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PubMed1988

Cholinergic synaptic vesicle heterogeneity: evidence for regulation of acetylcholine transport.

Crude cholinergic synaptic vesicles from a homogenate of the electric organ of Torpedo californica were centrifuged to equilibrium in an isosmotic sucrose density gradient. The classical VP1 synaptic vesicles banding at 1.055 g/mL actively transported [3H]acetylcholine (AcCh). An organelle banding at about 1.071 g/mL transported even more [3H]AcCh. Transport by both organelles was inhibited by the known AcCh storage blockers trans-2-(4-phenylpiperidino)cyclohexanol (vesamicol, formerly AH5183) and nigericin. Relative to VP1 vesicles the denser organelle was slightly smaller as shown by size-exclusion chromatography. It is concluded that the denser organelle corresponds to the recycling VP2 synaptic vesicle originally described in intact Torpedo marmorata electric organ [Zimmermann, H., & Denston, C.R. (1977) Neuroscience (Oxford) 2, 695-714; Zimmermann, H., & Denston, C.R. (1977) Neuroscience (Oxford) 2, 715-730]. The properties of the receptor for vesamicol were studied by measuring binding of [3H]vesamicol, and the amount of SV2 antigen characteristic of secretory vesicles was assayed with a monoclonal antibody directed against it. Relative to VP1 vesicles the VP2 vesicles had a ratio of [3H]AcCh transport activity to vesamicol receptor concentration that typically was 4-7-fold higher, whereas the ratio of SV2 antigen concentration to vesamicol receptor concentration was about 2-fold higher. Based on an antibody standardization, in a typical preparation the VP1 vesicles contained 237 +/- 15 pmol of receptor/mg of protein whereas VP2 vesicles contained 102 +/- 3 pmol of receptor/mg of protein, and VP2 vesicles transported AcCh 2-3-fold more actively than VP1 vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1980

Electrokinetic properties of synaptic vesicles and synaptosomal membranes.

Using the technique of electrophoretic light scattering, we have measured the electrophoretic mobilities of synaptic vesicles and synaptosomal plasma membranes isolated from guinea-pig cerebral cortex. The electrophoretic mobility of synaptic vesicles is slightly greater than that of synaptosomal plasma membranes. Ca+2 and Mg+2 reduced the mobility of both species to the same extent at physiologically relevant concentrations (0-1 mM) and near-physiologic ionic strength. The extent of the reduction was not large (approximately 6% for synaptic vesicles in the presence of 100 mM KCl) at 1 mM divalent cation concentrations. At concentrations of approximately 2 mM and higher, Ca+2 reduced the mobility of synaptic vesicles more than did Mg/2. A similar but much smaller effect was observed in the case of synaptosomal plasma membranes. The addition of 1 mM Mg+2-ATP had no effect upon synaptic vesicle mobility either in the presence or absence of the ionophores nigericin or valinomycin. These data, together with earlier work (Siegel et al., 1978, Biophys. J. 22:341-346), demonstrate that substantial reduction of the average electrostatic surface charge density is not the most important role of divalent cations in promoting close approach of secretory granules and secretory cell membranes, and that it is certainly not the Ca+2-specific step in exocytosis.

Animals↗
PubMed2004

Characterization of synaptogyrin 3 as a new synaptic vesicle protein.

