprisingly, however, the N-peptide binding mode seems dispensable for SNARE-mediated exocytosis per se, as long as the protein is trafficked to the plasma membrane.

Gβγ Binds to the Extreme C Terminus of SNAP25 to Mediate the Action of Gi/o-Coupled G Protein-Coupled Receptors.

Gi/o-coupled G protein-coupled receptors can exert an inhibitory effect on vesicle release through several G protein-driven mechanisms, more than one of which may be concurrently present in individual presynaptic terminals. The synaptosomal-associated protein of 25 kDa (SNAP25) is a key downstream effector of Gβγ subunits. It has previously been shown that proteolytic cleavage of SNAP25 by botulinum toxin A reduces the ability of Gβγ to compete with the calcium sensor synaptotagmin 1 (Syt1) for binding to SNAP25 in a calcium-dependent manner. These truncated SNAP25 proteins sustain a low level of exocytosis but are unable to support serotonin-mediated inhibition of exocytosis in lamprey spinal neurons. Here, we generate a SNAP25 extreme C-terminal mutant that is deficient in its ability to bind Gβγ while retaining normal calcium-dependent Syt1 binding to soluble N-ethylmaleimide attachment protein receptor (SNARE) and vesicle release. The SNAP25Δ3 mutant, in which residue G204 is replaced by a stop codon, features a partial reduction in Gβ1γ2 binding in vitro as well as a partial reduction in the ability of the lamprey 5-hydroxytryptamine1b-type serotonin receptor to reduce excitatory postsynaptic current amplitudes, an effect previously shown to be mediated through the interaction of Gβγ with SNAP25. Syt1 calcium-dependent binding to SNAP25Δ3 was reduced by a small extent compared with the wild type. We conclude that the extreme C terminus of SNAP25 is a critical region for the Gβγ-SNARE interaction.

Sex-specific regulation of follicle-stimulating hormone secretion by synaptotagmin 9.

The anterior pituitary releases six different hormones that control virtually all aspects of vertebrate physiology, yet the molecular mechanisms underlying their Ca2+-triggered release remain unknown. A subset of the synaptotagmin (syt) family of proteins serve as Ca2+ sensors for exocytosis in neurons and neuroendocrine cells, and are thus likely to regulate pituitary hormone secretion. Here we show that numerous syt isoforms are highly expressed in the pituitary gland in a lobe, and sex-specific manner. We further investigated a Ca2+-activated isoform, syt-9, and found that it is expressed in a subpopulation of anterior pituitary cells, the gonadotropes. Follicle-stimulating hormone (FSH) and syt-9 are highly co-localized in female, but not male, mice. Loss of syt-9 results in diminished basal and stimulated FSH secretion only in females, resulting in alterations in the oestrus cycle. This work uncovers a new function for syt-9 and reveals a novel sex difference in reproductive hormone secretion.

The small GTPase Rab33A participates in regulation of amylase release from parotid acinar cells

Amylase is released from exocrine parotid acinar cells via typical exocytosis. Exocytosis of amylase-containing granules occurs through several steps, including formation, maturation, and transport of granules. These steps are thought to be regulated by members of the small GTPase Rab family. We previously demonstrated that Rab27 and its effectors mediate amylase release from parotid acinar cells, but the functional involvement of other Rab proteins in exocrine granule exocytosis remains largely unknown. Here, we studied isoproterenol (IPR)-induced amylase release from parotid acinar cells to investigate the possible involvement of Rab33A, which was recently suggested to regulate exocytosis in hippocampal neurons and PC12 cells. Rab33A was endogenously expressed in parotid acinar cells and present in secretory granules and the Golgi body. Functional ablation of Rab33A with anti-Rab33A antibody or a dominant-negative Rab33A-T50N mutant significantly reduced IPR-induced amylase release. Our results indicated that Rab33A is a novel component of IPR-stimulated amylase secretion from parotid acinar cells.

Lambert-Eaton syndrome IgG inhibits transmitter release via P/Q Ca2+ channels.

