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.     

Putative Synaptic Vesicle Nucleotide Transporter Identified as Glyceraldehyde-3-Phosphate Dehydrogenase

Abstract: Synaptic vesicles isolated from electric ray electric organ have been shown previously to contain a 34-kDa protein that binds azido-ATP, azido-AMP, and N -ethylmaleimide. The protein was found to share similarities with the mitochondrial ADP/ATP carrier and assumed to represent the synaptic vesicle nucleotide transporter. Synaptic vesicles were purified by sucrose density gradient centrifugation and subsequent chromatography on Sephacryl S-1000 from both Torpedo electric organ and bovine brain cerebral cortex. They contained ATP-binding proteins of 35 kDa and 34 kDa, respectively. ATP binding was inhibited by AMP. Both proteins were highly enriched after column chromatography of vesicle proteins of AMP-Sepharose. Antibodies were obtained against both proteins. Antibodies against the bovine brain synaptic vesicle protein of 34 kDa bound specifically to the 35-kDa protein of Torpedo vesicles. An N-terminal sequence obtained against the 34-kDa protein of bovine brain synaptic vesicles identified it as glyceraldehyde-3-phosphate dehydrogenase. The previously observed molecular characteristics of the putative vesicular nucleotide transporter in Torpedo fit those of glyceraldehyde-3-phosphate dehydrogenase. We, therefore, suggest that the protein previously identified as putative nucleotide transporter is, in fact, glyceraldehyde-3-phosphate dehydrogenase.     

Simultaneous Release of Acetylcholine and ATP from Stimulated Cholinergic Synaptosomes

Abstract: The release of acetylcholine (ACh) and ATP from pure cholinergic synaptosomes isolated from the electric organ of Torpedo was studied in the same perfused sample. A presynaptic ATP release was demonstrated either by depolarization with KCl or after the action of a venom extracted from the annelid Glycera convoluta (GV). The release of ATP exhibited similar kinetics to that of ACh release and was therefore probably closely related to the latter. The ACh/ATP ratio in perfusates after KCl depolarization was 45; this was much higher than the ACh/ATP ratio in cholinergic synaptic vesicles, which was 5. The ACh/ATP ratio released after the action of GV was also higher than that of synaptic vesicles. These differences are discussed. The stoichiometry of ACh and ATP release is not consistent with the view that the whole synaptic vesicle content is released by exocytosis after KCl depolarization, as is the case for chromatin cells in the adrenal medulla.     

Identification of a Synaptic Vesicle Antigen (Mr 86,000) Conserved Between Torpedo and Rat

Antisera were raised in guinea pigs to synaptic vesicles purified from the electric organ of Torpedo marmorata. In cholinergic nerve terminals from Torpedo the major antigens identified had Mr 300,000-150,000, 86,000, and 18,000. The Mr 86,000 antigen was conserved between Torpedo and rat, where it is neuron-specific and concentrated in nerve terminals. When rat brain synaptosomes are subfractionated the antigen is associated with synaptic vesicles. The antigen is not found in the cytoskeleton and in the vesicle-free cytosol. Immunohistochemical localization of the antigen in rat shows it to be associated with synapses in diaphragm, cerebellum, hippocampus, and cerebral cortex. The staining pattern of the antigen indicates that the antigen is not cholinergic-specific. The function of the Mr 86,000 antigen remains to be identified.     

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.     

Purification and Characterization of a Nonvesicular Vesamicol-Binding Protein from Electric Organ and Demonstration of a Related Protein in Mammalian Brain

A protein that binds vesamicol has been purified from a soluble fraction of the Torpedo electric organ homogenate that does not contain synaptic vesicles. The purified vesamicol-binding protein (VBP) has a molecular mass of 470 kDa composed of 30- and 24-kDa subunits. Chemical deglycosylation yielded a single, heterogeneous protein of 24 kDa. The 30-kDa subunit is also sensitive to endo-beta-galactosidase. The dissociation constant of the VBP.vesamicol complex is 0.9 microM, and the Bmax is 5,500 pmol/mg. Antiserum raised to the 30-kDa subunit cross-reacts with the 24-kDa subunit, but not with synaptic vesicles. Drug binding studies and Western blot analysis show that VBP is present in other Torpedo tissues as well as mammalian brain. Immunofluorescence microscopy demonstrates that VBP-like immunoreactivity is not localized exclusively to the nerve terminal regions of the electric organ. Thermal stability, the pH dependence of vesamicol binding, and pharmacological comparisons demonstrate that the VBP is not the cholinergic synaptic vesicle receptor for vesamicol. The implications of this finding for current efforts to develop in vivo diagnostics of cholinergic nerve terminal status based on vesamicol are discussed.     

