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PubMed1997

Age-related abnormalities in expression of mRNAs encoding synapsin 1A, synapsin 1B, and synaptophysin in the temporal cortex of schizophrenics.

Synaptic abnormalities have been implicated in schizophrenia. In order to investigate synaptic pathology in schizophrenia, we examined levels of mRNAs encoding synaptophysin, synapsin 1A and synapsin 1B in the left temporal cortex from schizophrenics (n = 24) and from normal control individuals with no history of psychiatric illness (n = 10). Levels of synaptic mRNAs in the left superior temporal and left middle temporal gyrus declined significantly with age in schizophrenics, but not in controls. Dividing the diagnostic groups according to age (below and above 75 years), the data revealed that in "young" schizophrenics (age <75 years) levels of the three synaptic mRNAs in the left superior and left middle temporal gyri were approximately two times higher than in the age-matched controls. In the "old" schizophrenics (age >75 years) the levels of synaptic mRNAs in temporal cortex did not differ from age-matched controls. These findings further support the hypothesis that developmental synaptic abnormalities may be involved in the pathophysiology of schizophrenia.

Age Factors↗
PubMed1999

Homo- and heterodimerization of synapsins.

In vertebrates, synapsins constitute a family of synaptic vesicle proteins encoded by three genes. Synapsins contain a central ATP-binding domain, the C-domain, that is highly homologous between synapsins and evolutionarily conserved in invertebrates. The crystal structure of the C-domain from synapsin I revealed that it constitutes a large (>300 amino acids), independently folded domain that forms a tight dimer with or without bound ATP. We now show that the C-domains of all synapsins form homodimers, and that in addition, C-domains from different synapsins associate into heterodimers. This conclusion is based on four findings: 1) in yeast two-hybrid screens with full-length synapsin IIa as a bait, the most frequently isolated prey cDNAs encoded the C-domain of synapsins; 2) quantitative yeast two-hybrid protein-protein binding assays demonstrated pairwise strong interactions between all synapsins; 3) immunoprecipitations from transfected COS cells confirmed that synapsin II heteromultimerizes with synapsins I and III in intact cells, and similar results were obtained with bacterial expression systems; and 4) quantification of the synapsin III level in synapsin I/II double knockout mice showed that the level of synapsin III is decreased by 50%, indicating that heteromultimerization of synapsin III with synapsins I or II occurs in vivo and is required for protein stabilization. These data suggest that synapsins coat the surface of synaptic vesicles as homo- and heterodimers in which the C-domains of the various subunits have distinct regulatory properties and are flanked by variable C-terminal sequences. The data also imply that synapsin III does not compensate for the loss of synapsins I and II in the double knockout mice.

Animals↗
PubMed1987

Nearest neighbor analysis for brain synapsin I. Evidence from in vitro reassociation assays for association with membrane protein(s) and the Mr = 68,000 neurofilament subunit.

Synapsin I, a major neuron-specific substrate for cAMP-dependent and Ca2+/calmodulin-dependent protein kinases, associates in in vitro assays with brain integral membrane protein site(s) distinct from secretory vesicles and with the neurofilament Mr = 68,000 subunit. The membrane sites for synapsin involve protein(s) and are likely to have physiological relevance since the binding of 125I-labeled synapsin is abolished by digestion with chymotrypsin, is displaced by unlabeled synapsin, is of high affinity (KD = 10 nM), and has a capacity (42 pmol/mg membrane protein) that is comparable to the amount of synapsin in brain, optimal binding occurs at physiological pH (6.8-7.2) and salt concentrations (50 mM), and synapsin binding to membranes is inhibited by phosphorylation with Ca2+/calmodulin-dependent protein kinase. The brain membrane protein sites for synapsin are not due to synaptic vesicles, since synaptic vesicles do not sediment under the conditions of the binding assay. Association between synapsin and the Mr = 68,000 neurofilament subunit has also been demonstrated. The binding of synapsin with the neurofilament subunit is specific since this binding interaction is saturable, with a 1:1 stoichiometry, the binding involves only certain proteolytically derived domains of synapsin, and is therefore not a simple electrostatic interaction between the basic domains of synapsin and the acidic regions in the neurofilament subunit, and Ca2+/calmodulin-dependent phosphorylation of synapsin inhibits this interaction. Synapsin promotes cross-linking of synaptic vesicles to brain membranes, and these complexes are reduced by phosphorylation of synapsin. This interconnecting function of synapsin may be a general characteristic of synapsin binding, with a membrane (synaptic vesicle or nonsecretory vesicle)-bound synapsin associating with microtubules, neurofilaments, or spectrin.

Animals↗
PubMed1994

Differential distribution of the synapsins in the rat olfactory bulb.

