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PubMed1983

Asymmetry of lipid organization in cholinergic synaptic vesicle membranes.

The lipid composition of purified Torpedo cholinergic synaptic vesicles was determined and their distribution between the inner and outer leaflets of the vesicular membrane was investigated. The vesicles contain cholesterol and phospholipids at a molar ratio of 0.63. The vesicular phospholipids are (mol% of total phospholipids): phosphatidylcholine (40.9); phosphatidylethanolamine (24.6); plasmenylethanolamine (11.5); sphingomyelin (12); phosphatidylserine (7.3); phosphatidylinositol (3.7). The asymmetry of the synaptic vesicle membranes was investigated by two independent approaches: (a) determining accessibility of the amino lipids to the chemical label trinitrobenzenesulphonic acid (TNBS); (b) determining accessibility of the vesicular glycerophospholipids to phospholipase C (Bacillus cereus). TNBS was found to render the vesicles leaky and thus cannot be used reliably to determine the asymmetry of Torpedo synaptic vesicle membranes. Incubation of the vesicles with phospholipase C (Bacillus cereus) results in biphasic hydrolysis of the vesicular glycerophospholipids. About 45% of the phospholipids are hydrolysed in less than 1 min, during which no vesicular acetylcholine is released. In the second phase, the hydrolysis of the phospholipids slows down markedly and is accompanied by loss of all the vesicular acetylcholine. These findings suggest that the lipids hydrolysed during the first phase are those comprising the outer leaflet. Analysis of the results thus obtained indicate that the vesicular membrane is asymmetric: all the phosphatidylinositol, 77% of the phosphatidylethanolamine, 47% of the plasmenylethanolamine and 58% of the phosphatidylcholine were found to reside in the outer leaflet. Since phosphatidylserine is a poor substrate for phospholipase C (B. cereus), its distribution between the two leaflets of the synaptic vesicle membrane is only suggestive.

Acetylcholine↗

PubMed2002

Synaptic vesicle endocytosis: the races, places, and molecular faces.

The classical experiments on synaptic vesicle recycling in the 1970s by Heuser and Reese, Ceccarelli, and their colleagues raised opposing theories regarding the speed, mechanisms, and locations of membrane retrieval at the synapse. The Heuser and Reese experiments supported a model in which synaptic vesicle recycling is mediated by the formation of coated vesicles, is relatively slow, and occurs distally from active zones, the sites of neurotransmitter release. Because heavy levels of stimulation were needed to visualize the coated vesicles, Ceccarelli's experiments argued that synaptic vesicle recycling does not require the formation of coated vesicles, is relatively fast, and occurs directly at the active zone in a "kiss-and-run" reversal of exocytosis under more physiological conditions. For the next thirty years, these models have provided the foundation for studies of the rates, locations, and molecular elements involved in synaptic vesicle endocytosis. Here, we describe the evidence supporting each model and argue that the coated vesicle pathway is the most predominant physiological mechanism for recycling synaptic vesicles.

Animals↗

PubMed1993

The SV2 protein of synaptic vesicles is a keratan sulfate proteoglycan.

We have determined that synaptic vesicles contain a vesicle-specific keratan sulfate integral membrane proteoglycan. This is a major proteoglycan in electric organ synaptic vesicles. It exists in two forms on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, i.e., the L form, which migrates like a protein with an M(r) of 100,000, and the H form, with a lower mobility that migrates with an M(r) of approximately 250,000. Both forms contain SV2, an epitope located on the cytoplasmic side of the vesicle membrane. In addition to electric organ, we have analyzed the SV2 proteoglycan in vesicle fractions from two other sources, electric fish brain and rat brain. Both the H and L forms of SV2 are present in these vesicles and all are keratan sulfate proteoglycans. Unlike previously studied synaptic vesicle proteins, this proteoglycan contains a marker specific for a single group of neurons. This marker is an antigenically unique keratan sulfate side chain that is specific for the cells innervating the electric organ; it is not found on the synaptic vesicle keratan sulfate proteoglycan in other neurons of the electric fish brain.

Animals↗

PubMed1992

The synaptic vesicle protein SV2 is a novel type of transmembrane transporter.

The primary function of synaptic vesicles is to store and release neurotransmitter. Synaptic vesicles are locally recycled following exocytosis and rapidly refilled with neurotransmitter from the cytoplasm by a process that depends on the electrochemical gradient generated by a proton pump. Little is known about the molecules that import neurotransmitter into synaptic vesicles. We report here that the sequence of the synaptic vesicle protein SV2 identifies this protein as a novel type of transmembrane transporter. The deduced amino acid sequence of SV2 contains two sets of six predicted transmembrane domains: the six most N-terminal transmembrane domains are highly homologous to a subfamily of transporters that includes the human glucose transporter, while the six most C-terminal domains are homologous to the plasma membrane transporters for neurotransmitters. We propose that SV2 mediates transport of neurotransmitters into synaptic vesicles.

