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.     

Isoosmotic isolation of rat brain synaptic vesicles, some of which contain tyrosine hydroxylase

Rat brain synaptic vesicles were isoosmotically isolated and examined for Mg(2+)-ATPase [EC 3.6.1.3.] and tyrosine hydroxylase [EC 1.14.16.2.] associated with the synaptic vesicles. Synaptosomes in 0.32 M sucrose were disrupted by freezing and thawing treatment, and the cytosol fraction was fractionated on a Sephacryl S-500 column with a mean exclusion size of 200 nm. Peak I at the void volume was a mixture of large vesicular membranes, small amounts of synaptic vesicles and coated vesicles, etc. Peak II consisted of non- and granulated synaptic vesicles of 35-40 nm diameter, and peak III of soluble proteins. The synaptic vesicles in peak II reacted with antibodies against the H(+)-ATPase A-subunit, vesicular acetylcholine transporter, and vesicular monoamine transporter. However, they showed little Mg(2+)-ATPase activity. Tyrosine hydroxylase was observed in either peak II or III on blotting with an anti-tyrosine hydroxylase antibody. These results imply that tyrosine hydroxylase exists in soluble and bound forms to synaptic vesicles in nerve terminals.     

Ca2+-dependent mobility of vesicles capturing anti-VGLUT1 antibodies

Several aspects of secretory vesicle cycle have been studied in the past, but vesicle trafficking in relation to the fusion site is less well understood. In particular, the mobility of recaptured vesicles that traffic back toward the central cytoplasm is still poorly defined. We exposed astrocytes to antibodies against the vesicular glutamate transporter 1 (VGLUT1), a marker of glutamatergic vesicles, to fluorescently label vesicles undergoing Ca(2+)-dependent exocytosis and examined their number, fluorescence intensity, and mobility by confocal microscopy. In nonstimulated cells, immunolabeling revealed discrete fluorescent puncta, indicating that VGLUT1 vesicles, which are approximately 50 nm in diameter, cycle slowly between the plasma membrane and the cytoplasm. When the cytosolic Ca(2+) level was raised with ionomycin, the number and fluorescence intensity of the puncta increased, likely because the VGLUT1 epitopes were more accessible to the extracellularly applied antibodies following Ca(2+)-triggered exocytosis. In nonstimulated cells, the mobility of labeled vesicles was limited. In stimulated cells, many vesicles exhibited directional mobility that was abolished by cytoskeleton-disrupting agents, indicating dependence on intact cytoskeleton. Our findings show that postfusion vesicle mobility is regulated and may likely play a role in synaptic vesicle cycle, and also more generally in the genesis and removal of endocytic vesicles.     

Isolation of small agranular synaptic vesicles of rat brain by gel filtration chromatography

I attempted to isolate synaptic vesicles by gel filtration. The rat brain synaptic vesicles in a synaptosomal lysate were collected by ammonium sulfate salting-out and fractionated on a Sephacryl S-500 with a mean exclusion size of 200 nm. Peak I at the void volume contained large vesicular membranes and coated vesicles besides synaptic vesicles; Peak II consisted almost entirely of small agranular synaptic vesicles of 40-50 nm diameter; and Peak III comprised soluble proteins. Western blotting revealed that components of 72 kDa in peaks I and II reacted with an anti-H(+)-ATPase A-subunit antibody [Moriyama et al. (1995) FEBS Lett. 367, 233-236]. When examined for Mg(2+)-ATPase activity, peak I showed specific activity of 4.52 ( micromol ATP hydrolyzed/mg protein/30 min), while that of peak II was as low as 0.22. As estimated from the inhibition by bafilomycin A(1) [Bowman et al. (1988) PROC: Natl. Acad. Sci. USA 85, 7972-7976], the percentage of H(+)-ATPase as to total Mg(2+)-ATPase, 18-22%, was unchanged, indicating no accumulation of the H(+)-ATPase in peak II even on the chromatography. In brief, the small agranular synaptic vesicles in peak II showed little or no Mg(2+)-ATPase activity, although they reacted with the H(+)-ATPase antibody. The reason for this is obscure. Mg(2+)-ATPase might not be a constituent of small agranular synaptic vesicles of rat brain.     