The distribution of the different forms of synapsin in the rat olfactory bulb was investigated by biochemical and immunocytochemical methods. Western blots of tissue derived from microdissection of the surface and core regions of the olfactory bulb were performed using antibodies to synapsin I and synapsin II. The relative levels of the synapsins in the core region of the olfactory bulb were similar to the cerebral cortex. In contrast, the surface region of the olfactory bulb had significantly higher levels of synapsin IIa and significantly lower levels of synapsin I, relative to the cortex. Immunocytochemical localization of synapsin I and synapsin II revealed that synapsin I immunoreactivity was the most dense in the external plexiform layer and in the glomeruli; immunoreactivity was also present in the granule cell layer and the periglomerular regions. Synapsin II immunoreactivity was the most dense in the glomeruli. The external plexiform layer, internal plexiform layer, and granule cell layer exhibited much lower immunoreactivity. To determine the source of synapsin II immunoreactivity in the glomeruli, the olfactory epithelium was damaged to decrease the primary afferent input to the bulb. Three to four days later, olfactory bulb sections were double labeled with anti-olfactory marker protein (OMP) antibodies and anti-synapsin II antibodies. Following denervation, both OMP and synapsin II immunoreactivities were diminished, and continued to colocalize in regions retaining immunoreactivity. Individual puncta were immunoreactive for both OMP and synapsin II. Occasional puncta contained only synapsin II immunoreactivity. These results indicate that the distribution of the synapsins in the olfactory bulb differs from most other brain regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1998

A third member of the synapsin gene family.

Synapsins are a family of neuron-specific synaptic vesicle-associated phosphoproteins that have been implicated in synaptogenesis and in the modulation of neurotransmitter release. In mammals, distinct genes for synapsins I and II have been identified, each of which gives rise to two alternatively spliced isoforms. We have now cloned and characterized a third member of the synapsin gene family, synapsin III, from human DNA. Synapsin III gives rise to at least one protein isoform, designated synapsin IIIa, in several mammalian species. Synapsin IIIa is associated with synaptic vesicles, and its expression appears to be neuron-specific. The primary structure of synapsin IIIa conforms to the domain model previously described for the synapsin family, with domains A, C, and E exhibiting the highest degree of conservation. Synapsin IIIa contains a novel domain, termed domain J, located between domains C and E. The similarities among synapsins I, II, and III in domain organization, neuron-specific expression, and subcellular localization suggest a possible role for synapsin III in the regulation of neurotransmitter release and synaptogenesis. The human synapsin III gene is located on chromosome 22q12-13, which has been identified as a possible schizophrenia susceptibility locus. On the basis of this localization and the well established neurobiological roles of the synapsins, synapsin III represents a candidate gene for schizophrenia.

Alternative Splicing↗
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↗
PubMed1992

Synapsin IIa: expression in insect cells, purification, and characterization.

Synapsin IIa belongs to a family of neuron-specific phosphoproteins called synapsins, which are associated with synaptic vesicles in presynaptic nerve terminals. In order to examine the biochemical properties of synapsin IIa, and ultimately its physiological function, purified protein is required. Since attempts to purify significant quantities of synapsin IIa, an isoform of the synapsins, from mammalian brain have proven difficult, we undertook the production of recombinant synapsin IIa by utilizing the baculovirus expression system. Rat synapsin IIa cDNA was introduced into the baculovirus genome via homologous recombination, and the recombinant baculovirus was purified. Spodoptera frugiperda (Sf9) cells infected with this virus expressed synapsin IIa as 5% of the total cellular protein. The recombinant protein was extracted from the particulate fraction of the infected Sf9 cells with salt and a nonionic detergent and purified by immunoaffinity chromatography. The purified synapsin IIa was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase to a stoichiometry of 0.8 mol of phosphate/mol of protein. Metabolic labeling with [32P]Pi demonstrated synapsin IIa phosphorylation in infected Sf9 cells. Using a homogenate of uninfected Sf9 cells, a cAMP-dependent protein kinase activity which can phosphorylate synapsin IIa was detected. Limited proteolysis of recombinant synapsin IIa phosphorylated in vitro and in vivo resulted in identical phosphopeptide maps. Further, synapsin IIa, like synapsin I, binds with high affinity in a saturable manner to synaptic vesicles purified from rat cortex.

Animals↗
PubMed1992

Differential expression of synapsins I and II among rat retinal synapses.