Amino Acid Sequence↗

PubMed1994

A role for synaptic vesicles in non-neuronal cells: clues from pancreatic beta cells and from chromaffin cells.

Synaptic vesicles, vesicular carriers highly specialized for the secretion of fast nonpeptide neurotransmitters, until recently were considered neuron-specific organelles. The identification and characterization of several of the most abundant synaptic vesicle proteins have led to the identification in peptide-secreting endocrine cells of a class of microvesicles, referred to as synaptic-like microvesicles (SLMVs), which are similar to synaptic vesicles in membrane composition, biogenesis, and life cycle. Studies of pancreatic beta cells and of a chromaffin cell-derived cell line (PC12 cells) have suggested that SLMVs, like synaptic vesicles, store and secrete neurotransmitter-like substances that act as paracrine/endocrine signaling molecules. SLMVs of pancreatic beta cells store GABA; SLMVs of PC12 cells contain acetylcholine. Both synaptic vesicles and SLMVs are recycling organelles that represent a specialized subcompartment of the receptor-mediated recycling pathway of all cells. Recently, homologues of membrane proteins of synaptic vesicles and SLMVs have been identified in the endocytic/recycling compartment of non-neuronal, nonendocrine cells. Tracing the evolution of synaptic vesicles from vesicular carriers present in all cells will help to advance our understanding of neurotransmitter release as well as of molecular mechanisms of vesicular traffic.

Animals↗

PubMed1981

[The effect of conditioning on the ultrastructural distribution of synaptic vesicles in the hippocampus of the rat].

In three months old male Wistar-rats the influence of conditioning (brightness discrimination reaction) on the number and distribution of vesicles of the synapses in the stratum radiatum (CA 3) of the hippocampus were investigated. A total of 15 rats (5 trained rats, 5 passive and 5 active controls) were studied by the aid of electron microscopic and morphometric methods. The number of synaptic vesicles was evaluated for the passive controls by 27 vesicles per synaptic area. In the CA 3-region 70 minutes after training the calculation of the number of synaptic vesicles revealed a significant decrease in comparison with passive and active controls (16 and 32 vesicles per synaptic area, respectively; significant difference at p less than 0.05). The volume of the synaptic vesicles was reduced for the trained rats by 20-25% in comparison with controls. The morphological results after training could be correlated with the changes of the Ach fractions, and seem to be of great significance at the initial stages of memory formation.

Acetylcholine↗

PubMed2003

The molecular machinery of synaptic vesicle exocytosis.

At the synapse, neurotransmitters are released via Ca(2+)-triggered exocytotic fusion of synaptic vesicles with the presynaptic plasma membrane. Synaptic vesicle exocytosis seems to share many basic principles and homologous proteins with other membrane fusion events. Conserved components of the general fusion machinery that participate in synaptic vesicle exocytosis include soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), ATPase N-ethylmaleimide-sensitive factor, Munc18/nSec1, Rab3 GTPase, and the exocyst proteins. In addition, synaptic vesicle exocytosis uses a set of unique components, such as synaptotagmin, complexin, Munc13, and RIM, to meet the special needs of fast Ca(2+)-triggered neurotransmitter release. This review summarizes present knowledge about the molecular mechanisms by which these components mediate and/or regulate synaptic vesicle exocytosis.

Animals↗

PubMed1994

Okadaic acid disrupts clusters of synaptic vesicles in frog motor nerve terminals.

The fluorophore FM1-43 appears to stain membranes of recycled synaptic vesicles. We used FM1-43 to study mechanisms of synaptic vesicle clustering and mobilization in living frog motor nerve terminals. FM1-43 staining of these terminals produces a linear series of fluorescent spots, each spot marking the cluster of several hundred synaptic vesicles at an active zone. Most agents we tested did not affect staining, but the phosphatase inhibitor okadaic acid (OA) disrupted the fluorescent spots, causing dye to spread throughout the terminal. Consistent with this, electron microscopy showed that vesicle clusters were disrupted by OA treatment. However, dye did not spread passively to a uniform spatial distribution. Instead, time lapse movies showed clear evidence of active dye movements, as if synaptic vesicles were being swept along by an active translocation mechanism. Large dye accumulations sometimes occurred at sites of Schwann cell nuclei. These effects of OA were not significantly affected by pretreatment with colchicine or cytochalasin D. Electrophysiological recordings showed that OA treatment reduced the amount of acetylcholine released in response to nerve stimulation. The results suggest that an increased level of protein phosphorylation induced by OA treatment mobilizes synaptic vesicles and unmasks a powerful vesicle translocation mechanism, which may function normally to distribute synaptic vesicles between active zones.