The behaviour of lung surfactant in electrolyte solutions

Surface and electrokinetic properties of purified calf lung surfactant in various electrolyte solutions were determined. Surface properties were pH dependent in distilled water and the surfactant performed as a good lung surfactant only below pH 4. In more physiological media it was pH insensitive over the range 2-8.5. In distilled water at pH 6 its surface properties improved when NaCl was added up to 20 mM; above this concentration it had the surface properties required to stabilise alveoli. The surface properties of surfactant in distilled water were also restored by certain cations (Ca2+, Mg2+, Mn2+, Cd2+ and Ni2+) but not others (Na+, K+, La3+ and Fe3+) when added to an ionic strength of 5.6 mM. Cations that restored its surface activity also reduced the surface charge density on the surfactant particles. Aggregation of surfactant by various metal chlorides was studied by light scattering measurements and bore no relation to surface activity or the charge on the particles. Aggregation of surfactant particles by Ca2+, Cd2+ and Mn2+ was instantly reversed by addition of excess EGTA. The influence of electrolytes on the surface properties of lung surfactant is explained in terms of the electrostatic forces operating in the system.     

Electron-microscope cytochemistry of acetylcholine-like cation by means of low-temperature "ionic fixation"

A fresh preparation of frog neuromuscle was fixed at low temperatures (0 degree-4 degrees C) by means of an "ionic-fixation" procedure which is based on the precipitation of quaternary ammonium cations, such as choline and acetylcholine, with molybdic or tungstic heteropolyanions. A low temperature was used to slow down drastically the biological processus of vesicular exocytosis so that ionic fixation, the speed of which is only slightly influenced by temperature variation, could be performed efficiently. In addition to the conventional point-like precipitate in the synaptic vesicle which is considered to be vesicular acetylcholine, numerous spot-like precipitates were observed in the synaptic cleft. Most of these were contiguous to the active zone, and some were in a paired form and corresponded to the double rows of the synaptic vesicles in contact with active zones. It is concluded that these spot-like precipitates were acetylcholine-like cations of the synaptic vesicles which had been discharged into the synaptic cleft by exocytosis and captured by the ionic fixation procedure. The results are discussed in relation to the vesicular and non-vesicular hypothesis of acetylcholine release.     

Neurotoxic phospholipases directly affect synaptic vesicle function

Snake neurotoxic phospholipases (SPAN) exclusively affect pre-synaptic nerve terminals where they lead to a block of neurotransmission by not fully understood mechanisms. Here, we report that the SPANs, taipoxin and paradoxin, in nanomolar concentrations directly dissociate the synaptophysin/synaptobrevin (Syp/Syb) complex on isolated synaptic vesicles in the presence of synaptosomal cytosol. The phospholipase activity of SPANs depends on Ca(2+) but the dissociation of the Syp/Syb complex does not require Ca(2+). Ca(2+) (100 μM free) alone also dissociates the Syp/Syb complex in the presence of cytosol. Treatment with SPANs disturbs the lipid raft association of synaptophysin and synaptobrevin comparable to cholesterol depletion by β-methyl-cyclodextrin while Ca(2+) alone has no effect. SPANs but not Ca(2+) directly inhibit vesicular uptake of serotonin and glutamate. It is concluded that SPANs directly affect vesicular properties independent from their Ca(2+) -dependent phospholipase activity. SPANs and Ca(2+) dissociate the Syp/Syb complex as a prerequisite for exocytosis. SPANs also prevent the filling of synaptic vesicles thereby adding to the inhibition of neurotransmission.     

A phosphatidylinositol 4,5-bisphosphate-sensitive casein kinase I alpha associates with synaptic vesicles and phosphorylates a subset of vesicle proteins

In interphase cells, alpha-casein kinase I (alpha-CKI) is found associated with cytosolic vesicular structures, the centrosome, and within the nucleus. To identify the specific vesicular structures with which alpha-CKI is associated, established cell lines and primary rat neurons were immunofluorescently labeled with an antibody raised to alpha-CKI. In nonneuronal cells, alpha-CKI colocalizes with vesicular structures which align with microtubules and are partially coincident with both Golgi and endoplasmic reticulum markers. In neurons, alpha- CKI colocalizes with synaptic vesicle markers. When synaptic vesicles were purified from rat brain, they were highly enriched in a CKI, based on activity and immunoreactivity. The synaptic vesicle-associated CKI is an extrinsic kinase and was eluted from synaptic vesicles and purified. This purified CKI has properties most similar to alpha-CKI. When the activities of casein kinase I or II were specifically inhibited on isolated synaptic vesicles, CKI was shown to phosphorylate a specific subset of vesicle proteins, one of which was identified as the synaptic vesicle-specific protein SV2. As with alpha-CKI, the synaptic vesicle CKI is inhibited by phosphatidylinositol 4,5- bisphosphate (PIP2). However, synthesis of PIP2 was detected only in plasma membrane-containing fractions. Therefore, PIP2 may spatially regulate CKI. Since PIP2 synthesis is required for secretion, this inhibition of CKI may be important for the regulation of secretion.     

