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PubMed1992

The synaptic vesicle protein synaptotagmin associates with calcium channels and is a putative Lambert-Eaton myasthenic syndrome antigen.

Immunoglobulin G fractions from patients with Lambert-Eaton myasthenic syndrome (LEMS), an autoimmune disease of neuromuscular transmission, immunoprecipitate 125I-labeled omega-conotoxin GVIA-labeled calcium channels solubilized from rat brain. A 58-kDa antigen was detected by probing Western blots of partially purified calcium channels with LEMS plasma and IgG and was shown to be the relevant antigen in omega-conotoxin receptor immunoprecipitation. Monoclonal antibody 1D12, produced by immunizing mice with synaptic membranes, has properties similar to these autoimmune IgGs in both immunoprecipitation and Western blotting assays. 1D12 antigen was purified by immunoaffinity chromatography and shown to bind LEMS IgG. The antigen was identified by screening a rat brain cDNA library with 1D12 and was found to have strong homology to the synaptic vesicle membrane protein synaptotagmin. Our results indicate therefore that these antibodies immunoprecipitate omega-conotoxin receptors by binding to synaptotagmin that is associated with calcium channels. We suggest that the interaction between synaptotagmin and the voltage-gated calcium channel plays a role in docking synaptic vesicles at the plasma membrane prior to rapid neurotransmitter release and that autoantibody binding to a synaptotagmin-calcium-channel complex may be involved in the etiology of LEMS.

Antibodies, Monoclonal↗

PubMed2000

Detection of thiol modification following generation of reactive nitrogen species: analysis of synaptic vesicle proteins.

S-nitrosylation is an important means of regulating the activity of proteins. We have developed a method which allows unbiased identification of thiol modified proteins within a complex mixture following NO generation, by taking advantage of the fact that prior nitrosylation will block subsequent modification of cysteine residues with 1-biotinamido-4-[4'-(maleimidomethyl)-cyclohexane-carboxamid o] butane (biotin-BMCC). Thiol modified proteins are reduced in intensity when revealed by blotting and overlay with avidin-horseradish peroxidase. In the case of a purified synaptic vesicle fraction we observe a high degree of enrichment of specific biotinylated proteins relative to homogenate. We find that thiol modification of proteins in the presence of NO donors is widespread, occurring in the majority of proteins that will react with biotin-BMCC. In a further development of this technique we have depleted the biotinylated proteins from solubilised synaptic vesicles using avidin-agarose and analysed the supernatants with a panel of antibodies. This has allowed us to identify SNARE proteins (soluble NSF attachment protein receptors) as potential targets for S-nitrosylation.

Animals↗

PubMed2006

Na+, Cl-, and pH dependence of the human choline transporter (hCHT) in Xenopus oocytes: the proton inactivation hypothesis of hCHT in synaptic vesicles.

The recent cloning of the human choline transporter (hCHT) has allowed its expression in Xenopus laevis oocytes and the simultaneous measurement of choline transport and choline-induced current under voltage clamp. hCHT currents and choline transport are evident in cRNA-injected oocytes and significantly enhanced by the hCHT trafficking mutant L530A/V531A. The charge/choline ratio of hCHT varies from 10e/choline at -80 mV to 3e/choline at -20 mV, in contrast with the reported fixed stoichiometry of the Na+-coupled glucose transporter in the same gene family. Ion substitution shows that the choline uptake and choline-induced current are Na+ and Cl- dependent; however, the reversal potential of the induced current suggests a Na+-selective mechanism, consigning Cl- to a regulatory role rather than a coupled, cotransported-ion role. The hCHT-specific inhibitor hemicholinium-3 (HC-3) blocks choline uptake and choline-induced current; in addition, HC-3 alone reveals a constitutive, depolarizing leak current through hCHT. We show that external protons reduce hCHT current, transport, and binding with a similar pKa of 7.4, suggesting proton titration of residue(s) that support choline binding and transport. Given the localization of the choline transporter to synaptic vesicles, we propose that proton inactivation of hCHT prevents acetylcholine and proton leakage from the acidic interior of cholinergic synaptic vesicles. This mechanism would allow cholinergic, activity-triggered delivery of silent choline transporters to the plasma membrane, in which normal pH would reactivate the transporters for choline uptake and subsequent acetylcholine synthesis.

Acetylcholine↗

PubMed1992

Redistribution of secretory granule components precedes that of synaptic vesicle proteins during differentiation of a neuronal cell line in serum-free medium.