Animals↗

PubMed1996

Phosphocreatine-dependent glutamate uptake by synaptic vesicles. A comparison with atp-dependent glutamate uptake.

ATP-dependent uptake of glutamate into synaptic vesicles has been well documented. Stimulation of glutamate uptake into synaptic vesicles by other high-energy phosphates has not been described. In this paper, we examine the stimulation of phosphocreatine (PCr)-induced glutamate uptake and determine whether this stimulation is secondary to conversion of PCr to ATP. We found the following. 1) PCr stimulates glutamate uptake into synaptic vesicles in the absence of added ATP. 2) At a glutamate concentration of 50 microM, no concentration of added ATP could produce the degree of stimulation seen in the presence of PCr. 3) 0.5 mM iodoacetamide completely inhibits synaptic vesicle creatine kinase activity but does not inhibit PCr-stimulated glutamate uptake. 4) PCr-dependent glutamate uptake, unlike ATP-dependent uptake, is not magnesium- or chloride-dependent. 5) 0.5 mM N-ethylmaleimide, a selective H+-ATPase inhibitor, completely inhibits ATP-dependent glutamate uptake but only slightly inhibits PCr-dependent glutamate uptake. 6) PCr-dependent glutamate uptake is sensitive to valinomycin, a K+/H+ translocator, whereas the ATP-dependent uptake is not. Therefore, it appears that in addition to the well-known ATP-dependent glutamate uptake system, there is a previously unreported PCr-dependent glutamate uptake system in synaptic vesicles. The total glutamate uptake by synaptic vesicles is likely the sum of both ATP- and PCr-dependent glutamate uptake.

Adenosine Triphosphate↗

PubMed2007

Fast synaptic vesicle reuse slows the rate of synaptic depression in the CA1 region of hippocampus.

During short-term synaptic depression, neurotransmission rapidly decreases in response to repetitive action potential firing. Here, by blocking the vacuolar ATPase, alkalinizing the extracellular pH, or exposing hippocampal slices to pH buffers, we impaired neurotransmitter refilling, and electrophysiologically tested the role of vesicle reuse in synaptic depression. Under all conditions, synapses onto hippocampal CA1 pyramidal cells showed faster depression with increasing stimulation frequencies. At 20 Hz, compromising neurotransmitter refilling increased depression within 300 ms reaching completion within 2 s, suggesting a minimal contribution of reserve vesicles to neurotransmission. In contrast, at 1 Hz, depression emerged gradually and became significant within 100 s. Moreover, the depression induced by pH buffers was reversible with a similar frequency dependence, suggesting that the frequency-dependent increase in depression was caused by impairment of rapid synaptic vesicle reuse. These results indicate that synaptic vesicle trafficking impacts the kinetics of short-term synaptic plasticity at an extremely rapid time scale.

Animals↗

PubMed1996

Isoform-specific, calcium-regulated interaction of the synaptic vesicle proteins SV2 and synaptotagmin.

The identification and functional characterization of proteins localized to synaptic vesicles has contributed significantly to our understanding of neurotransmission. Studies of synaptic vesicle protein interactions have both led to the identification of novel synaptic proteins and suggested hypotheses of protein function. Synaptic vesicle protein 2 (SV2), is an integral membrane glycoprotein present in all synaptic vesicles. There are two characterized isoforms, SV2A and SV2B. Despite their homology to transporter proteins, the function of the SV2s remains unknown. In an effort to determine SV2 function and identify cofactors required for SV2 activity, we examined the protein interactions of SV2 using a combination of cross-linking, immunoprecipitation, and recombinant protein affinity chromatography. We report that SV2 is part of a large protein complex that contains the synaptic vesicle protein synaptotagmin. The interaction between SV2 and synaptotagmin is direct, specific to SV2A, and inhibited by calcium with an EC50 of approximately 10 microM. Interaction is mediated by the cytoplasmic amino terminus of SV2A and the C2B domain of synaptotagmin. Our observations suggest a regulatory relationship between these two proteins.

Animals↗

PubMed1993

VAT-1 from Torpedo electric organ forms a high-molecular-mass protein complex within the synaptic vesicle membrane.