An antiserum specific for cholinergic synaptic vesicles from electric organ

Rabbit antisera to highly purified synaptic vesicles from the electric organ of Narcine brasiliensis, an electric ray, reveal a unique population of synaptic vesicle antigens in addition to a population shared with other electric organ membranes. Synaptic vesicle antigens were detected by binding successively rabbit antivesicle serum and radioactive goat anti-rabbit serum. To remove antibodies directed against antigens common to synaptic vesicles and other electric organ fractions, the antivesicle serum was extensively preadsorbed against an electric organ membrane fraction that was essentially free of synaptic vesicles. The adsorbed serum retained 40% of its ability to bind to synaptic vesicles, suggesting that about half of the antigenic determinants are unique. Vesicle antigens were quantified with a radioimmunoassay (RIA) that utilized precipitation of antibody-antigen complexes with Staphylococcus aureus cells. By this assay, the vesicles, detected by their acetylcholine (ACh) content and the antigens detected by the RIA, have the same buoyant density after isopycnic centrifugation of crude membrane fractions on sucrose and glycerol density gradients. The ratio of ACh to antigenicity was constant across the vesicle peaks and was close to that observed for vesicles purified to homogeneity. Even though the vesicles make up only approximately 0.5% of the material in the original homogenate, the ratio of acetylcholine to vesicle antigenicity could still be measured and also was indistinguishable from that of pure vesicles. We conclude that synaptic vesicles contain unique antigenic determinants not present to any measurable extent in other fractions of the electric organ. Consequently, it is possible to raise a synaptic vesicle- specific antiserum that allows vesicles to be detected and quantified. These findings are consistent with earlier immunohistochemical observations of specific antibody binding to motor nerve terminals.     

The Synaptic Vesicle-Associated G Protein o-rab3 Is Expressed in Subpopulations of Neurons

Abstract: The distribution of o-rab3—a synaptic vesicle-associated low-molecular-weight GTP-binding protein—was studied in various neural tissues of the electric ray Torpedo marmorata. o-rab3 was shown to be associated selectively with isolated cholinergic synaptic vesicles derived from the electric organ. Gel filtration of cholinergic synaptic vesicles using Sephacryl S-1000 column chromatography demonstrated a copurification of o-rab3 with the synaptic vesicle content marker ATP and with SV2—a synaptic vesicle transmembrane glycoprotein. Indirect immunofluorescence using antibodies against o-rab3 and SV2 and a double labeling protocol revealed an identical distribution of both antigens in the cholinergic nerve terminals within the electric organ and at neuromuscular junctions. An immunoelectron microscopic analysis demonstrated the presence of o-rab3 at the surface of the synaptic vesicle membrane. In the CNS immunofluorescence of o-rab3 and SV2 overlap only in small and distinct areas. Whereas SV2 has an overall distribution in nerve terminals of the entire CNS, o-rab3 is restricted to a subpopulation of nerve terminals in the dorsolateral neuropile of the rhombencephalon and in the dorsal horn of the spinal cord. Our results demonstrate that the synaptic vesicle-associated G protein o-rab3 is specifically expressed only in subpopulations of neurons in the Torpedo CNS.     

The release of acetylcholine: from a cellular towards a molecular mechanism

The isolation of synaptic vesicles rich in acetylcholine (ACh) from the electric organ of Torpedo has indeed strengthened the hypothesis of transmitter exocytosis, but soon after it was found that non-vesicular free ACh was released and renewed upon stimulation. In contrast, vesicular ACh and the number of vesicles remained stable during physiological stimulations. In addition free ACh variations (representing the cytoplasmic pool) were correlated to the release kinetics as measured by the electroplaque discharge. Consequently, the mechanism releasing ACh from the cytoplasm in a packet form was searched at the presynaptic membrane itself. With synaptosomes isolated from the electric organ of Torpedo, it became possible to freeze them rapidly at the peak of ACh release and study their membrane and contents after cryofracture. A statistical analysis showed that the main structural change was the occurrence of large intramembrane particles at the peak of ACh release and under all release conditions. This impressive change contrasted with the stability in the number of vesicles. Another role for the vesicle was envisaged during intense stimulations when the cytoplasmic ACh and ATP pools become exhausted. The decrease in ATP leads to an increase in calcium and protons in the cytoplasm; this signals the depletion of vesicular ACh and ATP stores in the cytoplasm. Release can go on, while ATP promotes the uptake of calcium by vesicles. At the end of its cycle the vesicle will be full of calcium and will perhaps release it. As far as the mechanism of ACh release is concerned it probably depends on a membrane component (perhaps the large particles) activated by calcium and able to translocate ACh in a quantal or subquantal form. In most recent work we showed that if a lyophilized presynaptic membrane was used to make proteoliposomes filled with ACh, they released ACh upon calcium action.     

