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At least 793 records · Page 44Linked to original sources
PubMed2003

Cloning and sequence analysis of the human cDNA encoding the synaptoporin (delta), a highly conservative synaptic vesicle protein.

Synaptoporin is a membrane protein of synaptic vesicles, which belongs to the synaptophysin family. It was first isolated from rat brain. Here we report the cloning and characterization of the human cDNA that encodes the human homologue of synaptoporin. It has two splicing variants, which is in accordance with its ortholog in chicken. Its deduced amino acid sequence displays 97% and 81% identity with the synaptoporin of rat and chicken, respectively. This gene was mapped to the chromosome 3p14.3 by matching against the Human Genome Sequence Database. 16-tissue RT-PCR analysis shows that human synaptoporin is specifically expressed in the brain.

Amino Acid Sequence↗
PubMed1986

Stoichiometries of acetylcholine uptake, release, and drug inhibition in Torpedo synaptic vesicles: heterogeneity in acetylcholine transport and storage.

Highly purified Torpedo electric organ synaptic vesicles form a 49 nM suspension at 1 mg protein/ml. Under active transport conditions hundreds of molecules of [3H]acetylcholine ([3H]ACh) can be accumulated per vesicle, which requires the ACh transporter to undergo multiple turnovers. The transport blocker trans-2-(4-phenylpiperidino)cyclohexanol (AH5183) has no effect on storage of endogenous ACh by vesicles. In contrast, AH5183, other blocking drugs, and nonradioactive ACh caused a rapid release of at least 30-63 molecules of newly transported [3H]ACh per vesicle. Thus AH5183 distinguishes recently transported "new" vesicular ACh from "old" endogenous ACh. l-AH5183 inhibits transport of ACh with a half-inhibitory concentration of 16 +/- 7 nM at 12 nM vesicles and 115 +/- 34 nM at 120 nM vesicles. With the assumption that AH5183 acts on a receptor in an unamplified manner about 2.7 or fewer receptors per vesicle need to be occupied to cause inhibition of ACh transport. The apparent amplification in the number of [3H]ACh molecules per vesicle that are released by AH5183 suggests that AH5183 inhibits ACh storage by an indirect mechanism that distinguishes new from old ACh.

Acetylcholine↗
PubMed2001

The neuronal form of adaptor protein-3 is required for synaptic vesicle formation from endosomes.

Heterotetrameric adaptor complexes vesiculate donor membranes. One of the adaptor protein complexes, AP-3, is present in two forms; one form is expressed in all tissues of the body, whereas the other is restricted to brain. Mice lacking both the ubiquitous and neuronal forms of AP-3 exhibit neurological disorders that are not observed in mice that are mutant only in the ubiquitous form. To begin to understand the role of neuronal AP-3 in neurological disease, we investigated its function in in vitro assays as well as its localization in neural tissue. In the presence of GTPgammaS both ubiquitous and neuronal forms of AP-3 can bind to purified synaptic vesicles. However, only the neuronal form of AP-3 can produce synaptic vesicles from endosomes in vitro. We also identified that the expression of neuronal AP-3 is limited to varicosities of neuronal-like processes and is expressed in most axons of the brain. Although the AP-2/clathrin pathway is the major route of vesicle production and the relatively minor neuronal AP-3 pathway is not necessary for viability, the absence of the latter could lead to the neurological abnormalities seen in mice lacking the expression of AP-3 in brain. In this study we have identified the first brain-specific function for a neuronal adaptor complex.

Adaptor Proteins, Vesicular Transport↗
PubMed1975

Enlargement of synaptic vesicles in degenerating nerve endings: a comparison between cat and monkey.

The synaptic vesicles of optic nerve endings in the lateral geniculate nucleus of cats and monkeys were measured following eye enucleation. A notable increase in the mean vesicle volume was found in both species. Increments in the values of standard deviation were also noted. Comparison of the results obtained indicate that changes are faster in cats than in monkeys. An explanation for difference is proposed.

Animals↗
PubMed1978

Synaptic vesicles and microtubules in frog motor endplates.

Motor endplates of the cutaneous pectoris skeletal muscle of the frog have been examined by electron microscopy using a new technique. This involves pretreatment with an albumin solution, followed by fixation with 4% unbuffered tetroxide. A small proportion of the endplate axonal ramifications show microtubules clothed in synaptic vesicles and focused on the presynaptic membrane, in particular on the active zones. The microtubules run in the presynaptic cytoplasm either parallel to or across the active zones. These two sets of microtubules cross each other at the active zones, which lie opposite the dips in the post-junctional folds. The possibility that the microtubules are involved in the translocation of synaptic vesicles to the active zone is discussed.

Albumins↗
PubMed1983

Glucocorticoid-induced changes in synaptic vesicles of rat phrenic nerve terminals.

