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PubMed1980

ATP-dependent calcium uptake by cholinergic synaptic vesicles isolated from Torpedo electric organ.

Cholinergic synaptic vesicles were purified from Torpedo electric organ to near morphological homogeneity. They were isolated in a K+ environment. A method is described for the preparation of concentrated synaptic vesicles that allows uptake studies by conventional techniques. An ATP-Mg-dependent calcium uptake associated with synaptic vesicles is characterized. The uptake system transports calcium against a high concentration gradient. The maximum accumulation rate is obtained for the calcium, Mg++ and ATP concentrations likely to be found in the nerve terminal cytoplasm. It is suggested that synaptic vesicles are implicated in the removal of the calcium entering the nerve terminal during synaptic activity.

Acetylcholine↗

PubMed2003

Synaptotagmin 7 splice variants differentially regulate synaptic vesicle recycling.

The speed of synaptic vesicle recycling determines the efficacy of neurotransmission during repetitive stimulation. Synaptotagmins are synaptic C(2)-domain proteins that are involved in exocytosis, but have also been linked to endocytosis. We now demonstrate that upon expression in transfected neurons, a short splice variant of synaptotagmin 7 that lacks C(2)-domains accelerates endocytic recycling of synaptic vesicles, whereas a longer splice variant that contains C(2)-domains decelerates recycling. These results suggest that alternative splicing of synaptotagmin 7 acts as a molecular switch, which targets vesicles to fast and slow recycling pathways.

Alternative Splicing↗

PubMed2004

Cycling of synaptic vesicles: how far? How fast!

Synaptic transmission is based on the regulated exocytotic fusion of synaptic vesicles filled with neurotransmitter. In order to sustain neurotransmitter release, these vesicles need to be recycled locally. Recent data suggest that two tracks for the cycling of synaptic vesicles coexist: a slow track in which vesicles fuse completely with the presynaptic plasma membrane, followed by clathrin-mediated recycling of the vesicular components, and a fast track that may correspond to the transient opening and closing of a fusion pore. In this review, we attempt to provide an overview of the components involved in both tracks of vesicle cycling, as well as to identify possible mechanistic links between these two pathways.

Animals↗

PubMed1983

A highly antigenic proteoglycan-like component of cholinergic synaptic vesicles.

A monoclonal antibody, tor70, recognizes an antigenic determinant on the inside surface of synaptic vesicles, purified from the electric organ of Narcine brasiliensis. The antigenic determinant appears to be unique to vesicles since it co-purifies with vesicle content and is blocked by an antiserum specific for synaptic vesicle antigens. Immunoblotting of vesicle proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the antigen has a low heterogeneous electrophoretic mobility and corresponds to a major protein component of pure synaptic vesicles. Synaptic vesicles contain a proteoglycan-like material since proteolytic digestion yields a ruthenium red-binding material that migrates during electrophoresis with a mammalian heparin standard. The only major vesicle component with which the proteoglycan-like material co-elutes during chromatography on Sepharose 6B is the material recognized by tor70. The antigen adsorbs specifically to beads coated with the lectin wheat germ agglutinin. Isolation of the tor70 antigen by velocity sedimentation in sodium dodecyl sulfate-sucrose gradients shows it to contain glucosamine (0.75 nmol/microgram of protein) and uronic acid but no galactosamine. Earlier work has shown that specific antiserum to pure synaptic vesicles could be used to identify nerve terminals, quantitate vesicle components, purify membranes, and monitor exocytosis. We now know that one of the components recognized by the antiserum is a molecule with properties of a proteoglycan, attached to the inside surface of vesicle membranes.

Animals↗

PubMed2004

Syntaxin-1A is excluded from recycling synaptic vesicles at nerve terminals.