Incubation of the rat sensory neuron-derived cell line ND7 in serum-free medium results in the arrest of mitosis and the appearance of numerous neuronal processes. During this differentiation event, secretory granule components such as chromogranins, neuropeptide Y and the C-flanking peptide of pro-neuropeptide Y move to the tips of the majority of the neuronal processes regardless of process length. In contrast, the synaptic vesicle component, synaptophysin, is found only at the tips of the very long processes which appear following prolonged periods of culture in serum-free medium. A similar restriction of synaptophysin to long processes is also observed following differentiation and process formation induced by other treatments such as incubation in reduced serum or treatment with cyclic AMP or phorbol myristate acetate. Hence the regulated secretory pathway associated with the chromogranins and neuropeptides appears to be segregated into the processes at an earlier stage of ND7 differentiation than the synaptophysin-associated synaptic vesicle pathway. ND7 cells therefore provide a model system for studying the processes regulating these pathways and the redistribution of their components during neuronal differentiation.

Animals↗

PubMed2001

The Eps15 C. elegans homologue EHS-1 is implicated in synaptic vesicle recycling.

Eps15 represents the prototype of a family of evolutionarily conserved proteins that are characterized by the presence of the EH domain, a protein-protein interaction module, and that are involved in many aspects of intracellular vesicular sorting. Although biochemical and functional studies have implicated Eps15 in endocytosis, its function in the endocytic machinery remains unclear. Here we show that the Caenorhabditis elegans gene, zk1248.3 (ehs-1), is the orthologue of Eps15 in nematodes, and that its product, EHS-1, localizes to synaptic-rich regions. ehs-1-impaired worms showed temperature-dependent depletion of synaptic vesicles and uncoordinated movement. These phenotypes could be correlated with a presynaptic defect in neurotransmission. Impairment of EHS-1 function in dyn-1(ky51) worms, which express a mutant form of dynamin and display a temperature-sensitive locomotion defect, resulted in a worsening of the dyn-1 phenotype and uncoordination at the permissive temperature. Thus, ehs-1 and dyn-1 interact genetically. Moreover, mammalian Eps15 and dynamin protein were shown to interact in vivo. Taken together, our results indicate that EHS-1 acts in synaptic vesicle recycling and that its function might be linked to that of dynamin.

Aldicarb↗

PubMed2002

KIF1Bbeta2, capable of interacting with CHP, is localized to synaptic vesicles.

Kinesin family proteins are microtubule-dependent molecular motors involved in the intracellular motile process. Using a Ca2+ -binding protein, CHP (calcineurin B homologous protein), as a bait for yeast two hybrid screening, we identified a novel kinesin-related protein, KIF1Bbeta2. KIF1Bbeta2 is a member of the KIF1 subfamily of kinesin-related proteins, and consists of an amino terminal KIF1B-type motor domain followed by a tail region highly similar to that of KIF1A. CHP binds to regions adjacent to the motor domains of KIF1Bbeta2 and KIF1B, but not to those of the other KIF1 family members, KIF1A and KIF1C. Immunostaining of neuronal cells showed that a significant portion of KIF1Bbeta2 is co-localized with synaptophysin, a marker protein for synaptic vesicles, but not with a mitochondria-staining dye. Subcellular fractionation analysis indicated the co-localization of KIF1Bbeta2 with synaptophysin. These results suggest that KIF1Bbeta2, a novel CHP-interacting molecular motor, mediates the transport of synaptic vesicles in neuronal cells.

Amino Acid Sequence↗

PubMed1986

Formation of synaptic vesicles in the superior cervical ganglion of cat: choline dependency.

De novo formation of synaptic vesicles was studied electron microscopically in synapses of the superior cervical ganglion of cat. Following prolonged electrical stimulation in a choline deficient condition, almost complete loss of vesicle content was observed. After choline administration, together with the partial restoration of transmission, the reappearance of vesicles could be seen in nerve terminals. In interpreting our observations, the double role of choline (transmitter precursor and membrane constituent) is emphasized.

Animals↗

PubMed1981

Smooth endoplasmic reticulum and synaptic vesicles in cultured murine neuroblastoma cells.