Objective:To determine whether immunoglobulin G (IgG) from patients with Lambert-Eaton myasthenic syndrome (LEMS) decreases action potential–evoked synaptic vesicle exocytosis, and whether the effect is mediated by P/Q-type voltage-gated calcium channels (VGCCs).Methods:IgG was obtained from 4 patients with LEMS (3 males, 1 female), including 2 patients with lung malignancy. Antibodies against P/Q-type VGCCs were detected in all 4 patients, and against N-type VGCCs in 2. We incubated neuronal cultures with LEMS IgG and determined the size of the total recycling pool of synaptic vesicles and the rate of action potential–evoked exocytosis using fluorescence imaging of the amphiphilic dye SynaptoRed C1. Pooled IgG from healthy volunteers was used as a control. We repeated the experiments on synapses lacking P/Q-type calcium channels from a Cacna1a knockout mouse to determine whether these channels account for the pathogenic effect of LEMS IgG.Results:LEMS IgG had no effect on the total recycling pool size but significantly reduced the rate of action potential–evoked synaptic exocytosis in wild-type neurons when compared with neurons treated with control IgG. In contrast, LEMS IgG had no effect on the rate of synaptic vesicle exocytosis in neurons lacking P/Q-type channels.Conclusions:These data provide direct evidence that LEMS IgG inhibits neurotransmitter release by acting on P/Q-type VGCCs.

Fusion of Lysosomes with Secretory Vesicle Leads to Excessive Uncontrolled Exocytosis in Mucolipidosis Type IV

Mutations in TRPML1 cause the lysosomal storage disease (LSD) Mucolipidosis type IV (MLIV), which is characterized by neurodegeneration, muscular hypotonia corneal opacity, achlorhydia, anemia and severe psychomotor deficiency with impaired neuromuscular junctions. The role of the TRP channel TRPML1 in cell function and how mutations in TRPML1 cause MLIV are not well understood. Studies on the causes of MLIV are focused on the potential role of TRPML1 in constitutive membrane trafficking to and from the lysosomes. However, the role of TRPML1 in secretory cells that their cardinal function is regulated exocytosis has not been studied. Here, we analyzed several forms of regulated exocytosis in neurons and secretory epithelia in a knockout mouse model of TRPML1 and, unexpectedly, found excessive exocytosis due to marked enlargement of synaptic vesicles and secretory granules due to fusion with lysosomes. As a result, Trpml1−/− neurons maintain high basal and stimulated exocytosis of the neurotransmitter and neurotoxin glutamate, which may account for the neurodegeneration in ML4. Trpml1−/− pancreatic and salivary glands acini show high Ca2+− and cAMP-stimulated exocytosis, resulting in pancreatitis. These features were not found in another model of LSD, the Niemann-Pick disease type C1 mouse model. These findings indicate that a major role of TRPML1 is to guard against pathological fusion of lysosomes with other intracellular organelles, including secretory vesicles, and suggest a new approach towards developing treatment for MLIV.

Functional distribution of synapsin I in human sperm

Proteins known to function during cell–cell communication and exocytosis in neurons and other secretory cells have recently been reported in human sperm. Synapsins are a group of proteins that have been very well characterized in neurons, but little is known about synapsin function in other cell types. Based upon previous findings and the known function of synapsin, we tested the hypothesis that synapsin I was present in human sperm. Washed, capacitated, and acrosome induced sperm preparations were used to evaluate the functional distribution of synapsin I using immunocytochemistry. Protein extracts from mouse brain, mouse testis/epididymis, and human semen were used for protein blotting techniques. Immunolocalization revealed synapsin I was enriched in the sperm equatorial segment. Protein extracts from mouse brain, mouse testis/epididymis, and human semen were positive for synapsin I using several different antibodies, and dot blot results were confirmed by Western blot analyses. Finally, treatment of capacitated and acrosome reaction induced samples with anti-synapsin antibodies significantly reduced sperm motility. Localization of synapsin I in human sperm is a novel finding. The association of synapsin I with the sperm equatorial segment and effects on motility are suggestive of a role associated with capacitation and/or acrosome reaction, processes that render sperm capable of fertilizing an oocyte.