Synaptotagmin VII as a plasma membrane Ca(2+) sensor in exocytosis

Synaptotagmins I and II are Ca(2+) binding proteins of synaptic vesicles essential for fast Ca(2+)-triggered neurotransmitter release. However, central synapses and neuroendocrine cells lacking these synaptotagmins still exhibit Ca(2+)-evoked exocytosis. We now propose that synaptotagmin VII functions as a plasma membrane Ca(2+) sensor in synaptic exocytosis complementary to vesicular synaptotagmins. We show that alternatively spliced forms of synaptotagmin VII are expressed in a developmentally regulated pattern in brain and are concentrated in presynaptic active zones of central synapses. In neuroendocrine PC12 cells, the C(2)A and C(2)B domains of synaptotagmin VII are potent inhibitors of Ca(2+)-dependent exocytosis, but only when they bind Ca(2+). Our data suggest that in synaptic vesicle exocytosis, distinct synaptotagmins function as independent Ca(2+) sensors on the two fusion partners, the plasma membrane (synaptotagmin VII) versus synaptic vesicles (synaptotagmins I and II).     

Surface potential and surface charge density of the cerebral-cortex synaptic vesicle and stability of vesicle suspension

Using a microelectrophoresis instrument employing the Lazer-Zee system, the electrophoretic mobility of synaptic vesicles isolated from Guinea-pig brain cortex was measured under several conditions. The mobility was found to depend on both pH and ionic concentration of the solution. The surface of the synaptic vesicle was shown to be negatively charged under physiological conditions. The isoelectric point was observed at pH 4.0 in 0.01 M NaCl solution. Effects of divalent cations were examined and reversal of surface charge was observed in 0.1 M CaCl₂ solution. Interaction of vesicles was also considered on the basis of the DLVO theory of colloid stability by using calculated values of surface charge density and surface potential of the synaptic vesicle.     

Inhibition of vesicular glutamate storage and exocytotic release by Rose Bengal

It had been thought that quantal size in synaptic transmission is invariable. Evidence has been emerging, however, that quantal size can be varied under certain conditions. We present evidence that alteration in vesicular [(3)H]L-glutamate (Glu) content within the synaptosome (a pinched-off nerve ending preparation) leads to a change in the amount of exocytotically released [(3)H]Glu. We found that Rose Bengal, a polyhalogenated fluorescein derivative, is a quite potent membrane-permeant inhibitor (K(i) = 19 nM) of glutamate uptake into isolated synaptic vesicles. This vesicular Glu uptake inhibition was achieved largely without affecting H(+)-pump ATPase. We show that various degrees of reduction elicited by Rose Bengal in [(3)H]Glu in synaptic vesicles inside the synaptosome result in a corresponding decrease in the amount of [(3)H]Glu released in a depolarization- (induced by 4-aminopyridine) and Ca(2+)-dependent manner. In contrast, fluorescein, the halogen-free analog of Rose Bengal, which is devoid of inhibitory activity on vesicular [(3)H]Glu uptake, failed to change the amount of exocytotically released [(3)H]Glu. These observations suggest that glutamate synaptic transmission could be altered by pharmacological intervention of glutamate uptake into synaptic vesicles in the nerve terminal, a new mode of synaptic manipulation for glutamate transmission.     

Self-aggregation of surfactant protein A

Environmental factors of physiological relevance such as pH, calcium, ionic strength, and temperature can affect the state of self-aggregation of surfactant protein A (SP-A). We have studied the secondary structure of different SP-A aggregates and analyzed their fluorescence characteristics. (a) We found that self-aggregation of SP-A can be Ca(2+)-dependent. The concentration of Ca(2+) needed for half-maximal self-association (K(a)(Ca)()2+) depended on the presence of salts. Thus, at low ionic strength, K(a)(Ca)()2+ was 2.3 mM, whereas at physiological ionic strength, K(a)(Ca)()2+ was 2.35 microM. Circular dichroism and fluorescence measurements of Ca(2+)-dependent SP-A aggregates indicated that those protein aggregates formed in the absence of NaCl are structurally different from those formed in its presence. (b) We found that self-aggregation of SP-A can be pH-dependent. Self-aggregation of SP-A induced by H(+) was highly influenced by the presence of salts, which reduced the extent of self-association of the protein. The presence of both salts and Ca(2+) attenuated even more the effects of acidic media on SP-A self-aggregation. (c) We found that self-aggregation of SP-A can be temperature-dependent. At 20 degrees C, SP-A underwent self-aggregation at physiological but not at low ionic strength, in the presence of EDTA. All of these aggregates were dissociated by either adding EDTA (a), increasing the pH to neutral pH (b), or increasing the temperature to 37 degrees C (c). Dissociation of Ca(2+)-induced protein aggregates at low ionic strength was accompanied by an irreversible loss of both SP-A secondary structure and SP-A-dependent lipid aggregation properties. On the other hand, temperature-dependent experiments indicated that a structurally intact collagen-like domain was required for either Ca(2+)- or Ca(2+)/Na(+)-induced SP-A self-aggregation but not for H(+)-induced protein aggregation.     