Synaptogyrins comprise a family of tyrosine-phosphorylated proteins with two neuronal (synaptogyrins 1 and 3) and one ubiquitous (cellugyrin) isoform. Previous studies have indicated that synaptogyrins are involved in the regulation of neurotransmitter release. Synaptogyrin 1 is a synaptic vesicle protein; cellugyrin, by contrast, is absent from synaptic vesicles. In an effort to further characterize the synaptogyrin family, we studied the distribution of the synaptogyrin 3 protein in the nervous system. Subcellular fractionation and immunoprecipitation of synaptic vesicles from mouse brain showed that synaptogyrin 3 is associated with synaptic vesicles and that synaptogyrins 1 and 3 can reside on the same synaptic vesicle. Immunofluorescent staining of cultured hippocampal neurons confirmed the synaptic localization of synaptogyrin 3. Analysis of the relative distributions of synaptogyrins 1 and 3 in mouse brain revealed a more restricted expression pattern for synaptogyrin 3 compared to the ubiquitous distribution of synaptogyrin 1. Strong synaptogyrin 3 labeling was observed in the mossy fiber region of the hippocampus, substantia nigra pars reticulata, pallidum, and deep cerebellar nuclei. By comparison, the striatum and reticular and ventral posterolateral thalamic nuclei, which all showed synaptogyrin 1 labeling, contained significantly less synaptogyrin 3. Finally, we used in situ hybridization experiments to correlate synaptogyrin 3 mRNA in cell bodies with synaptogyrin 3 protein at synapses. Altogether, our data indicate that neuronal synaptogyrins are differentially expressed protein isoforms that may represent functionally distinct populations of synapses and/or synaptic vesicles.

Animals↗
PubMed1991

A synaptic vesicle specific GTP-binding protein from ray electric organ.

A cDNA encoding a synaptic vesicle associated GTP-binding protein was identified by screening a lambda gt11 expression library derived from the electric lobe of Discopyge ommata with polyclonal antibodies recognizing vesicle-specific proteins of Mr 25,000. Nucleotide sequence analysis defines an open reading frame of 218 amino acids. The protein belongs to the ras superfamily and shares about 75% amino acid identity with smg-25A, B and C identified in bovine brain and rab3A characterized in rat brain. Northern blot analysis revealed a 4.5 kb transcript present only in neural tissues, the highest level of expression being observed in electric lobe. Western blot analysis of total tissue homogenates derived from D. ommata detected the protein in electric organ, forebrain and to a lesser extent in electric lobe and spinal cord. No immunoreactivity was detected in non-neuronal tissues. Blotting of subcellular fractions derived from electric ray electric organ revealed that the GTP-binding protein co-purifies with synaptic vesicles. The neural specific expression and the localization to synaptic vesicles suggest a role of this protein in synaptic vesicle trafficking and targeting.

Amino Acid Sequence↗
PubMed2002

Synaptic vesicle depletion correlates with attenuated synaptic responses to prolonged repetitive stimulation in mice lacking alpha-synuclein.

Although the mutation of alpha-synuclein, a protein associated with presynaptic vesicles, is implicated in the etiology and pathogenesis of Parkinson's disease, the biological function of the normal protein is unknown. Mice that lack alpha-synuclein have been generated by homologous recombination in embryonic stem cells. Electron microscopic examination of hippocampal synapses revealed a striking selective deficiency of undocked vesicles without affecting docked vesicles. Field recording of CA1 synapses in hippocampal slices from the mutant mice demonstrated normal basal synaptic transmission, paired-pulse facilitation, and response to a brief train of high-frequency stimulation (100 Hz, 40 pulses) that exhausts only docked vesicles. In contrast, the alpha-synuclein knock-out mice exhibited significant impairments in synaptic response to a prolonged train of repetitive stimulation (12.5 Hz, 300 pulses) capable of depleting docked as well as reserve pool vesicles. Moreover, the replenishment of the docked vesicles by reserve pool vesicles after depletion was slower in the mutant synapses. Thus, alpha-synuclein may be required for the genesis and/or maintenance of a subset of presynaptic vesicles, those in the "reserve" or "resting" pools. These results reveal, for the first time, the normal function of endogenous alpha-synuclein in regulating synaptic vesicle mobilization at nerve terminals.

Amphetamine↗
PubMed1982

Synaptic vesicle recycling at the neuromuscular junction in the presence of a presynaptic membrane marker.