The mean cross sectional area of synaptic vesicle profiles of the myoneural junction of rat diaphragm is increased significantly when rats are injected i.p. with single doses of 200 micrograms/kg dexamethasone 1/2 hr to 8 hr previously to a control injection of 0.9% NaCl. A maximum effect is observed at 8 hr after pretreatment with dexamethasone. The treatment of rats with hemicholinium-3 (300 micrograms/kg) induces a significant reduction of the vesicle size. Complete prevention of this decrease in vesicle size is already observed when the animals are pretreated with a single dose of 200 micrograms/kg dexamethasone 1/2 hr before treatment with 300 micrograms/kg hemicholinium-3. Dexamethasone in the same dose causes an optimum increase of the mean cross sectional area when it is given 1 hr before treatment of the animals with hemicholinium-3. It is concluded that glucocorticoids have direct actions in both "normal" motor nerve terminals and in motor nerve terminals with a deficient choline transport system, which may contribute to their proposed beneficial effects in certain cases of muscle weakness.

Animals↗
PubMed1982

Cyclic AMP independent protein kinase activity in rat cerebral cortex synaptic vesicles--partial characterization.

The intrinsic protein kinase activity of a highly purified synaptic vesicle preparation was characterized. The time-course of the reaction was found to be rapid and linear for about 1 min, but plateaued after 30 min by which time approximately 1 nmol of 32P per mg protein was incorporated into trichloroacetic acid precipitated vesicular protein. The enzyme was optimally active at pH 6.0 (37 degrees C), and had apparent Km values of 40 and 88 microM for ATP and GTP respectively. The enzyme was not stimulated by cAMP or cGMP. Mg2+ was required for maximal activity. The reaction was inhibited by free Ca2+, and non-selectively by Na+, K+, and NH4+.

Animals↗
PubMed2001

A novel all helix fold of the AP180 amino-terminal domain for phosphoinositide binding and clathrin assembly in synaptic vesicle endocytosis.

Clathrin-mediated endocytosis plays a major role in retrieving synaptic vesicles from the plasma membrane following exocytosis. This endocytic process requires AP180 (or a homolog), which promotes the assembly and restricts the size of clathrin-coated vesicles. The highly conserved 33 kDa amino-terminal domain of AP180 plays a critical role in binding to phosphoinositides and in regulating the clathrin assembly activity of AP180. The crystal structure of the amino-terminal domain reported herein reveals a novel fold consisting of a large double layer of sheets of ten alpha helices and a unique site for binding phosphoinositides. The finding that the clathrin-box motif is mostly buried and lies in a helix indicates a different site and mechanism for binding of the domain to clathrins than previously assumed.

Adaptor Proteins, Vesicular Transport↗
PubMed1999

Calcium-dependent oligomerization of synaptotagmins I and II. Synaptotagmins I and II are localized on the same synaptic vesicle and heterodimerize in the presence of calcium.

Synaptotagmins constitute a large family of membrane proteins characterized by their distinct distributions and different biochemical features. Genetic evidence suggests that members of this protein family are likely to function as calcium sensors in calcium-regulated events in neurons, although the precise molecular mechanism remains ill defined. Here we demonstrate that different synaptotagmin isoforms (Syt I, II, and IV) are present in the same synaptic vesicle population from rat brain cortex. In addition, Syt I and II co-localize on the same small synaptic vesicle (SSV), and they heterodimerize in the presence of calcium with a concentration dependence resembling that of the starting phase of SSV exocytosis (EC50 = 6 +/- 4 microM). The association between Syt I and Syt II was demonstrated by immunoprecipitation of the native proteins and the recombinant cytoplasmic domains and by using fluorescence resonance energy transfer (FRET). Although a subpopulation of SSV containing Syt I and IV can be isolated, these two isoforms do not show a calcium-dependent interaction. These results suggest that the self-association of synaptotagmins with different calcium binding features may create a variety of calcium sensors characterized by distinct calcium sensitivities. This combinatorial hypothesis predicts that the probability of a single SSV exocytic event is determined, in addition to the gating properties of the presynaptic calcium channels, by the repertoire and relative abundance of distinct synaptotagmin isoforms present on the SSV surface.

Amino Acid Sequence↗
PubMed2006

A complexin/synaptotagmin 1 switch controls fast synaptic vesicle exocytosis.