Environmental Perturbations that Cause Structural Changes in the SNARE Protein SNAP-25

The SNARE protein SNAP-25 plays a critical role in neuronal exocytosis by providing 2 of the 4 helical regions (SNARE domains) that form a coiled-coil quaternary structure. SNAP-25B contains four cysteines, one tryptophan and one tyrosine all in the linker region. Previously using molecular dynamics simulations, we showed that cysteine oxidation (disulfide bond formation) de-stabilizes the SNARE complex (Bock et al., 2010. Biophysical. J. 99:1221-1230). To test this prediction in vitro, we used Circular Dichroism and Fluorescence Spectroscopy to measure structural changes in SNAP-25B as a function of pH.Using Circular Dichroism to measure the melting of SNARE domains, we show that oxidation destabilizes both the SNARE complex and SNAP-25B. A similar loss in helical structure is observed with an increase in pH. Using the fluorescent signal from tryptophan and tyrosine, an increase in pH changes the peak emission with a pK of ∼8.5, similar to the expected pKa for the sulfhydryl of cysteine. Replacing all cysteines with serines prevents this shift. These data support the hypothesis that cysteine modification significantly alters SNAP-25 structure, possibly affecting exocytosis.In vivo, cysteines are the sites of various posttranslational modifications, such as palmitoylation and oxidation. Palmitoylation anchors SNAP-25 to the membrane (and determines its distribution within the cell), while the level of oxidation may regulate neurotransmitter release.Using an in vitro assay developed in our lab, we have measured the extent of non-enzymatic palmitoylation of SNAP-25B. Only 3 of the 4 cysteines are palmitoylated; the process is cooperative with a Hill Coefficient >2. However, all four cysteines are easily oxidized with a lower Hill coefficient. Through additional experimentation we hope to determine how oxidation and palmitoylation alter SNARE complex formation to modulate neurotransmitter release.

Chasing the Functional Asymmetry between C2A and C2B in Full-Length Synaptotagmin 1 during Ca2+-Dependent Membrane Binding

Synaptotagmin 1 (Syt1) acts as the major calcium sensor in neuronal exocytosis. Syt1 is a synaptic vesicle-anchored membrane protein that contains two tandem Ca2+ binding C2 domains, named C2A and C2B. There is evidence that Syt1 interacts with membrane lipids and SNARE proteins simultaneously to facilitate two key steps during Ca2+-triggered membrane fusion: vesicle binding and content release. Although the overall function of Syt1 has been extensively investigated, the detailed molecular behavior of the single C2A and C2B domains in the neural regulatory process remains unclear. In particular, the differential function of the two C2 domains and how they cooperate in membrane fusion has not been determined.Here we employed EPR spectroscopy, fluorescence interference contrast (FLIC) microscopy and total internal reflection fluorescence (TIRF) microscopy to dissect the state of C2A and C2B domains in full-length Syt1 during Ca2+-dependent membrane binding. CW-EPR lineshapes and power saturation of spin-labeled positions in calcium binding loops of C2A in full-length Syt1 (1-421) and truncated Syt1 (1-266) without C2B domain suggest that C2A domain alone in Syt1 has a stronger membrane binding ability. A TIRF liposome capture assay further reveals that truncated Syt1 has a significant higher initial rate and extent in trans-binding with liposomes than full-length Syt1. Our data indicate that the C2B domain might be involved in some cis-binding thereby reducing total liposome binding. FLIC microscopy validates the strong involvement of C2A during Ca2+-dependent liposome binding.Our detailed structural and functional information thus provides a clue to differential regulatory mechanisms employed by C2A and C2B domains in full-length Syt1 interacting with Ca2+ and key membrane lipids, such as PS and PIP2.

Synaptic vesicle glycoprotein 2A modulates vesicular release and calcium channel function at peripheral sympathetic synapses.

Synaptic vesicle glycoprotein (SV)2A is a transmembrane protein found in secretory vesicles and is critical for Ca(2+) -dependent exocytosis in central neurons, although its mechanism of action remains uncertain. Previous studies have proposed, variously, a role of SV2 in the maintenance and formation of the readily releasable pool (RRP) or in the regulation of Ca(2+) responsiveness of primed vesicles. Such previous studies have typically used genetic approaches to ablate SV2 levels; here, we used a strategy involving small interference RNA (siRNA) injection to knockdown solely presynaptic SV2A levels in rat superior cervical ganglion (SCG) neuron synapses. Moreover, we investigated the effects of SV2A knockdown on voltage-dependent Ca(2+) channel (VDCC) function in SCG neurons. Thus, we extended the studies of SV2A mechanisms by investigating the effects on vesicular transmitter release and VDCC function in peripheral sympathetic neurons. We first demonstrated an siRNA-mediated SV2A knockdown. We showed that this SV2A knockdown markedly affected presynaptic function, causing an attenuated RRP size, increased paired-pulse depression and delayed RRP recovery after stimulus-dependent depletion. We further demonstrated that the SV2A-siRNA-mediated effects on vesicular release were accompanied by a reduction in VDCC current density in isolated SCG neurons. Together, our data showed that SV2A is required for correct transmitter release at sympathetic neurons. Mechanistically, we demonstrated that presynaptic SV2A: (i) acted to direct normal synaptic transmission by maintaining RRP size, (ii) had a facilitatory role in recovery from synaptic depression, and that (iii) SV2A deficits were associated with aberrant Ca(2+) current density, which may contribute to the secretory phenotype in sympathetic peripheral neurons.