At presynaptic terminals, intermixing during cycles of exocytosis and endocytosis challenges the molecular identity of the plasma and synaptic vesicle membranes. Although synaptic vesicle components are retrieved during recycling, the extent to which plasma membrane proteins enter the synaptic vesicle recycling pathway has not been examined. The target-SNARE (N-ethylmaleimide-sensitive factor attachment protein receptor) syntaxin-1 was shown previously to be present on putative synaptic vesicular membranes (Koh et al., 1993; Walch-Solimena et al., 1995; Kretzschmar et al., 1996), suggesting that syntaxin may cycle between the synaptic vesicle pool and the cell surface (Walch-Solimena et al., 1995). This implies that the molecular identity of the two membranes is not maintained during synaptic activity. Because the main role of syntaxin-1 is as a target-SNARE for vesicle fusion, appearance on synaptic vesicles could lead to futile interactions with vesicle-SNARE proteins. We investigated whether the subcellular localization of syntaxin-1A, tagged with the pH-sensitive fluorescent tag pHluorin, is regulated during neurotransmission using laser-scanning microscopy. We report here that syntaxin-1A is predominantly localized to the plasma membrane, with a small proportion present in an intracellular compartment with a lumenal pH consistent with synaptic vesicles. However, the internal fraction of syntaxin-1A is excluded from synaptic vesicles that undergo action potential-dependent recycling. These data indicate that the molecular identity of opposing exocytotic membranes is preserved by the sorting of syntaxin-1A from recycling synaptic vesicles.

Action Potentials↗

PubMed2001

Cycling of synaptic vesicles: how far? How fast!

Animals↗

PubMed1992

Members of the VAMP family of synaptic vesicle proteins are components of glucose transporter-containing vesicles from rat adipocytes.

Existing data support the hypothesis that insulin triggers the exocytosis of small vesicles containing the GluT4 isoform of the glucose transporter. The data also suggest that these vesicles reform through endocytosis of GluT4. These processes resemble those described for synaptic vesicles after depolarization of nerve cells. To determine whether GluT4 vesicles are related to synaptic vesicles, rat adipocyte low density microsomes (LDM), which are rich in GluT4 vesicles, were screened for the synaptic vesicle proteins synaptotagmin, synaptophysin, SV2, p29, rab3, and VAMP (synaptobrevin) by immunoblotting. Two polypeptides that reacted with antibodies against the VAMPs were identified, one with the same apparent size as the two isoforms of VAMP in the brain (18 kDa) and one that was slightly smaller (17 kDa). These members of the VAMP family were highly enriched in GluT4 vesicles isolated by immunoadsorption and translocated from the LDM to the plasma membrane in response to insulin. With the exception of rab3, which was observed in the LDM but was not localized in the GluT4 vesicles, the other synaptic vesicle proteins were not detected. The presence of the VAMPs in both GluT4 and synaptic vesicles suggests that the genesis and/or exocytosis of these two types of vesicles involve shared processes.

Adipose Tissue↗

PubMed1980

Tubulin: an integral protein of mammalian synaptic vesicle membranes.

The major protein in isolated synaptic vesicles from bovine cerebral cortex has been compared to tubulin by sodium dodecyl sulphate-urea polyacrylamide gel electrophoresis, by two-dimensional gel electrophoresis, and by peptide mapping following limited proteolysis of the protein by Staphylococcus aureus protease. The results establish in purified synaptic vesicles the presence of tubulin, which is composed of the alpha and beta subunits. In the presence of ethyleneglycolbis)aminoethyl ether)-N,N'-tetraacetic acid (EGTA) or magnesium in the isolation buffers, the synaptic vesicles contained mainly the alpha-tubulin whereas the beta subunit was less abundant. Similarly, synaptosomal plasma membranes that were prepared in the presence of EGTA also contained more of alpha-tubulin than of the beta subunit. Non-ionic detergents such as Triton X-100 or Nonidet P-40 failed to solubilize the tubulin from the synaptic vesicles. Ionic detergents such as deoxycholate and sodium dodecyl sulphate solubilized all the vesicle proteins, including tubulin. The results indicate that alpha-tubulin is an integral vesicle membrane protein, whereas most of the beta subunit is peripherally attached and can be easily dissociated from the vesicle membrane with EGTA.

Animals↗

PubMed2000

Distinction between Ca(2+) pump and Ca(2+)/H(+) antiport activities in synaptic vesicles of sheep brain cortex.