Ca(2+) binding to synaptotagmin 1 triggers fast exocytosis of synaptic vesicles that have been primed for release by SNARE-complex assembly. Besides synaptotagmin 1, fast Ca(2+)-triggered exocytosis requires complexins. Synaptotagmin 1 and complexins both bind to assembled SNARE complexes, but it is unclear how their functions are coupled. Here we propose that complexin binding activates SNARE complexes into a metastable state and that Ca(2+) binding to synaptotagmin 1 triggers fast exocytosis by displacing complexin from metastable SNARE complexes. Specifically, we demonstrate that, biochemically, synaptotagmin 1 competes with complexin for SNARE-complex binding, thereby dislodging complexin from SNARE complexes in a Ca(2+)-dependent manner. Physiologically, increasing the local concentration of complexin selectively impairs fast Ca(2+)-triggered exocytosis but retains other forms of SNARE-dependent fusion. The hypothesis that Ca(2+)-induced displacement of complexins from SNARE complexes triggers fast exocytosis accounts for the loss-of-function and gain-of-function phenotypes of complexins and provides a molecular explanation for the high speed and synchronicity of fast Ca(2+)-triggered neurotransmitter release.

Adaptor Proteins, Vesicular Transport↗
PubMed1997

Peptide mapping of the [125I]Iodoazidoketanserin and [125I]2-N-[(3'-iodo-4'-azidophenyl)propionyl]tetrabenazine binding sites for the synaptic vesicle monoamine transporter.

The full-length cDNA for the rat recombinant synaptic vesicle monoamine transporter (rVMAT2) containing a COOH-terminal polyhistidine epitope was engineered into baculovirus DNA for expression in Spodoptera frugiperda (Sf9) cells. Using this recombinant baculovirus and cultured Sf9 cells, rVMAT2 has been expressed to high levels and purified to >95% homogeneity using immobilized Ni2+-affinity chromatography followed by lectin (concanavalin A) chromatography. Purified transporter was photolabeled using [125I]-7-azido-8-iodoketanserin ([125I]AZIK) and [125I]2-N-[(3'-iodo-4'-azidophenyl)propionyl]tetrabenazine ([125I]TBZ-AIPP). Both [125I]AZIK and [125I]TBZ-AIPP photoaffinity labeling of purified rVMAT2 were protectable by 10 microM tetrabenazine (TBZ), 10 microM 7-aminoketanserin, and 1 mM concentrations of the transporter substrates dopamine, norepinephrine, and serotonin. Radiolabeled peptides were generated using enzymatic and chemical methods, purified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and NH2-terminal microsequenced. Radiosequencing of [125I]AZIK-labeled rVMAT2 indicated derivatization of Lys-20 in the NH2 terminus, just prior to putative transmembrane domain 1 (TMD1). [125I]TBZ-AIPP derivatized a segment of rVMAT2 between Gly-408 and Cys-431 in TMD10 and 11. These data implicate juxtaposition of TMD1 and 10/11.

Affinity Labels↗
PubMed1987

Synapsin I is associated with cholinergic nerve terminals in the electric organs of Torpedo, Electrophorus, and Malapterurus and copurifies with Torpedo synaptic vesicles.

Using an affinity-purified monospecific polyclonal antibody against bovine brain synapsin I, the distribution of antigenically related proteins was investigated in the electric organs of the three strongly electric fish Torpedo marmorata, Electrophorus electricus, Malapterurus electricus and in the rat diaphragm. On application of indirect fluorescein isothiocyanate-immunofluorescence and using alpha-bungarotoxin for identification of synaptic sites, intense and very selective staining of nerve terminals was found in all of these tissues. Immunotransfer blots of tissue homogenates revealed specific bands whose molecular weights are similar to those of synapsin Ia and synapsin Ib. Moreover, synapsin I-like proteins are still attached to the synaptic vesicles that were isolated in isotonic glycine solution from Torpedo electric organ by density gradient centrifugation and chromatography on Sephacryl-1000. Our results suggest that synapsin I-like proteins are also associated with cholinergic synaptic vesicles of electric organs and that the electric organ may be an ideal source for studying further the functional and molecular properties of synapsin.

Animals↗
PubMedDate not supplied

Genetic and molecular analysis of synaptic vesicle recycling in Drosophila.

Following exocytosis, one of the major presynaptic events is replenishing synaptic vesicles (SVs) to ensure the possibility of continuous synaptic transmission. The nerve terminal is thought to recycle SVs through clathrin-mediated endocytosis and by a clathrin-independent pathway called "kiss and run". This review highlights the use of the genetic model organism, the fruit fly (Drosophila melanogaster), in dissecting the molecular mechanisms of clathrin-mediated endocytosis in recycling SVs at neuromuscular junctions (NMJs). Analyses of endocytotic mutants in Drosophila indicate that clathrin-mediated endocytosis may be essential for SV recycling, including a putative fast recycling mechanism uncovered recently. Further, a rather complex picture begins to emerge suggesting that clathrin-mediated endocytosis involves several sequential steps mediated by a large number of proteins. Finally, these studies also reveal that SV proteins may be selectively retrieved into nascent SVs by clathrin accessory proteins and defects in protein retrieval have significant impacts on synaptic transmission. Following the completion of the Drosophila Genome Project and the development of gene targeting and RNAi approaches, genetic studies in Drosophila have become increasingly efficient. Hence, Drosophila is expected to continue to serve as an important model organism for studies of SV recycling.