Cultured murine neuroblastoma cells contain an extensive smooth endoplasmic reticulum (SER), also within their numerous processes and their terminals, consisting of a complex network of sacs and tubules. Both the perikaryal and the SER found within the processes and endings resemble one another morphologically. From the SER whether it is within the perikaryon or within the terminals of the neural processes, round coated vesicles with clear centers appear to form. These clusters of vesicles are 40...60nm in diameter and indistinguishable from the synaptic vesicles of normal neurons. In addition a few vesicles with clear centers with a diameter of 20...40 nm and length of 80...200 nm are encountered and appear to originate from the SER. The dense-cored vesicles do not seem to develop from the SER. There is no detectable association of the vesicles with any other cell organelle.

Animals↗

PubMed1998

Neuroendocrine synaptic vesicles are formed in vitro by both clathrin-dependent and clathrin-independent pathways.

In the neuroendocrine cell line, PC12, synaptic vesicles can be generated from endosomes by a sorting and vesiculation process that requires the heterotetrameric adaptor protein AP3 and a small molecular weight GTPase of the ADP ribosylation factor (ARF) family. We have now discovered a second pathway that sorts the synaptic vesicle-associated membrane protein (VAMP) into similarly sized vesicles. For this pathway the plasma membrane is the precursor rather than endosomes. Both pathways require cytosol and ATP and are inhibited by GTPgammaS. The second pathway, however, uses AP2 instead of AP3 and is brefeldin A insensitive. The AP2-dependent pathway is inhibited by depletion of clathrin or by inhibitors of clathrin binding, whereas the AP3 pathway is not. The VAMP-containing, plasma membrane-derived vesicles can be readily separated on sucrose gradients from transferrin (Tf)-containing vesicles generated by incubating Tf-labeled plasma membrane preparations at 37 degreesC. Dynamin- interacting proteins are required for the AP2-mediated vesiculation from the plasma membrane, but not from endosomes. Thus, VAMP is sorted into small vesicles by AP3 and ARF1 at endosomes and by AP2 and clathrin at the plasma membrane.

ADP-Ribosylation Factor 1↗

PubMed2004

The Caenorhabditis elegans UNC-14 RUN domain protein binds to the kinesin-1 and UNC-16 complex and regulates synaptic vesicle localization.

Kinesin-1 is a heterotetramer composed of kinesin heavy chain (KHC) and kinesin light chain (KLC). The Caenorhabditis elegans genome has a single KHC, encoded by the unc-116 gene, and two KLCs, encoded by the klc-1 and klc-2 genes. We show here that UNC-116/KHC and KLC-2 form a complex orthologous to conventional kinesin-1. KLC-2 also binds UNC-16, the C. elegans JIP3/JSAP1 JNK-signaling scaffold protein, and the UNC-14 RUN domain protein. The localization of UNC-16 and UNC-14 depends on kinesin-1 (UNC-116 and KLC-2). Furthermore, mutations in unc-16, klc-2, unc-116, and unc-14 all alter the localization of cargos containing synaptic vesicle markers. Double mutant analysis is consistent with these four genes functioning in the same pathway. Our data support a model whereby UNC-16 and UNC-14 function together as kinesin-1 cargos and regulators for the transport or localization of synaptic vesicle components.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Two components of transmitter release in retinal bipolar cells: exocytosis and mobilization of synaptic vesicles.

Ca2+-transmitter release coupling was examined using bipolar cells with large presynaptic terminals dissociated from the goldfish retina. Presynaptic Ca2+ current (I(Ca)) was recorded under the whole-cell voltage clamp. Release of excitatory amino acid transmitter was simultaneously monitored as the current through N-methyl-D-asperate (NMDA) receptors of reporter cells or as the membrane capacitance (C(m)) change associated with exocytosis. When I(Ca) was activated by a long depolarizing pulse, a double-peaked transmitter-induced current (I(tr)) was elicited in reporter cells. The rapid component of I(tr) was evoked immediately after the onset of depolarization, and was affected only slightly by intracellularly applied Ca2+ chelators. The delayed slow component of I(tr) was elicited during depolarization once a fixed amount of Ca2+ was accumulated in presynaptic terminals, and its appearance was suppressed or retarded by Ca2+ chelators. Two components of transmitter release were also recognized by monitoring C(m) changes elicited by the activation of I(Ca). These results suggest that bipolar cells have at least two pools of synaptic vesicles; a small, immediately releasable pool and a large releasable pool. The rapid and the delayed slow components of transmitter release may reflect exocytosis and mobilization of synaptic vesicles, respectively.