The Arabidopsis synaptotagmin SYTA regulates the cell-to-cell movement of diverse plant viruses.

Synaptotagmins are a large gene family in animals that have been extensively characterized due to their role as calcium sensors to regulate synaptic vesicle exocytosis and endocytosis in neurons, and dense core vesicle exocytosis for hormone secretion from neuroendocrine cells. Thought to be exclusive to animals, synaptotagmins have recently been characterized in Arabidopsis thaliana, in which they comprise a five gene family. Using infectivity and leaf-based functional assays, we have shown that Arabidopsis SYTA regulates endocytosis and marks an endosomal vesicle recycling pathway to regulate movement protein-mediated trafficking of the Begomovirus Cabbage leaf curl virus (CaLCuV) and the Tobamovirus Tobacco mosaic virus (TMV) through plasmodesmata (Lewis and Lazarowitz, 2010). To determine whether SYTA has a central role in regulating the cell-to-cell trafficking of a wider range of diverse plant viruses, we extended our studies here to examine the role of SYTA in the cell-to-cell movement of additional plant viruses that employ different modes of movement, namely the Potyvirus Turnip mosaic virus (TuMV), the Caulimovirus Cauliflower mosaic virus (CaMV) and the Tobamovirus Turnip vein clearing virus (TVCV), which in contrast to TMV does efficiently infect Arabidopsis. We found that both TuMV and TVCV systemic infection, and the cell-to-cell trafficking of the their movement proteins, were delayed in the Arabidopsis Col-0 syta-1 knockdown mutant. In contrast, CaMV systemic infection was not inhibited in syta-1. Our studies show that SYTA is a key regulator of plant virus intercellular movement, being necessary for the ability of diverse cell-to-cell movement proteins encoded by Begomoviruses (CaLCuV MP), Tobamoviruses (TVCV and TMV 30K protein) and Potyviruses (TuMV P3N-PIPO) to alter PD and thereby mediate virus cell-to-cell spread.

A Highly Tilted Membrane Configuration for the Prefusion State of Synaptobrevin

The SNARE complex plays a vital role in vesicle fusion arising during neuronal exocytosis. Key components in the regulation of SNARE complex formation, and ultimately fusion, are the transmembrane and linker regions of the vesicle-associated protein, synaptobrevin. However, the membrane-embedded structure of synaptobrevin in its prefusion state, which determines its interaction with other SNARE proteins during fusion, is largely unknown. This study reports all-atom molecular-dynamics simulations of the prefusion configuration of synaptobrevin in a lipid bilayer, aimed at characterizing the insertion depth and the orientation of the protein in the membrane, as well as the nature of the amino acids involved in determining these properties. By characterizing the structural properties of both wild-type and mutant synaptobrevin, the effects of C-terminal additions on tilt and insertion depth of membrane-embedded synaptobrevin are determined. The simulations suggest a robust, highly tilted state for membrane-embedded synaptobrevin with a helical connection between the transmembrane and linker regions, leading to an apparently new characterization of structural elements in prefusion synaptobrevin and providing a framework for interpreting past mutation experiments.

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.

DOC2 isoforms play dual roles in insulin secretion and insulin-stimulated glucose uptake.