Animals↗
PubMed1996

Dynamics of synaptic vesicles in cultured spinal cord neurons in relationship to synaptogenesis.

The dynamics of synaptic vesicles (SVs) during the development of presynaptic specializations in cultured Xenopus spinal cord neurons was studied with the fluorescent vesicular probe FM1-43. In naive neurons that have not contacted synaptic targets, packets of SVs are distributed along the entire neurite and are quite mobile. The interaction with the synaptic target, such as a muscle cell or a latex bead coated with basic fibroblast growth factor, results in the localization and immobilization of SV packets at the contact site. Depolarization resulted in exocytosis of SVs in both naive and target-contacted neurites. Okadaic acid, a phosphatase inhibitor, caused a dispersal of SV packets in both naive and target-contacted neurites. Thus, prior to target contact, SVs are already organized into packets capable of release and recycling by a phosphorylation-dependent mechanism. Target interaction then recruits and anchors these functional SV packets into forming the presynaptic nerve terminal. With fluorescent phalloidin as a probe, F-actin was found to colocalize with SV clusters at bead-neurite contacts. Although okadaic acid caused a dispersal of SVs at the beads, F-actin localization there was relatively resistant to this drug treatment. This suggests that SVs become localized at the target by interacting with an actin-based cytoskeletal specialization in a phosphorylation-sensitive manner. The induction of this cytoskeletal specialization by the target may be an early event in presynaptic differentiation.

Actins↗
PubMed1995

The neuron-specific kinesin superfamily protein KIF1A is a unique monomeric motor for anterograde axonal transport of synaptic vesicle precursors.

Axonal transport has been intensively examined as a good model for studying the mechanism of organelle transport in cells, but it is still unclear how different types of membrane organelles are transported through the nerve axon. To elucidate the function of this mechanism, we have cloned KIF1A, a novel neuron-specific kinesin superfamily motor that was discovered to be a monomeric, globular molecule and that had the fastest reported anterograde motor activity (1.2 microns/s). To identify its cargo, membranous organelles were isolated from the axon. KIF1A was associated with organelles that contained synaptic vesicle proteins such as synaptotagmin, synaptophysin, and Rab3A. However, this organelle did not contain SV2, another synaptic vesicle protein, nor did it contain presynaptic membrane proteins, such as syntaxin 1A or SNAP-25, or other known anterograde motor proteins, such as kinesin and KIF3. Thus, we suggest that the membrane proteins are sorted into different classes of transport organelles in the cell body and are transported by their specific motor proteins through the axon.

Amino Acid Sequence↗
PubMed1999

Inhibitors of myosin light chain kinase block synaptic vesicle pool mobilization during action potential firing.

During repetitive action potential firing the maintenance of synaptic transmission relies on a continued supply of synaptic vesicles for fusion with the presynaptic plasma membrane. The mechanism of transport by which vesicles are delivered to the site of fusion from a reserve pool is unknown, as are the biochemical pathways linking intracellular Ca2+ elevation with vesicle mobilization. Here, using the fluorescent tracer FM1-43 in hippocampal synaptic terminals, I show that inhibitors of myosin light chain kinase can block mobilization of the reserve pool and not the immediately releasable pool.

Action Potentials↗
PubMed1983

A synaptic vesicle antigen is restricted to the junctional region of the presynaptic plasma membrane.

The plasma membrane of electric organ nerve terminals has two domains that can be distinguished by monoclonal antibodies. A library of 111 mouse monoclonal antibodies raised to nerve terminals from Torpedo californica contains 4 antibodies that bind specifically to the outside of intact synaptosomes. The distribution of the binding sites of these monoclonal antibodies on the outside of intact nerve terminals was examined by immunofluorescence and immunoelectron microscopy. The binding sites of 3 (tor23, 25, and 132) are distributed uniformly over nerve trunks and fine terminal branches. The binding site of the fourth (tor70) is restricted to synaptic junctional regions. This antibody, but not the other 3, recognizes a major component of synaptic vesicles, a proteoglycan associated with the inner surface of the vesicle membrane. The difference in the pattern of binding of these monoclonal antibodies suggests that the region of the plasma membrane containing active zones is antigenically distinguishable from other nerve terminal plasma membrane. We suggest that the antigen recognized by tor70 is externalized by exocytosis of synaptic vesicles while other plasma antigens take a different route to the surface. The unexpected observation that the vesicle antigen remains on the surface after exocytosis and is prevented from diffusion from the synaptic junctional region would be consistent with an interaction between the vesicle proteoglycan and elements of the synaptic cleft.

Animals↗