Attempts at localizing calcium in relation to synaptic transmission: an X-ray microanalytical study

1. In view of the importance of calcium in triggering the transmitter secretion during nerve stimulation or depolarization, the localization of calcium binding sites was studied in stimulated nerve endings that were fixed in calcium-containing s-collidine buffered paraformaldehyde solution. 2. In cholinergic synapses, such as the superior cervical ganglion of cat, the myoneural junction of rat diaphragm and the electric organ of Torpedo marmorata, a stimulation-dependent accumulation of calcium was found in the mitochondria of presynaptic nerve endings. 3. In synaptosomes prepared from rat cerebral cortex, the activity-dependent accumulation of calcium in mitochondria of pinched off nerve endings was also observed. The mitochondrial accumulation of calcium in synaptosomes was dependent on ATP, temperature and could be inhibited by quercetin. 4. In stimulated synapses, only the mitochondria seemed to accumulate calcium in appreciable amounts, whereas other intra-terminal structures, such as the smooth endoplasmic reticulum and synaptic vesicles were devoid of calcium.     

Identification of a Proteoglycan Antigen Characteristic of Cholinergic Synaptic Vesicles

An antiserum to cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata was purified by adsorption with fractions containing unwanted antigens. The adsorbed antiserum responds to the proteoglycan core material of the cholinergic synaptic vesicles. The major antigen migrates in an anomalous fashion on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), forming a broad band with an apparent molecular weight of approximately 120,000 - 300,000. The distribution of this antigen after sucrose density gradient centrifugation of synaptic vesicles is the same as that of vesicular ATP. The antigen comigrates with a substance that can be stained with Alcian-Blue after SDS-PAGE of highly purified synaptic vesicles. This substance is related to the low-molecular-weight, Alcian-Blue-positive glycosaminoglycan vesiculin, which is formed from the high-molecular-weight proteoglycan by prolonged dialysis against water or by protease treatment. No antibodies were detected against vesiculin itself, indicating that the antigenic determinants are restricted to the proteoglycan.     

Presence of a Membrane-Bound Acetylcholinesterase Form in a Preparation of Nerve Endings from Torpedo marmorata Electric Organ

Abstract: We adapted a method, originally described by Israel et al. (1976) for the preparation of cholinergic nerve endings from Torpedo , to deal with a larger quantity of electric tissue. We followed the distribution of acetylcholine (ACh), ATP, acetylcholine receptor (AChR), choline acetyltransferase (ChAT), ouabainresistant and -sensitive ATPase, lactate dehydrogenase (LDH) and acetylcholinesterase (AChE) and obtained a nerve ending fraction, without detectable contamination by postsynaptic components. This preparation consisted of closed structures of 1–5 μm diameter, containing synaptic vesicles. It had the capacity to synthetize and release ACh. This preparation is therefore quite suitable for biochemical analysis of presynaptic elements. We particularly investigated its content of AChE: it consists exclusively of the 6S dimeric, hydrophobic form of the enzyme. This enzyme is enriched in the nerve ending preparation, by a factor higher than that obtained for ChAT. The yields obtained for the two enzymes suggest that the hydrophobic 6S AChE form may be mostly presynaptic in Torpedo electric organs. We characterized this form as a membrane-bound, externally active enzyme in the nerve ending preparation. It may thus participate in the hydrolysis of extracellularly liberated AChE and its abundance suggests that presynaptic AChE could play an essential role in cholinergic transmission in Torpedo electric organs and perhaps also in other cholinergic synapses.     