VAT-1 is an abundant 41-kDa protein from Torpedo cholinergic synaptic vesicles. Most of VAT-1 immunoreactivity (70%) is localized to the synaptic vesicle membrane while the rest (30%) copurifies with larger membranous fragments. VAT-1 forms a high-molecular-mass complex within the synaptic vesicle membrane. The Stokes radius of the VAT-1 complex is 4.85 nm and the sedimentation coefficient is 8.0 x 10(-13) S. Using these values, the calculated apparent mass of the VAT-1 complex is 176 kDa and the friction coefficient is consistent with that for a globular protein. Electrophoresis of solubilized synaptic vesicle proteins following cross-linking resulted in a 40-kDa ladder which was detected by VAT-1 antibodies. This is in accord with VAT-1 protein complex being composed primarily of VAT-1 subunits. The hydrodynamic characteristics of the VAT-1 protein complex suggest that it is composed of three or four VAT-1 subunits. Synaptophysin, an abundant component of Torpedo synaptic vesicle membranes, which has a similar apparent size as VAT-1, is not part of the VAT-1 protein complex. Interactions between the subunits within the protein complex do not depend on disulfide bonds or on lowering the ionic strength. However, partial dissociation of VAT-1 subunits from the complex occurs by chelating calcium ions.

Animals↗

PubMed1997

Targeting of the synaptic vesicle protein synaptobrevin in the axon of cultured hippocampal neurons: evidence for two distinct sorting steps.

Synaptic vesicles are concentrated in the distal axon, far from the site of protein synthesis. Integral membrane proteins destined for this organelle must therefore make complex targeting decisions. Short amino acid sequences have been shown to act as targeting signals directing proteins to a variety of intracellular locations. To identify synaptic vesicle targeting sequences and to follow the path that proteins travel en route to the synaptic vesicle, we have used a defective herpes virus amplicon expression system to study the targeting of a synaptobrevin-transferrin receptor (SB-TfR) chimera in cultured hippocampal neurons. Addition of the cytoplasmic domain of synaptobrevin onto human transferrin receptor was sufficient to retarget the transferrin receptor from the dendrites to presynaptic sites in the axon. At the synapse, the SB-TfR chimera did not localize to synaptic vesicles, but was instead found in an organelle with biochemical and functional characteristics of an endosome. The chimera recycled in parallel with synaptic vesicle proteins demonstrating that the nerve terminal efficiently sorts transmembrane proteins into different pathways. The synaptobrevin sequence that controls targeting to the presynaptic endosome was not localized to a single, 10- amino acid region of the molecule, indicating that this targeting signal may be encoded by a more distributed structural conformation. However, the chimera could be shifted to synaptic vesicles by deletion of amino acids 61-70 in synaptobrevin, suggesting that separate signals encode the localization of synaptobrevin to the synapse and to the synaptic vesicle.

Animals↗

PubMed1996

Ion channels from synaptic vesicle membrane fragments reconstituted into lipid bilayers.

Cholinergic synaptic vesicles were isolated from the electric organ of Torpedo californica. Vesicle membrane proteins were reconstituted into planar lipid bilayers by the nystatin/ergosterol fusion technique. After fusion, a variety of ion channels were observed. Here we identify four channels and describe two of them in detail. The two channels share a conductance of 13 pS. The first is anion selective and strongly voltage dependent, with a 50% open probability at membrane potentials of -15 mV. The second channel is slightly cation selective and voltage independent. It has a high open probability and a subconductance state. A third channel has a conductance of 4-7 pS, similar to the subconductance state of the second channel. This channel is fairly nonselective and has gating kinetics different from those of the cation channel. Finally, an approximately 10-pS, slightly cation selective channel was also observed. The data indicate that there are one or two copies of each of the above channels in every synaptic vesicle, for a total of six channels per vesicle. These observations confirm the existence of ion channels in synaptic vesicle membranes. It is hypothesized that these channels are involved in vesicle recycling and filling.

Animals↗

PubMed1988

Ganglioside composition of synaptic vesicles from Torpedo electric organ.