Glucose-stimulated insulin secretion (GSIS) and insulin-stimulated glucose uptake are processes that rely on regulated intracellular vesicle transport and vesicle fusion with the plasma membrane. DOC2A and DOC2B are calcium-sensitive proteins that were identified as key components of vesicle exocytosis in neurons. Our aim was to investigate the role of DOC2 isoforms in glucose homeostasis, insulin secretion and insulin action. DOC2 expression was measured by RT-PCR and western blotting. Body weight, glucose tolerance, insulin action and GSIS were assessed in wild-type (WT), Doc2a (-/-) (Doc2aKO), Doc2b (-/-) (Doc2bKO) and Doc2a (-/-)/Doc2b (-/-) (Doc2a/Doc2bKO) mice in vivo. In vitro GSIS and glucose uptake were assessed in isolated tissues, and exocytotic proteins measured by western blotting. GLUT4 translocation was assessed by epifluorescence microscopy. Doc2b mRNA was detected in all tissues tested, whereas Doc2a was only detected in islets and the brain. Doc2aKO and Doc2bKO mice had minor glucose intolerance, while Doc2a/Doc2bKO mice showed pronounced glucose intolerance. GSIS was markedly impaired in Doc2a/Doc2bKO mice in vivo, and in isolated Doc2a/Doc2bKO islets in vitro. In contrast, Doc2bKO mice had only subtle defects in insulin secretion in vivo. Insulin action was impaired to a similar degree in both Doc2bKO and Doc2a/Doc2bKO mice. In vitro insulin-stimulated glucose transport and GLUT4 vesicle fusion were defective in adipocytes derived from Doc2bKO mice. Surprisingly, insulin action was not altered in muscle isolated from DOC2-null mice. Our study identifies a critical role for DOC2B in insulin-stimulated glucose uptake in adipocytes, and for the synergistic regulation of GSIS by DOC2A and DOC2B in beta cells.

α-SNAP Interferes with the Zippering of the SNARE Protein Membrane Fusion Machinery

Neuronal exocytosis is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Before fusion, SNARE proteins form complexes bridging the membrane followed by assembly toward the C-terminal membrane anchors, thus initiating membrane fusion. After fusion, the SNARE complex is disassembled by the AAA-ATPase N-ethylmaleimide-sensitive factor that requires the cofactor α-SNAP to first bind to the assembled SNARE complex. Using chromaffin granules and liposomes we now show that α-SNAP on its own interferes with the zippering of membrane-anchored SNARE complexes midway through the zippering reaction, arresting SNAREs in a partially assembled trans-complex and preventing fusion. Intriguingly, the interference does not result in an inhibitory effect on synaptic vesicles, suggesting that membrane properties also influence the final outcome of α-SNAP interference with SNARE zippering. We suggest that binding of α-SNAP to the SNARE complex affects the ability of the SNARE complex to harness energy or transmit force to the membrane.

Differential Interaction of Tomosyn with Syntaxin and SNAP25 Depends on Domains in the WD40 β-Propeller Core and Determines Its Inhibitory Activity

Neuronal exocytosis depends on efficient formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes and is regulated by tomosyn, a SNARE-binding protein. To gain new information about tomosyn's activity, we characterized its mobility and organization on the plasma membrane (PM) in relation to other SNARE proteins and inhibition of exocytosis. By using direct stochastic optical reconstruction microscopy (dSTORM), we found tomosyn to be organized in small clusters adjacent to syntaxin clusters. In addition, we show that tomosyn is present in both syntaxin-tomosyn complexes and syntaxin-SNAP25-tomosyn complexes. Tomosyn mutants that lack residues 537-578 or 897-917 from its β-propeller core diffused faster on the PM and exhibited reduced binding to SNAP25, suggesting that these mutants shift the equilibrium between tomosyn-syntaxin-SNAP25 complexes on the PM to tomosyn-syntaxin complexes. As these deletion mutants impose less inhibition on exocytosis, we suggest that tomosyn inhibition is mediated via tomosyn-syntaxin-SNAP25 complexes and not tomosyn-syntaxin complexes. These findings characterize, for the first time, tomosyn's dynamics at the PM and its relation to its inhibition of exocytosis.