Thiamine and Cholinergic Transmission in the Electric Organ of Torpedo

The electric organ of Torpedo marmorata was found to contain as much as 120 +/- 24 nmol of thiamine per g of fresh tissue. The vitamin was distributed as nonesterified thiamine (32%), thiamine monophosphate (22%), thiamine diphosphate (8%), and an important proportion of thiamine triphosphate (38%). A high level of thiamine triphosphate was found in synaptosomes isolated from the electric organ. In contrast, the synaptic vesicles did not show any enrichment in thiamine, whereas they contained a marked peak of acetylcholine (ACh) and ATP. Thus thiamine seems to be very abundant in cholinergic nerve terminals; its localization is apparently extravesicular, either in the axoplasm or in association with plasma membrane. When calcium was reduced and magnesium increased in the external medium, the efficiency of transmission was diminished, owing to inhibition of ACh release; in a parallel manner the degree of thiamine phosphorylation was found to increase--this condition is known to modify the repartition of ACh between vesicular and extravesicular compartments. Electrical stimulation, which causes periodic variations of the level of ACh and ATP, also caused significant changes in thiamine esters. In addition, related changes of the vitamin and the transmitter were observed under other conditions, suggesting a functional link between the metabolism of thiamine and that of ACh in cholinergic nerve terminals.     

Cholinergic Vesicle Specific Proteoglycan: Stability in Isolated Vesicles and in Synaptosomes During Induced Transmitter Release

Exposure of synaptosomes isolated from the electric organ of Torpedo marmorata to conditions that promote the release of acetylcholine does not cause the co-release of a vesicle specific proteoglycan. Proteoglycan within synaptosomes is quite stable during various incubation conditions as measured by immune dot blotting. Isolated vesicles from Torpedo also retain their proteoglycan immunoreactivity when exposed to a variety of incubation conditions. Lysis of vesicles in H2O, treatment with pH 11.5 buffer, or exposure to high ionic strength (2 M KCl) results in the loss of acetylcholine or ATP while the proteoglycan is retained by vesicle membranes. Only treatment with Nonidet P-40 releases proteoglycan from vesicles or synaptosomes and free proteoglycan immunoreactivity is then susceptible to degradation by trypsin or heparinase. These results suggest that the proteoglycan is an integral component of vesicle membranes and is at least in the synaptosomal preparation not subject to extensive co-release with acetylcholine or ATP.     

Uncoupling of Cholinergic Synaptic Vesicles by the Presynaptic Toxin β-Bungarotoxin

The effect of the presynaptic neurotoxin beta-bungarotoxin (beta-BuTx) on the acetylcholine (ACh) storage system of synaptic vesicles isolated from the electric organ of Torpedo californica was studied. The toxin can totally inhibit active transport of [3H]ACh by the vesicles in a Ca2+-, time-, and concentration-dependent manner. Correlated with these effects is a 50-60% stimulation of the vesicle proton-pumping ATPase activity. The beta-BuTx-mediated transport inhibition and ATPase stimulation are antagonized by delipidated bovine serum albumin, not reversed by excess EGTA, and not mimicked by other cationic proteins or soybean or pancreatic trypsin inhibitors. The behavior is consistent with phospholipase A2 (PLA2)-dependent damage to the vesicle membrane caused by beta-BuTx, which results in uncoupling of the ATPase and ACh transporter systems. The nonneurotoxic Naja naja venom PLA2 causes similar effects, except that it is slightly more potent on a molar basis. About 100-fold more beta-BuTx is required to effect lysis of synaptic vesicles than to uncouple them. ATP is a strong inhibitor of beta-BuTx- but not of N. naja PLA2-mediated uncoupling. The observations suggest that a component of beta-BuTx toxicity in the cholinergic terminal might involve attack on synaptic vesicles or vesicle-like structures and that a nucleotide-like factor might modulate the toxicity.     

Cholinergic Synaptic Vesicles Contain a V-Type and a P-Type ATPase

Fifty to eighty-five percent of the ATPase activity in different preparations of cholinergic synaptic vesicles isolated from Torpedo electric organ was half-inhibited by 7 microM vanadate. This activity is due to a recently purified phosphointermediate, or P-type, ATPase, Acetylcholine (ACh) active transport by the vesicles was stimulated about 35% by vanadate, demonstrating that the P-type enzyme is not the proton pump responsible for ACh active transport. Nearly all of the vesicle ATPase activity was inhibited by N-ethylmaleimide. The P-type ATPase could be protected from N-ethylmaleimide inactivation by vanadate, and subsequently reactivated by complexation of vanadate with deferoxamine. The inactivation-protection pattern suggests the presence of a vanadate-insensitive, N-ethylmaleimide-sensitive ATPase consistent with a vacuolar, or V-type, activity expected to drive ACh active transport. ACh active transport was half-inhibited by 5 microM N-ethylmaleimide, even in the presence of vanadate. The presence of a V-type ATPase was confirmed by Western blots using antisera raised against three separate subunits of chromaffin granule vacuolar ATPase I. Both ATPase activities, the P-type polypeptides, and the 38-kilodalton polypeptide of the V-type ATPase precisely copurify with the synaptic vesicles. Solubilization of synaptic vesicles in octaethyleneglycol dodecyl ether detergent results in several-fold stimulation of the P-type activity and inactivation of the V-type activity, thus explaining why the V-type activity was not detected previously during purification of the P-type ATPase. It is concluded that cholinergic vesicles contain a P-type ATPase of unknown function and a V-type ATPase which is the proton pump.     