Calcium-ion binding to the chromaffin-granule surface

1. Ca(2+) ions decreased the surface charge of isolated adrenal-medullary chromaffin granules whether the granules were untreated or previously incubated with neuraminidase. 2. Ca(2+) binding in both cases followed a Langmuir adsorption isotherm. 3. The chromaffin-granule surface was essentially polyanionic with about 10800 anionic sites per granule, of which 4200 sites are capable of binding Ca(2+) ions with an electrochemical free energy of adsorption of -1.32kJ.mol(-1). 4. The surface region of the chromaffin granule was calculated to bind 1306 Ca(2+) ions at 2.2mm-Ca(2+) (ionic strength 0.16mol.litre(-1)). 5. The importance of Ca(2+) binding to the chromaffin-granule surface is discussed in relation to the hypothesis of secretion by exocytosis.     

Normal biogenesis and cycling of empty synaptic vesicles in dopamine neurons of vesicular monoamine transporter 2 knockout mice

The neuronal isoform of vesicular monoamine transporter, VMAT2, is responsible for packaging dopamine and other monoamines into synaptic vesicles and thereby plays an essential role in dopamine neurotransmission. Dopamine neurons in mice lacking VMAT2 are unable to store or release dopamine from their synaptic vesicles. To determine how VMAT2-mediated filling influences synaptic vesicle morphology and function, we examined dopamine terminals from VMAT2 knockout mice. In contrast to the abnormalities reported in glutamatergic terminals of mice lacking VGLUT1, the corresponding vesicular transporter for glutamate, we found that the ultrastructure of dopamine terminals and synaptic vesicles in VMAT2 knockout mice were indistinguishable from wild type. Using the activity-dependent dyes FM1-43 and FM2-10, we also found that synaptic vesicles in dopamine neurons lacking VMAT2 undergo endocytosis and exocytosis with kinetics identical to those seen in wild-type neurons. Together, these results demonstrate that dopamine synaptic vesicle biogenesis and cycling are independent of vesicle filling with transmitter. By demonstrating that such empty synaptic vesicles can cycle at the nerve terminal, our study suggests that physiological changes in VMAT2 levels or trafficking at the synapse may regulate dopamine release by altering the ratio of fillable-to-empty synaptic vesicles, as both continue to cycle in response to neural activity.     

CAPS-1 and CAPS-2 are essential synaptic vesicle priming proteins

Before transmitter-filled synaptic vesicles can fuse with the plasma membrane upon stimulation they have to be primed to fusion competence. The regulation of this priming process controls the strength and plasticity of synaptic transmission between neurons, which in turn determines many complex brain functions. We show that CAPS-1 and CAPS-2 are essential components of the synaptic vesicle priming machinery. CAPS-deficient neurons contain no or very few fusion competent synaptic vesicles, which causes a selective impairment of fast phasic transmitter release. Increases in the intracellular Ca(2+) levels can transiently revert this defect. Our findings demonstrate that CAPS proteins generate and maintain a highly fusion competent synaptic vesicle pool that supports phasic Ca(2+) triggered release of transmitters.     

Nearly neutral secretory vesicles in Drosophila nerve terminals

The acidity of mammalian secretory vesicles drives concentration and processing of their contents. Here, pH-sensitive green fluorescent protein (GFP) variants show that the > or =30-fold (H+) difference between secretory vesicles (pH < or = 5.7) and the cytoplasm (pH = 7.2) in mammalian cells is not present in peptidergic and small synaptic vesicles of the Drosophila neuromuscular junction. First, we find that fluorescence from Topaz-tagged atrial natriuretic factor, a peptidergic vesicle pH indicator, is only modestly affected by collapsing the H+ gradient in type III synaptic boutons. Quantitation shows that peptidergic vesicles are nearly neutral (pH = 6.74 +/- 0.05), even when temperature is elevated. Furthermore, small synaptic vesicles in glutamatergic synaptic boutons, studied with synaptophluorin, are as alkaline as peptidergic vesicles. Finally, yellow fluorescent protein measurements show that cytoplasmic pH is only slightly different than in mammals (pH = 7.4). Thus, the marked acidity of mammalian secretory vesicles is not conserved in evolution, and a modest vesicular H+ gradient is sufficient for supporting neurotransmission.     