Staining of the presynaptic axonal membrane of the neuromuscular junction with horseradish peroxidase-labeled alpha-bungarotoxin was utilized as a marker for observing directly the fate of this membrane during the process of synaptic vesicle release and recycling. The neuromuscular junctions of frog sartorius-sciatic nerve preparations were stained with horseradish peroxidase-alpha-bungarotoxin and stimulated by electrical stimulation of the nerve, high concentration of external potassium ions, and black widow spider venom. Some preparations were stimulated in the presence of exogenous horseradish peroxidase tracer after incubation in the conjugate and were found to contain horseradish peroxidase within many synaptic vesicles, indicating that the conjugate did not affect the process of synaptic vesicle recycling. Stimulation was followed by depletion of synaptic vesicles and appearance of axolemmal infoldings and membranous cisternae. With the rest after electrical and potassium stimulation, synaptic vesicles were reconstituted and terminals assumed a more normal appearance. Membrane staining after stimulation occurred in the axolemmal infoldings, some of the intra-axonal cisternae, and in a few coated vesicles. However, all synaptic vesicles were unreactive, in either rested or unrested terminals. Thus, axonal membrane labeled with horseradish peroxidase-alpha-bungarotoxin did not become incorporated into new synaptic vesicles. These observations support a mechanism of recycling of synaptic retrieval of vesicle membrane or constituents from the axolemma.

Animals↗
PubMed2000

Phosphorylation of synaptic vesicle protein 2 modulates binding to synaptotagmin.

Synaptic vesicle protein 2 (SV2) is a component of all synaptic vesicles that is required for normal neurotransmission. Here we report that in intact synaptic terminals SV2 is a phosphoprotein. Phosphopeptide mapping studies indicate that a major site of phosphorylation is located on the cytoplasmic amino terminus. SV2 is phosphorylated on serine and threonine but not on tyrosine residues, indicating that it is a substrate for serine/threonine kinases. Phosphopeptide mapping, in gel kinase assays, and surveys of kinase inhibitors suggest that casein kinase I is a primary SV2 kinase. The amino terminus of SV2 was previously shown to mediate its interaction with synaptotagmin, a calcium-binding protein also required for normal neurotransmission. Comparison of synaptotagmin binding with phosphorylated and unphosphorylated SV2 amino-terminal peptides reveals an increase in binding with phosphorylation. These results suggest that the affinity of SV2 for synaptotagmin is modulated by phosphorylation of SV2.

Animals↗
PubMed2004

Amphiphysin is a component of clathrin coats formed during synaptic vesicle recycling at the lamprey giant synapse.

Amphiphysin is a protein enriched at mammalian synapses thought to function as a clathrin accessory factor in synaptic vesicle endocytosis. Here we examine the involvement of amphiphysin in synaptic vesicle recycling at the giant synapse in the lamprey. We show that amphiphysin resides in the synaptic vesicle cluster at rest and relocates to sites of endocytosis during synaptic activity. It accumulates at coated pits where its SH3 domain, but not its central clathrin/AP-2-binding (CLAP) region, is accessible for antibody binding. Microinjection of antibodies specifically directed against the CLAP region inhibited recycling of synaptic vesicles and caused accumulation of clathrin-coated intermediates with distorted morphology, including flat patches of coated presynaptic membrane. Our data provide evidence for an activity-dependent redistribution of amphiphysin in intact nerve terminals and show that amphiphysin is a component of presynaptic clathrin-coated intermediates formed during synaptic vesicle recycling.

Amino Acid Sequence↗
PubMed1982

Proton gradient linkage to active uptake of [3H]acetylcholine by Torpedo electric organ synaptic vesicles.