Synaptic vesicles, isolated from a sheep brain cortex, accumulate Ca(2+) in a manner that depends on the pH and pCa values. In the presence of 100 microM CaCl(2), most of the Ca(2+) taken up by the vesicles was vanadate-inhibited (86%) at pH 7.4, whereas at pH 8.5, part of the Ca(2+) accumulated (36%) was DeltapH-dependent (bafilomycin and CCCP inhibited) and part was insensitive to those drugs (31%). We also observed that both vanadate-sensitive and bafilomycin-sensitive Ca(2+) accumulations were completely released by the Ca(2+) ionophore, ionomycin, and that these processes work with high (K(0.5)=0.6 microM) and low (K(0.5)=217 microM) affinity for Ca(2+), respectively. The DeltapH-dependent Ca(2+) transport appears to be largely operative at Ca(2+) concentrations (>100 microM) which completely inhibited the vanadate-sensitive Ca(2+) uptake. These Ca(2+) effects on the Ca(2+) accumulation were well correlated with those observed on the vanadate-inhibited Ca(2+)-ATPase and bafilomycin-inhibited H(+)-ATPase, respectively. The Ca(2+)-ATPase activity reached a maximum at about 25 microM (pH 7.4) and sharply declined at higher Ca(2+) concentrations. In contrast, Ca(2+) had a significant stimulatory effect on the H(+)-ATPase between 250 and 500 microM Ca(2+) concentration. Furthermore, we found that DeltapH-sensitive Ca(2+) transport was associated with proton release from the vesicles. About 21% of the maximal proton gradient was dissipated by addition of 607.7 microM CaCl(2) to the reaction medium and, if CaCl(2) was present before the proton accumulation, lower pH gradients were reached. Both vanadate-inhibited and bafilomycin-inhibited systems transported Ca(2+) into the same vesicle pool of our preparation, suggesting that they belong to the same cellular compartment. These results indicate that synaptic vesicles of the sheep brain cortex contain two distinct mechanisms of Ca(2+) transport: a high Ca(2+) affinity, proton gradient-independent Ca(2+) pump that has an optimal activity at pH 7.4, and a low Ca(2+) affinity, proton gradient-dependent Ca(2+)/H(+) antiport that works maximally at pH 8.5.

Adenosine Triphosphate↗

PubMed1980

Enhancement of synaptic vesicle attachment to the plasma membrane fraction by copper.

Synaptic vesicles from rat brain were labeled with 125I, and the association of the vesicles with other subcellular components of brain was examined using a centrifugation assay. Copper at micromolar concentrations enhances the binding of the vesicles to the synaptic membrane as well as other fractions. Magnesium, Ca2+, and calmodulin with Ca2+ are ineffective. There is virtually no binding of synaptic vesicles to the microtuble fraction and only a slight enhancement with Cu2+. These findings support the hypothesis that Cu may serve as a bridge between synaptic vesicles and the plasma membrane.

Animals↗

PubMed1999

Visualization of synaptic specializations in live C. elegans with synaptic vesicle protein-GFP fusions.

Synaptic specializations are difficult to visualize at the light microscope level in living preparations. To circumvent this problem, synaptic vesicle proteins were fused to green fluorescent protein (GFP) and expressed in C. elegans neurons. C. elegans synaptobrevin-GFP and synaptogyrin-GFP fusion proteins were observed to target to synaptic sites. This localization allowed the visualization of synaptic specializations in living animals with light microscopy. Restricted expression of synaptobrevin-GFP fusions in subsets of neurons enables the visualization of individual presynaptic varicosities. The cell biology underlying the sorting of synaptic vesicle proteins, trafficking of vesicles to terminals, and the development of presynaptic specializations is now more amenable to forward genetic analysis using these synaptic markers.

Amino Acid Sequence↗

PubMed1982

Identification of a heparan sulphate-containing proteoglycan as a specific core component of cholinergic synaptic vesicles from Torpedo marmorata.

Cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata were found to contain a proteoglycan in their core. The glycosaminoglycan part co-migrates upon thin layer electrophoresis with heparan sulphate and shows a chemical composition characteristic for this carbohydrate. [35S]Sulphate injected into the electric lobes of Torpedo, which contain the perikarya of the electromotor neurons innervating the electric organs, appeared 48 h later in covalently bound form in the synaptic vesicle fraction. The radiolabel had been incorporated into the vesicular heparan sulphate. Upon SDS-polyacrylamide gel electrophoresis fluorography of labelled vesicles a major and a minor band are formed both migrating above a protein standard of mol. wt. 200 000. Similarly, a major peak in the void volume and a minor peak in the included volume are seen upon gel filtration in Ultrogel AcA 34 in the presence of SDS. We interpret the minor fraction as being formed by the loss of glycosaminoglycan from the major fraction. The proteoglycan is located inside the vesicle since antibodies directed against it form immunoprecipitates only with vesicles lysed by detergent treatment. The experiments show that it is possible to label a synaptic organelle specifically by axonal transport.

Acetylcholine↗

PubMed1999

A phospho-switch controls the dynamic association of synapsins with synaptic vesicles.

Synapsins constitute a family of synaptic vesicle proteins essential for regulating neurotransmitter release. Only two domains are conserved in all synapsins: a short N-terminal A domain with a single phosphorylation site for cAMP-dependent protein kinase (PKA) and CaM Kinase I, and a large central C domain that binds ATP and may be enzymatic. We now demonstrate that synapsin phosphorylation in the A domain, at the only phosphorylation site shared by all synapsins, dissociates synapsins from synaptic vesicles. Furthermore, we show that the A domain binds phospholipids and is inhibited by phosphorylation. Our results suggest a novel mechanism by which proteins reversibly bind to membranes using a phosphorylation-dependent phospholipid-binding domain. The dynamic association of synapsins with synaptic vesicles correlates with their role in activity-dependent plasticity.

Amino Acid Sequence↗

PubMed2005

Developmental change in the calcium sensor for synaptic vesicle endocytosis in central nerve terminals.

Synaptic vesicle endocytosis is stimulated by calcium influx in mature central nerve terminals via activation of the calcium-dependent protein phosphatase, calcineurin. However, in different neuronal preparations calcineurin activity is either inhibitory, stimulatory or irrelevant to the process. We addressed this inconsistency by investigating the requirement for calcineurin activity in synaptic vesicle endocytosis during development, using vesicle recycling assays in isolated nerve terminals. We show that endocytosis occurs independently of calcineurin activity in immature nerve terminals, and that a calcineurin requirement develops 2-4 weeks after birth. Calcineurin-independent endocytosis is not due to the absence of calcineurin activity, since calcineurin is present in immature nerve terminals and its substrate, dynamin I, is dephosphorylated on depolarization. Calcineurin-independent endocytosis is calcium-dependent, since substitution of the divalent cation, barium, inhibits the process. Finally, we demonstrated that in primary neuronal cultures derived from neonatal rats, endocytosis that was initially calcineurin-independent developed a calcineurin requirement on maturation in culture. Our data account for the apparent inconsistencies regarding the role of calcineurin in synaptic vesicle endocytosis, and we propose that an unidentified calcium sensor exists to couple calcium influx to endocytosis in immature nerve terminals.

Age Factors↗

PubMed1988

Protein tyrosine phosphorylation in synaptic vesicles.

Protein tyrosine phosphorylation in purified synaptic vesicles from rat forebrain has been studied in the presence of Mn2+ and orthovanadate. High levels of endogenous protein tyrosine phosphorylation were observed. Four major phosphoproteins, with apparent molecular masses of 105, 94, 38, and 30 kDa, were shown to contain phosphotyrosine. The 38-kDa phosphoprotein was identified as synaptophysin (p38), a well-characterized integral membrane protein of synaptic vesicles. The three other phosphotyrosine-containing proteins distributed in the same manner as synaptophysin in all subcellular fractions. Like synaptophysin, the two high molecular weight phosphotyrosine proteins (105 and 94 kDa) were found to be glycoproteins by lectin chromatography. Tyrosine phosphorylation of synaptophysin was an intravesicular reaction and reached 50% of maximal level within 3 min. Triton X-100, a nonionic detergent, inhibited tyrosine phosphorylation of endogenous protein substrates but not the phosphorylation of an exogenous substrate, poly(Glu80,-Tyr20). Tyrosine phosphorylation of synaptophysin was also demonstrated in synaptosomes, indicating that tyrosine phosphorylation of synaptic vesicle proteins occurs in intact nerve terminals.