Differential sorting of the vesicular glutamate transporter 1 into a defined vesicular pool is regulated by light signaling involving the clock gene Period2

Synaptic strength depends on the amount of neurotransmitter stored in synaptic vesicles. The vesicular transmitter content has recently been shown to be directly dependent on the expression levels of vesicular neurotransmitter transporters indicating that the transport capacity of synaptic vesicles is a critical determinant for synaptic efficacy. Using synaptic vesicles prepared from whole brain at different times of the day we now show that the amount of vesicular glutamate transporter (VGLUT) 1 undergoes strong diurnal cycling. VGLUT1 protein levels are high before the start of the light period, decline at noon, increase again before start of the dark period, and decline again at midnight. Mice kept in complete darkness showed within a 24-h period only a single peak of VGLUT1 expression in the middle of the rest phase. In contrast, mice lacking the period gene Period 2, a core component of the circadian clock, did not show any light-cycle-dependent changes of VGLUT1 levels. No other of several synaptic vesicle proteins examined underwent circadian cycling. Circadian cycling of VGLUT1 was not seen when analyzing homogenate or synaptosomes, the starting fraction for vesicle preparation. Circadian cycling of VGLUT1 was also not reflected at the mRNA level. We conclude that nerve terminals are endowed with mechanisms that regulate quantal size by changing the copy number of transporters in synaptic vesicles. A reduced amount of VGLUT1 per vesicle is probably achieved by means of selective sorting controlled by clock genes.     

Translocation of hyaluronic acid in cell surface of cultured mammalian cells after X-irradiation and its recovery by added adenosine triphosphate

To investigate the mechanism of radiation-induced decrease in cell electrophoretic mobility and its recovery by added adenosine triphosphate, specific enzymes and buffer solutions of different ionic strength were utilized. Decrease in the mobility of irradiated cells was detected only with the buffer solution of ionic strengths higher than 0.100. In this range of ionic strengths, removal of hyaluronic acid from cell surface by hyaluronidase had no effect on the electrophoretic mobility of irradiated cells, while the enzyme treatment resulted in 27% mobility reduction in non-irradiated cells. The removal of sialic acid and chondroitin sulfate by their specific enzymes resulted in the similar decrease in mobility either in irradiated and non-irradiated cells. These results suggest that the X-ray induced translocation of hyaluronic acid from the peripheral zone of 0–7.5 Å into the deeper zone of about 10–17 Å, if we use the Debye-Hückel's thickness of ion atmosphere for an approximate estimate of effective depth of electrokinetic plane of shear. Hyaluronic acid reappeared to the peripheral zone by the subsequent incubation after small dose irradiation, or by the addition of 1 mM adenosine triphosphate with Ca²⁺.     

Fluorescent detection of Zn(2+)-rich vesicles with Zinquin: mechanism of action in lipid environments

High concentrations of free Zn2+ ions are found in certain glutamatergic synaptic vesicles in the mammalian brain. These terminals can be visualized histochemically with quinoline sulfonamide compounds that form fluorescent complexes with Zn2+. The present study was undertaken to examine the interaction of the water-soluble quinoline sulfonamide probe, Zinquin (2-methyl-8-(toluene-p-sulfonamido)-6-quinolyloxyacetic acid) with the complex heterogeneous cellular environment. Experiments on rat hippocampal and neocortical slices gave indications that Zinquin in its free acid form was able to diffuse across the plasma and synaptic vesicle membranes. Further experiments were undertaken on unilamellar liposomes to study the interaction of Zinquin and its metal complexes in membranes. These experiments confirmed that Zinquin is able to diffuse across lipid bilayers. Steady-state and time-resolved fluorimetric studies showed that Zinquin in aqueous solution mainly forms a 1:2 (metal:ligand) complex with small amounts of a 1:1 complex. Formation of the 1:1 complex was favored by the presence of lipid, suggesting that it partitions into membranes. Evidence is presented that Zinquin can act as a Zn(2+)-ionophore, exchanging Zn2+ for two protons. The presence of a pH gradient across vesicles traps the Zn(2+)-probe complex within the vesicles. Zinquin is useful as a qualitative probe for detecting the presence of vesicular Zn2+; however, its tendency to partition into membranes and to serve as an ionophore should be borne in mind.     

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.