Purification of the vesamicol receptor.

The vesamicol receptor (VR) present in cholinergic synaptic vesicles isolated from the electric organ of Torpedo was solubilized in cholate detergent and stabilized with glycerol and a phospholipid mixture. The receptor was purified in 7% yield by hydroxylapatite, wheat germ lectin affinity, DEAE anion-exchange, and size exclusion chromatographies based on a [3H]vesamicol binding assay. A final specific binding of 4400 pmol/mg of protein was obtained. The cholate-solubilized VR complex exhibited variable aggregation states with particle molecular masses of 210-3500 kDa in different experiments. The purified VR exhibited very heterogeneous electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with very diffuse protein staining at about 240 kDa. No "classical" polypeptide or glycopeptide band was detected. One form of the SV1 epitope, which is characteristic of cholinergic synaptic vesicle proteoglycan, copurified precisely with the VR. The SV2 epitope, which is found in most neuronal and endocrine secretory vesicles, also closely purified with the VR. Substantially purified VR retained both enantioselectivity for (-)-vesamicol and a linked AcCh-binding site. This confirms the allosteric model for the VR in the AcCh transporter. The physicochemical properties of the VR and copurification of it with the SV1 epitope strongly suggest that the VR is associated with cholinergic vesicle proteoglycan. A second proteoglycan that is not associated with the VR but which carries the SV1 and SV2 epitopes also was observed.     

Adenosine triphosphate. A constituent of cholinergic synaptic vesicles

1. Synaptic vesicles separated by density-gradient centrifugation from extracts of the cholinergic nerve terminals of the electric organ of Torpedo marmorata were found to contain appreciable amounts of ATP as well as acetylcholine. 2. Vesicular ATP was stable in the presence of concentrations of apyrase and myokinase that rapidly destroyed equivalent amounts of endogenous or added free ATP; pre-treatment of cytoplasmic extracts of electric tissue with these enzymes destroyed endogenous free ATP, but did not affect the vesicular ATP. 3. When [U-(14)C]ATP was added to electric tissue at the time of comminution and extraction of the vesicles, all the radioactivity was associated with soluble components in the subsequent fractionation: none was associated with vesicles or membrane fragments; thus it is unlikely that vesicular ATP can be accounted for by the sequestration of endogenous free ATP within any vesicles formed during comminution and extraction of the tissue. 4. When synaptic vesicles were passed through iso-osmotic columns of Bio-Gel A-5m, which separates vesicles from soluble proteins and small molecules, all the recovered ATP and acetylcholine passed through together in the void volume. 5. Regression analysis showed that vesicular ATP content was highly correlated with vesicular acetylcholine content in different experiments, the molar ratio acetylcholine/ATP being 5.32+/-(s.e.m.) 0.45 (21 expts.) for the peak density-gradient fraction. The ratio varied, however, somewhat across the density-gradient peak suggesting some degree of chemical heterogeneity in the vesicle population.     

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.     

In vitro binding of isolated synaptic vesicles to presynaptic plasma membranes: activation by Ca2+ and protein kinase C.

An in vitro model to study the molecular control of binding of highly purified synaptic vesicles to presynaptic plasma membranes has been developed. Presynaptic plasma membranes were immobilized by dotting onto nitrocellulose, and binding of iodinated synaptic vesicle membranes was studied under varying experimental conditions. Synaptic vesicles bind to presynaptic plasma membranes in the presence of Ca2+ and ATP. Binding is reduced in the presence of EGTA and abolished by the calmodulin antagonist trifluoperazine. Vesicle binding is stimulated 5-fold after incubation--prior to dotting--of presynaptic plasma membranes with ATP in the presence of the phorbol-ester 12-O-tetradecanoylphorbol-13-acetate (1 microM) and 2.5-fold after preincubation with Ca2+ (50 microM). Pretreatment of plasma membranes with alkaline phosphatase strongly reduces vesicle binding. Microsomes prepared from bovine liver did not bind to presynaptic plasma membranes. Our results suggest that activation of protein kinase C and Ca2+ stimulate binding of synaptic vesicles to the presynaptic membrane. In the intact nerve terminal this interaction may represent an initial step in synaptic vesicle exocytosis.