Animals↗

PubMed2001

Role of Rab3 GDP/GTP exchange protein in synaptic vesicle trafficking at the mouse neuromuscular junction.

The Rab3 small G protein family consists of four members, Rab3A, -3B, -3C, and -3D. Of these members, Rab3A regulates Ca(2+)-dependent neurotransmitter release. These small G proteins are activated by Rab3 GDP/GTP exchange protein (Rab3 GEP). To determine the function of Rab3 GEP during neurotransmitter release, we have knocked out Rab3 GEP in mice. Rab3 GEP-/- mice developed normally but died immediately after birth. Embryos at E18.5 showed no evoked action potentials of the diaphragm and gastrocnemius muscles in response to electrical stimulation of the phrenic and sciatic nerves, respectively. In contrast, axonal conduction of the spinal cord and the phrenic nerve was not impaired. Total numbers of synaptic vesicles, especially those docked at the presynaptic plasma membrane, were reduced at the neuromuscular junction approximately 10-fold compared with controls, whereas postsynaptic structures and functions appeared normal. Thus, Rab3 GEP is essential for neurotransmitter release and probably for formation and trafficking of the synaptic vesicles.

Action Potentials↗

PubMed1996

The V0 sector of the V-ATPase, synaptobrevin, and synaptophysin are associated on synaptic vesicles in a Triton X-100-resistant, freeze-thawing sensitive, complex.

Anti-synaptobrevin 2 immunoprecipitates obtained from freshly prepared Triton X-100 extracts of rat synaptosomes contained, in addition to synaptophysin, a 10-kDa band, which we identified by peptide sequencing and Western blotting as the c subunit of the vacuolar proton pump (V-ATPase) also called ductin or mediatophore. Ac39 and Ac116, two other transmembrane subunits of the V0 sector of the V-ATPase, were also found by Western blotting to be enriched in the immunoprecipitates. None of these V-ATPase subunits, or synaptophysin, was present in anti-synaptobrevin 2 immunoprecipitates obtained from frozen-thawed Triton X-100 extracts, which were greatly enriched, instead, in SNAP-25 and syntaxin 1. Accordingly, V-ATPase subunit c was found in anti-synaptophysin immunoprecipitates. Thus, the two complexes appear to be mutually exclusive. Subcellular fractionation of rat brain demonstrated that V-ATPase subunit c is localized with synaptobrevin 2 and synaptophysin in synaptic vesicles. The coprecipitation of V-ATPase subunit c with the synaptobrevin-synaptophysin complex suggests that this interaction may play a role in recruiting the proton pump into synaptic vesicles. Freeze-thawing, which involves a mild denaturing step, may produce a conformational change which dissociates the complex and mimics a change which occurs in vivo as a prerequisite to SNARE complex formation.

Amino Acid Sequence↗

PubMed1994

Increase in synaptic vesicle proteins accompanies long-term potentiation in the dentate gyrus.

Maintenance of long-term potentiation in synapses formed by the perforant path on to granule cells of the dentate gyrus is accompanied by a sustained increase in the extracellular concentration of glutamate, the presumed transmitter at this excitatory hippocampal pathway. Quantal analysis indicates that, at least in the first hour of induction, this reflects an increase in transmitter release rather than a decrease in glutamate uptake, while biochemical studies have suggested that the increase in release persists for several hours. Morphological studies have described early but persistent increases in the spine number and area. Increases in the number of segmented/perforated synapses persisting for at least 1 h after induction of long-term potentiation, have also been reported. These morphological changes suggest both presynaptic and postsynaptic modifications. Increases in synaptic vesicle number and distribution lasting for at least 1 h specifically indicate presynaptic changes. To explore further the role of the presynaptic terminal in long-term potentiation, we have investigated changes in three synaptic vesicle proteins, synapsin, synaptotagmin and synaptophysin, in control tissue and in tissue prepared from potentiated dentate gyrus 45 min and 3 h after induction of long-term potentiation. We found that there was an increase in the concentration of the three proteins 3 h after induction of long-term potentiation. No such increase was observed 45 min after induction or in tissue prepared from animals in which an intraventricular injection of the N-methyl-D-aspartate receptor antagonist, D(-)-2-amino-5-phosphonopentanoic acid, blocked induction of long-term potentiation.

2-Amino-5-phosphonovalerate↗

PubMed1997

Synaptic vesicle ultrastructural changes in the rat hippocampus induced by a combination of alpha-linolenate deficiency and a learning task.