It has been confirmed that cholinergic synaptic vesicles isolated from the electric organ of Torpedo californica exhibit adenosine 5'-triphosphate (ATP) dependent active uptake of [3H]acetylcholine. Active uptake can be completely inhibited by low concentrations of the mitochondrial uncouplers carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, nigericin, gramicidin, valinomycin, and A 23187. Under similar conditions uncouplers stimulate the vesicle adenosinetriphosphatase (ATPase) by from 40 to 80%. ATP-supported uptake of [3H]acetylcholine increases greatly as the external pH is increased from 6.6 to 7.6 and remains approximately constant from pH 7.8 to pH 8.6. The uptake also becomes more selective for [3H]acetylcholine compared to [14C]choline as the pH is increased from 6.6 to 7.6, achieving 12-fold selectively, in a manner similar to the increase in the amount of [3H]acetylcholine taken up. Bicarbonate stimulates both the amount and selectivity of [3H]acetylcholine uptake over the lower pH range, but it has no effect over the higher pH range. Exogenous ammonium ion completely inhibits active [3H]acetylcholine uptake, with lower concentrations of ammonium ion required at higher pH values in a manner consistent with ammonia being the active species. Adenosine 5'-diphosphate and a nonhydrolyzable ATP analogue do not support active [3H]acetylcholine uptake. It is concluded that an ATPase pumps protons into the cholinergic synaptic vesicle to produce an internally acidic and positively charged proton gradient that is linked to [3H]acetylcholine uptake.

Acetylcholine↗
PubMed1982

Cross-linking of synaptic vesicle proteins. Effect of ATP.

Synaptic vesicles isolated from bovine brain were subjected to cross-linking with the bifunctional amino group reagent dimethyl adipimidate. The resulting proteins were analyzed by SDS-polyacrylamide gel electrophoresis. The reagent (10 mM) caused partial or complete disappearance from the SDS gel of most of the major polypeptides of the vesicles and the formation of new polymeric species with molecular weights greater than 500000. Using lower concentrations of the adipimidate (2-5 mM) a more selective cross-linking occurred, with the disappearance of a group of protein bands having apparent molecular weight values of 60000-68000, 40000-41000 and 25000-30000. The extent of cross-linking was independent of vesicle concentration in the range 0.3-3.0 mg protein per ml. Addition of ATP or AMP to the cross-linking reaction mixture resulted in a marked reduction in cross-linking of all of the major vesicle polypeptides (apparent molecular weight values 160000, 77000, 55000, 42000, 32000, 28000 and 26000). Several proteins were less affected by ATP or AMP; these were mostly the same vesicle proteins as those which had become cross-linked with low concentrations (2 mM) of dimethyl adipimidate. The ATP effect was markedly reduced if the vesicles were pretreated prior to the cross-linking reaction with alkaline buffer (pH 8.5) in either the presence or the absence of ATP. In the presence of 32P-labeled ATP, several of the vesicle protein bands became phosphorylated, but the extent of their cross-linking did not depend upon the state of phosphorylation of the major phoshorylated proteins. The results are consistent with the presence of aggregated protein complexes and of stabilized arrays of the major proteins within the vesicle membrane.

Adenosine Monophosphate↗
PubMed1991

Properties of a synaptic vesicle protein binding plasma membranes.

Synaptic vesicles contain a protein (agglutinin) which binds cell plasma membranes. The protein activity is titrated measuring the agglutination of rabbit trypsinized-fixed red blood cells induced by serial dilutions of purified vesicles. The protein is highly concentrated in vesicles: the specific activity is 150-fold higher in vesicles compared to the brain homogenate, and 20-fold higher compared to the crude synaptosomal membrane fraction. The binding is specifically inhibited by the four major brain sialoglycosphingolipids, with a higher affinity for polysialo- rather than for monosialoganglioside (GM1) IC50 are 3.9 x 10(-5) M (GT1b), 8.9 x 10(-5) M (GD1a), 2.8 x 10(-4) M (GD1b) and 4.9 x 10(-4) M (GM1). Cells which are not agglutinated by the vesicles (human red blood cells) can be activated by incubation with gangliosides: insertion of the glycolipids in the plasma membranes makes them sensitive to the vesicle haemagglutinin.

Animals↗
PubMed1979

Characterization of a membrane protein from cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata.