We have studied the ganglioside content and pattern of synaptic vesicles isolated from the electric organs of two species of Torpedinidae, Torpedo californica and Torpedo marmorata. The ganglioside concentrations were high relative to protein content (77 and 58 micrograms of N-acetylneuraminic acid/mg of protein, respectively), owing to the low protein-to-lipid ratio; however, they were also appreciable in relation to phospholipid (15.6 and 10.0 micrograms of N-acetylneuraminic acid/mg of phospholipid). The fact that a membrane fraction that separated from synaptic vesicles of T. californica on a controlled-pore glass-bead column and constituted the main potential source of contamination in this preparation had a lower ganglioside content and a different TLC pattern than synaptic vesicles indicated the relatively high purity of the latter. Most of the gangliosides from synaptic vesicles of both species migrated on TLC in the vicinity of standards with three or more sialic acids. Synaptosomes from T. marmorata had a higher lipid N-acetylneuraminic acid/phospholipid ratio and a different TLC pattern than synaptic vesicles. Considering these results and other data appearing recently in the literature, we suggest that reexamination of synaptic vesicles from mammalian brain for the possible presence of gangliosides is warranted.

Animals↗

PubMed1989

The synaptic vesicle membrane: origin, axonal distribution, protein components, exocytosis and recycling.

The paper discusses functional and molecular aspects of the synaptic vesicle membrane during its life cycle. The distribution of the synaptic vesicle membrane compartment in an entire cholinergic neuron is monitored using colloidal gold labelling and a monoclonal antibody against the synaptic vesicle membrane protein SV2. This provides new insights concerning vesicle origin and fate in the various compartments of the neuron. A new synaptic vesicle membrane protein (svp25) of Mr 25,000 with properties similar to synaptophysin as well as a synaptic vesicle binding phosphoprotein of the presynaptic membrane (Mr 92,000) likely to be involved in vesicle exocytosis are described. The membrane compartment recycled on induced transmitter release contains synaptic vesicle but not plasma membrane markers and encloses both newly synthesized transmitter and a sample of extracellular medium.

Animals↗

PubMed1995

Phosphorylation of VAMP/synaptobrevin in synaptic vesicles by endogenous protein kinases.

VAMP/synaptobrevin (SYB), an integral membrane protein of small synaptic vesicles, is specifically cleaved by tetanus neurotoxin and botulinum neurotoxins B, D, F, and G is thought to play an important role in the docking and/or fusion of synaptic vesicles with the presynaptic membrane. Potential phosphorylation sites for various kinases are present in SYB sequence. We have studied whether SYB is a substrate for protein kinases that are present in nerve terminals and known to modulate neurotransmitter release. SYB can be phosphorylated within the same vesicle by endogenous Ca2+/calmodulin-dependent protein kinase II (CaMKII) associated with synaptic vesicles. This phosphorylation reaction occurs rapidly and involves serine and threonine residues in the cytoplasmic region of SYB. Similarly to CaMKII, a casein kinase II (CasKII) activity copurifying with synaptic vesicles is able to phosphorylate SYB selectively on serine residues of the cytoplasmic region. This phosphorylation reaction is markedly stimulated by sphingosine, a sphingolipid known to activate CasKII and to inhibit CaMKII and protein kinase C. The results show that SYB is a potential substrate for protein kinases involved in the regulation of neurotransmitter release and open the possibility that phosphorylation of SYB plays a role in modulating the molecular interactions between synaptic vesicles and the presynaptic membrane.

Amino Acid Sequence↗

PubMed1981

Studies on the uptake of [3H]dopamine by synaptic vesicle fraction isolated from bovine brain.

1. A synaptic vesicle fraction isolated from bovine caudatolenticular nuclei showed enzymic activities of tyrosine hydroxylase [EC 1.14.16.2] and dopamine beta-hydroxylase [EC 1.14.17.1]. Tyrosine hydroxylase, whose subcellular localization is uncertain, appeared to be associated with the synaptic vesicles. 2. The vesicle fraction took up [3H]dopamine increasingly with time without the aid of ATP (6.3 pmol of [3H]dopamine per mg of vesicle proteins, or 3-4% of the added dopamine, at 30 min). In the presence of ATP, a transient accumulation of the amine was observed, reaching the highest level at 7-10 min (5.6 pmol dopamine/mg protein), and then a rapid release of the amine took place, obeying first-order kinetics with respect to the amine concentration. The amine uptake was strongly inhibited with NEM, regardless of the presence or absence of ATP. 3. The vesicle fraction also exhibited a weak ability to translocate protons inward and ATP-dependently, as monitored by an increase in the fluorescence intensity of ANS. The fluorescence enhancement persisted for at least 30 min and this time-dependent change was not consistent with that of the transient accumulation of [3H]dopamine mentioned above. Since the present vesicle preparation contained a small amount of mitochondrial ATPase (18-20% of the total activity), the proton translocating ability could be attributable to contaminating submitochondrial particles. 4. Therefore these results made it impossible to conclude that the transient uptake of [3H]dopamine by the synaptic vesicles was coupled to ATP hydrolysis.

Adenosine Diphosphate↗