Rats fed either a safflower oil (alpha-linolenate-deficient) or a perilla oil (alpha-linolenate-sufficient) diet through two generations (F1) showed significant differences in the brightness-discrimination learning task. In this task, correct responses were lever-pressing responses, which were reinforced with dietary pellets, and incorrect responses were those with no reinforcement. The inferior learning performance in the safflower oil group was caused mainly by the inferior ability to rectify the incorrect responses through the learning sessions. In the safflower oil group after the learning task, the average densities of synaptic vesicles in the terminals of the hippocampus CA1 region were decreased by nearly 30% as compared with those in the perilla oil group, and it is notable that this difference was not detected without the learning task. These results suggest that dietary oil-induced morphological changes in synapses in the hippocampus of rats are related to the differential learning performance and that the turnover rate of synaptic vesicles in the hippocampus may be an important factor affecting learning performance.

Animals↗

PubMed2004

Nicotine enhancement of dopamine release by a calcium-dependent increase in the size of the readily releasable pool of synaptic vesicles.

A major factor underlying compulsive tobacco use is nicotine-induced modulation of dopamine release in the mesolimbic reward pathway (Wise and Rompre, 1989). An established biochemical mechanism for nicotine-enhanced dopamine release is by activating presynaptic nicotinic acetylcholine receptors (nAChRs) (Wonnacott, 1997). Prolonged application of 10(-7) to 10(-5) m nicotine to striatal synaptosomes promoted a sustained efflux of [3H]dopamine. This nicotine effect was mediated by non-alpha7 nAChRs, because it was blocked by 5 mum mecamylamine but was resistant to 100 nm alpha-bungarotoxin (alphaBgTx). Dopamine release was diminished by omitting Na+ or by applying peptide calcium channel blockers, indicating that nAChRs trigger release by depolarizing the nerve terminals. However, because alpha7 receptors rapidly desensitize in the continuous presence of agonists, a repetitive stimulation protocol was used to evaluate the possible significance of desensitization. This protocol produced a transient increase in [3H]dopamine released by depolarization and a significant increase in the response to hypertonic solutions that measure the size of the readily releasable pool (RRP) of synaptic vesicles. The nicotine-induced increase in the size of the readily releasable pool was blocked by alphaBgTx and by the calmodulin antagonist calmidazolium, suggesting that Ca2+ entry through alpha7 nAChRs specifically enhances synaptic vesicle mobilization at dopamine terminals. Thus, nicotine enhances dopamine release by two complementary actions mediated by discrete nAChR subtypes and suggest that the alpha7 nAChR-mediated pathway is tightly and specifically coupled to refilling of the RRP of vesicles in dopamine terminals.

Animals↗

PubMed2003

A chloride channel blocker reduces acetylcholine uptake into synaptic vesicles at the frog neuromuscular junction.

A key mechanism for loading acetylcholine (ACh(+)) into synaptic vesicles uses energy to transport H(+) into the vesicle interior and then exchanges H(+) for ACh(+). This mechanism requires anions to follow the H(+) into the vesicles to prevent the building up of an overwhelming electrical gradient across the vesicle membrane. Frog nerve-muscle preparations were treated with hypertonic solution in which sodium gluconate was the major constituent, which substantially increases the sizes of the quanta by increasing their ACh(+) content. The Cl(-) channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) antagonized the increases in quantal size, so it seems likely that Cl(-) follows H(+) to prevent the buildup of a potential gradient across the vesicular membrane.

Acetylcholine↗

PubMed1981

Acetylcholine incorporation by cholinergic synaptic vesicles from Torpedo marmorata.

The ability of cholinergic vesicles to incorporate acetylcholine (ACh) was studied using highly purified synaptic vesicles from Torpedo electric organ. Depleted vesicles were capable of rapidly taking up exogenous ACh. Evidence that this represented true incorporation was that labelled ACh comigrated with vesicular ATP on gel filtration and that vesicle-associated ACh was protected against enzymatic hydrolysis and was releasable under hypoosmotic conditions. The total amount of ACh incorporated depended on the ACh concentration up to 100 mM. A sudden fall in the external ACh concentration did not cause leakage of the ACh incorporated in vitro. Preliminary results indicated that retention of ACh inside the vesicle was pH-dependent. Choline was also taken up by vesicles, but the time pattern strongly suggested that it was not being retained. The magnitude of ach incorporation was estimated with respect to the intravesicular space.

Acetylcholine↗