Rabbits were immunized with cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata. The resultant antiserum had one major antibody activity against an antigen called the Torpedo vesicle antigen. This antigen could not be demonstrated in muscle, liver or blood and is therefore, suggested to be nervous-tissue specific. The vesicle antigen was quantified in various parts of the nervous system and in subcellular fractions of the electric organ of Torpedo marmorata and was found to be highly enriched in synaptic vesicle membranes. The antigen bound to concanavalin A, thereby demonstrating the presence of a carbohydrate moiety. By means of charge-shift electrophoresis, amphiphilicity was demonstrated, indicating that the Torpedo vesicle antigen is an intrinsic membrane protein. The antigen was immunochemically unrelated to other brain specific proteins such as 14-3-2, S-100, the glial fibrillary acidic protein and synaptin. Furthermore, it was unrelated to two other membrane proteins, the nicotinic acetylcholine receptor and acetylcholinesterase, present in Torpedo electric organ. The antiserum against Torpedo synaptic vesicles did not react with preparations of rat brain synaptic vesicles or ox adrenal medullary chromaffin granules.

Acetylcholine↗
PubMed2004

Synapsin is a novel Rab3 effector protein on small synaptic vesicles. I. Identification and characterization of the synapsin I-Rab3 interactions in vitro and in intact nerve terminals.

Synapsins, a family of neuron-specific phosphoproteins, have been demonstrated to regulate the availability of synaptic vesicles for exocytosis by binding to both synaptic vesicles and the actin cytoskeleton in a phosphorylation-dependent manner. Although the above-mentioned observations strongly support a pre-docking role of the synapsins in the assembly and maintenance of a reserve pool of synaptic vesicles, recent results suggest that the synapsins may also be involved in some later step of exocytosis. In order to investigate additional interactions of the synapsins with nerve terminal proteins, we have employed phage display library technology to select peptide sequences binding with high affinity to synapsin I. Antibodies raised against the peptide YQYIETSMQ (syn21) specifically recognized Rab3A, a synaptic vesicle-specific small G protein implicated in multiple steps of exocytosis. The interaction between synapsin I and Rab3A was confirmed by photoaffinity labeling experiments on purified synaptic vesicles and by the formation of a chemically cross-linked complex between synapsin I and Rab3A in intact nerve terminals. Synapsin I could be effectively co-precipitated from synaptosomal extracts by immobilized recombinant Rab3A in a GTP-dependent fashion. In vitro binding assays using purified proteins confirmed the binding preference of synapsin I for Rab3A-GTP and revealed that the COOH-terminal regions of synapsin I and the Rab3A effector domain are required for the interaction with Rab3A to occur. The data indicate that synapsin I is a novel Rab3 interactor on synaptic vesicles and suggest that the synapsin-Rab3 interaction may participate in the regulation of synaptic vesicle trafficking within the nerve terminals.

Amino Acid Sequence↗
PubMed1986

Characterization of synapsin I binding to small synaptic vesicles.

The binding of synapsin I, a synaptic vesicle-associated phosphoprotein, to small synaptic vesicles has been examined. For this study, synapsin I was purified under nondenaturing conditions from rat brain, using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and characterized. Small synaptic vesicles were purified from rat neocortex by controlled pore glass chromatography as the last purification step, and binding was characterized at an ionic strength equivalent to 40 mM NaCl. After removal of endogenous synapsin I, exogenous dephospho-synapsin I bound with high affinity (Kd, 10 +/- 6 nM) to synaptic vesicles. The binding saturated at 76 +/- 40 micrograms synapsin I/mg of vesicle protein, which corresponded to the amount found endogenously in purified vesicles. Synapsin I binding exhibited a broad pH optimum around pH 7. Other basic proteins, specifically myelin basic protein and histone H2b, did not compete with synapsin I for binding to vesicles. Other membranes purified from rat brain and membranes derived from human erythrocytes did not show the high affinity binding site for synapsin I found in vesicles. The binding of three different forms of phosphosynapsin I to vesicles was investigated. Synapsin I, phosphorylated at sites 2 and 3 by purified calcium/calmodulin-dependent protein kinase II, bound with a 5-fold lower affinity to the vesicles than did dephospho-synapsin I. In contrast, synapsin I, phosphorylated at site 1 by purified catalytic subunit of cAMP-dependent protein kinase, bound with an affinity close to that of dephospho-synapsin I. Synapsin I phosphorylated on all three sites bound to the vesicles with an affinity comparable to that of synapsin I phosphorylated on sites 2 and 3. Under conditions of higher ionic strength (150 mM NaCl equivalent), synapsin I bound with a 5-fold lower affinity to vesicles, and no effect of phosphorylation on binding was observed under these conditions.