Animals↗

PubMed1998

Ca2+-H+ antiport activity in synaptic vesicles isolated from sheep brain cortex.

Synaptic vesicles isolated from sheep brain cortex exhibit an ATP-dependent Ca2+ accumulation that is inhibited by the protonophore uncoupler carbonyl cyanide m-chorophenylhydrazone (CCCP) and completely released by the Ca2+ ionophore ionomycin. This transport activity was sensitive to the V-type ATPase inhibitor, bafilomycin, but not to the P-type ATPase inhibitor, vanadate. We also observed that the proton gradient, established across the synaptic vesicle membranes in the presence of ATP, is partially dissipated by the addition of Ca2+ (100-860 microM) in correlation to an increase of ATP hydrolysis by the H+-pumping ATPase. In contrast, the activity of the H+-ATPase, measured under uncoupling conditions (presence of CCCP), appears to be unaltered by the calcium ion. The Ca2+-induced H+ release visualized by fluorescence quenching of acridine orange correlates well with the Ca2+ uptake determined isotopically. These results indicate that synaptic vesicles accumulate Ca2+, via a low affinity Ca2+-H+ antiport system energized by the protonmotive force originated from the H+-pumping ATPase activity.

Adenosine Triphosphate↗

PubMed1999

Abnormal neurotransmission in mice lacking synaptic vesicle protein 2A (SV2A).

Synaptic vesicle protein 2 (SV2) is a membrane glycoprotein common to all synaptic and endocrine vesicles. Unlike many proteins involved in synaptic exocytosis, SV2 has no homolog in yeast, indicating that it performs a function unique to secretion in higher eukaryotes. Although the structure and protein interactions of SV2 suggest multiple possible functions, its role in synaptic events remains unknown. To explore the function of SV2 in an in vivo context, we generated mice that do not express the primary SV2 isoform, SV2A, by using targeted gene disruption. Animals homozygous for the SV2A gene disruption appear normal at birth. However, they fail to grow, experience severe seizures, and die within 3 weeks, suggesting multiple neural and endocrine deficits. Electrophysiological studies of spontaneous inhibitory neurotransmission in the CA3 region of the hippocampus revealed that loss of SV2A leads to a reduction in action potential-dependent gamma-aminobutyric acid (GABA)ergic neurotransmission. In contrast, action potential-independent neurotransmission was normal. Analyses of synapse ultrastructure suggest that altered neurotransmission is not caused by changes in synapse density or morphology. These findings demonstrate that SV2A is an essential protein and implicate it in the control of exocytosis.

Animals↗

PubMed1999

Neurotransmitter secretion along growing nerve processes: comparison with synaptic vesicle exocytosis.

In mature neurons, synaptic vesicles continuously recycle within the presynaptic nerve terminal. In developing axons which are free of contact with a postsynaptic target, constitutive membrane recycling is not localized to the nerve terminal; instead, plasma membrane components undergo cycles of exoendocytosis throughout the whole axonal surface (Matteoli et al., 1992; Kraszewski et al., 1995). Moreover, in growing Xenopus spinal cord neurons in culture, acetylcholine (ACh) is spontaneously secreted in the quantal fashion along the axonal shaft (Evers et al., 1989; Antonov et al., 1998). Here we demonstrate that in Xenopus neurons ACh secretion is mediated by vesicles which recycle locally within the axon. Similar to neurotransmitter release at the presynaptic nerve terminal, ACh secretion along the axon could be elicited by the action potential or by hypertonic solutions. We found that the parameters of neurotransmitter secretion at the nerve terminal and at the middle axon were strikingly similar. These results lead us to conclude that, as in the case of the presynaptic nerve terminal, synaptic vesicles involved in neurotransmitter release along the axon contain a complement of proteins for vesicle docking and Ca2+-dependent fusion. Taken together, our results support the idea that, in developing axons, the rudimentary machinery for quantal neurotransmitter secretion is distributed throughout the whole axonal surface. Maturation of this machinery in the process of synaptic development would improve the fidelity of synaptic transmission during high-frequency stimulation of the presynaptic cell.

ADP-Ribosylation Factors↗