Effects of isoflurane on dopaminergic neurotransmission (659.8)

Volatile anesthetics have poorly understood mechanisms of action, despite their wide usage in medicine. Current knowledge of the molecular and cellular mechanisms of anesthetic action is insufficient to explain amnesia, unconsciousness or immobilization, the principal features of general anesthesia. The volatile anesthetic isoflurane alters synaptic transmission by blocking ionotropic glutamate receptors postsynaptically and/or by enhancing fast inhibitory transmission and tonic currents mediated by GABAA receptors. However, dopaminergic neurons, which are primarily found in the ventral tegmental area and the substantia nigra pars compacta, play roles in cognition, attention, voluntary movement, among other functions, pose another potential site of anesthetic action. Here we test the hypothesis that isoflurane perturbs the stimulus‐secretion coupling between [Ca2+]i and exocytosis in dopaminergic neurons by probing synaptic vesicle exocytosis and nerve terminal [Ca2+]i. Laser‐ scanning fluorescence image measurements of [Ca2+]i in neonatal rat dopaminergic neurons were made using the cell permeant calcium indicator Fluo‐5N and action potential‐evoked exocytosis was measured with vesicular monoamine transporter‐ pHluorin (vMAT‐pH). The effects of isoflurane on [Ca2+]i and exocytosis at varying external [Ca2+] reveal a change in the Ca2+ sensitivity of exocytosis. These data suggest that isoflurane inhibits dopamine release from synaptic vesicles by shifting the coupling between [Ca2+]i and exocytosis.

Two modes of exocytosis in an artificial cell

The details of exocytosis, the vital cell process of neuronal communication, are still under debate with two generally accepted scenarios. The first mode of release involves secretory vesicles distending into the cell membrane to release the complete vesicle contents. The second involves partial release of the vesicle content through an intermittent fusion pore, or an opened or partially distended fusion pore. Here we show that both full and partial release can be mimicked with a single large-scale cell model for exocytosis composed of material from blebbing cell plasma membrane. The apparent switching mechanism for determining the mode of release is demonstrated to be related to membrane tension that can be differentially induced during artificial exocytosis. These results suggest that the partial distension mode might correspond to an extended kiss-and-run mechanism of release from secretory cells, which has been proposed as a major pathway of exocytosis in neurons and neuroendocrine cells.

Interference of Zippering of Snare Complexes by Alpha-Snap Arrest Fusion of Chromaffin Granule

Neuronal exocytosis is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Before fusion, SNARE proteins form complexes bridging the membrane followed by assembly toward the C-terminal membrane anchors, thus initiating membrane fusion. After fusion, the SNARE complex is disassembled by the AAA-ATPase NSF that requires the cofactor α-SNAP to first bind to the assembled SNARE complex. In vitro, SNARE-mediated fusion can be readily reconstituted, but it has been difficult to reproduce a docked state in which the SNAREs are arrested in a partially zippered state. Using both native and artificial vesicles we now show that α-SNAP on its own retards assembly of membrane-anchored SNARE complexes and can arrest SNAREs in a partially assembled trans-complex, preventing progression towards fusion, which can be rescued by active NSF. Intriguingly, the inhibitory effect of α-SNAP depends on the size of the vesicle, with inhibition being most profound in large vesicles. Our data suggest that binding of α-SNAP lowers the energy yield of the zippering reaction, inhibiting preferentially the fusion of large vesicles that exhibit low curvature strain and require more energy for overcoming the barrier for fusion.

In Vitro Palmitoylation and Oxidation of the Snare Protein SNAP-25

The SNARE protein SNAP-25 plays a critical role in neuronal exocytosis. The linker region of SNAP-25 contains 4 cysteines (cysteine-rich domain) which are the sites of various post-translational modifications, such as palmitoylation and oxidation. Palmitoylation anchors SNAP-25 to the membrane (and determines its distribution within the cell), while the level of oxidation may regulate neurotransmitter release.Quantifying the extent of oxidation and palmitoylation in vivo has proven elusive. Our in vitro data from SNAP-25 suggests that non-enzymatic palmitoylation is cooperative with a Hill Coefficient greater than 2, whereas oxidation appears to have a Hill coefficient of 1/2 (consistent with two cysteines being consumed for each oxidation event). Using Circular Dichroism to measure the melting of helical structures of SNAP-25, we show that oxidation destabilizes SNAP-25. Oxidation also destabilizes SNARE complex formation. During in vitro palmitoylation, we use Mass Spec analysis to confirm that multiple cysteines are palmitoylated under our conditions. In vitro palmitoylation and oxidation have an EC50 of about 50 μM. Through additional experimentation we hope to elucidate the EC50, Hill Coefficient, and extent of reaction for enzyme-catalyzed palmitoylation of the cysteine-rich domain of SNAP-25's linker region and also determine how oxidation and palmitoylation alter SNARE complex formation.