Animals↗
PubMed1993

The synaptic vesicle proteins SV2, synaptotagmin and synaptophysin are sorted to separate cellular compartments in CHO fibroblasts.

We expressed the synaptic vesicle proteins SV2, synaptotagmin, and synaptophysin in CHO fibroblasts to investigate the targeting information contained by each protein. All three proteins entered different cellular compartments. Synaptotagmin was found on the plasma membrane. Both SV2 and synaptophysin were sorted to small intracellular vesicles, but synaptophysin colocalized with early endosomal markers, while SV2 did not. SV2-containing vesicles did not have the same sedimentation characteristics as authentic synaptic vesicles, even though transfected SV2 was sorted from endosomal markers. We also created cell lines expressing both SV2 and synaptotagmin, both synaptotagmin and synaptophysin, and lines expressing all three synaptic vesicle proteins. In all cases, the proteins maintained their distinct compartmentalizations, were not found in the same organelle, and did not created synaptic vesicle-like structures. These results have important implications for models of synaptic vesicle biogenesis.

Animals↗
PubMed2004

Hydrogen ions control synaptic vesicle ion channel activity in Torpedo electromotor neurones.

During exocytosis the synaptic vesicle fuses with the surface membrane and undergoes a pH jump. When the synaptic vesicle is inside the presynaptic nerve terminal its internal pH is about 5.5 and after fusion, the inside of the vesicle comes in contact with the extracellular medium with a pH of about 7.25. We examined the effect of such pH jump on the opening of the non-specific ion channel in the synaptic vesicle membrane, in the context of the post-fusion hypothesis of transmitter release control. The vesicles were isolated from Torpedo ocellata electromotor neurones. The pH dependence of the opening of the non-specific ion channel was examined using the fused vesicle-attached configuration of the patch clamp technique. The rate of opening depends on both pH and voltage. Increasing the pH from 5.5 to 7.25 activated dramatically the non-specific ion channel of the vesicle membrane. The single channel conductance did not change significantly with the alteration in the pH, and neither did the mean channel open time. These results support the hypothesis that during partial fusion of the vesicle with the surface membrane, ion channels in the vesicle membrane open, admit ions and thus help in the ion exchange process mechanism, leading to the release of the transmitter from the intravesicular ion exchange matrix. This process may have also a pathophysiological significance in conditions of altered pH.

Animals↗
PubMed2002

Ca2+-dependent formation of a dynamin-synaptophysin complex: potential role in synaptic vesicle endocytosis.

Synaptophysin is a synaptic vesicle (SV) protein of unknown function. Here we show that a repeated sequence in the cytoplasmic tail of synaptophysin mediates the formation of a protein complex containing the GTPase dynamin. The formation of this complex requires a high Ca(2+) concentration, suggesting that it occurs preferentially at the sites of SV exocytosis. Coimmunoprecipitation of a dynamin-synaptophysin complex from brain extracts is promoted by dissociation of vesicle-associated membrane protein 2 from synaptophysin. This finding suggests that dynamin only associates with synaptophysin in vivo after vesicle-associated membrane protein 2 (VAMP2) enters the SNARE complex. GTP binding releases dynamin from synaptophysin, possibly serving to regulate dynamin selfassembly during endocytosis. Our results suggest that synaptophysin plays a role in SV recycling by recruiting dynamin to the vesicle membrane.

Amino Acid Sequence↗