The synapsins are a family of synaptic vesicle-associated phosphoproteins thought to regulate the availability of vesicles for neurotransmitter release. In order to assess variability of synapsin isoform expression, we compared the localization of synapsins Ia, Ib, IIa, and IIb in the inner plexiform layer of the rat retina. Double labeling in conjunction with confocal fluorescence and electron microscopy allowed imaging of synapsin I and II immunoreactivity within single presynaptic terminals. No qualitative differences were observed between expression of the a and b isoforms of synapsin I in individual terminals; likewise, the a and b isoforms of synapsin II were identically distributed. In contrast, marked differences were seen upon comparison of synapsin I and synapsin II expression in single terminals. Our results indicate the existence of three classes of presumed amacrine cell synaptic terminals: synapsin I+/synapsin II-, synapsin I-/synapsin II+, and synapsin I+/synapsin II+. Each class of synapse has a different distribution among five IPL sublayers, suggesting that they represent different subpopulations of amacrine cells. Double labeling with an antibody to choline acetyltransferase indicates that synapsin I-/II+ terminals may be those of cholinergic amacrine cells. Furthermore, all synapsin II+ terminals appear to be distinct from those expressing the GABA synthetic enzyme glutamic acid decarboxylase. The observed variations in synapsin content suggest the existence of presynaptic terminal heterogeneity that is not apparent from conventional morphological studies.

Animals↗
PubMed1997

Phosphorylation-dependent effects of synapsin IIa on actin polymerization and network formation.

The synapsins are a family of synaptic vesicle phosphoproteins which play a key role in the regulation of neurotransmitter release and synapse formation. In the case of synapsin I, these biological properties have been attributed to its ability to interact with both synaptic vesicles and the actin-based cytoskeleton. Although synapsin II shares some of the biological properties of synapsin I, much less is known of its molecular properties. We have investigated the interactions of recombinant rat synapsin Ila with monomeric and filamentous actin and the sensitivity of those interactions to phosphorylation, and found that: i) dephosphorylated synapsin II stimulates actin polymerization by binding to actin monomers and forming actively elongating nuclei and by facilitating the spontaneous nucleation/elongation processes; ii) dephosphorylated synapsin II induces the formation of thick and ordered bundles of actin filaments with greater potency than synapsin I; iii) phosphorylation by protein kinase A markedly inhibits the ability of synapsin II to interact with both actin monomers and filaments. The results indicate that the interactions of synapsin II with actin are similar but not identical to those of synapsin I and suggest that synapsin II may play a major structural role in mature and developing nerve terminals, which is only partially overlapping with the role played by synapsin I.

Actins↗
PubMed1994

Temporal onset of synapsin I gene expression coincides with neuronal differentiation during the development of the nervous system.

Synapsin I is the best characterized member of a family of nerve terminal-specific phosphoproteins implicated in the regulation of neurotransmitter release. During development, the expression of synapsin I correlates temporally and topographically with synapse formation, and recent physiological studies (Lu et al. [1992] Neuron 8:521-529.) have suggested that synapsin I may participate in the functional maturation of synapses. To better understand the temporal relationship between synapsin I gene expression and particular cellular events during neuronal development, we have used in situ hybridization histochemistry to localize synapsin I mRNA throughout the rat central and peripheral nervous systems during embryonic and postnatal development. From the earliest embryonic time points assayed (E12), the expression of the synapsin I gene was detectable in both the central and peripheral nervous systems. While, in general, levels of synapsin I mRNAs were high in utero, synapsin I cDNA probes revealed specific patterns of hybridization in different regions of the embryonic nervous system. To determine precisely the temporal onset of expression of the synapsin I gene during neuronal development, we examined in detail the appearance of synapsin I mRNA during the well characterized postnatal development of granule cells of the rat cerebellum and hippocampus. In both regions, the onset of synapsin I gene expression correlated with the period of stem cell commitment to terminal differentiation. Finally, our data demonstrate that, in a second phase, synapsin I gene expression increases to a maximum for a given neuronal population during a particular phase of differentiation, i.e., synaptogenesis.

Animals↗
PubMed1994

Synaptic targeting domains of synapsin I revealed by transgenic expression in photoreceptor cells.

Synapsins are abundant nerve terminal proteins present at all synapses except for ribbon synapses, e.g. photoreceptor cell synapses. Multiple functions have been proposed for synapsins, including clustering of synaptic vesicles and regulation of synaptic vesicle exocytosis. To investigate the physiological functions of synapsin and to ascertain which domains of synapsin are involved in synaptic targeting in vivo, we expressed synapsin Ib and its N- and C-terminal domains in the photoreceptor cells of transgenic mice. In these cells synapsin Ib is targeted efficiently to synaptic vesicles but has no significant effect on the development, structure or physiology of the synapses. This suggests that synapsin I does not have dominant physiological or morphoregulatory functions at these synapses. Full-length synapsin Ib and the N-terminal domains of synapsin Ib but not its C-terminal domains are transported to synapses, revealing that the molecular apparatus for synaptic targeting of synapsins is also present in cells which form ribbon synapses that normally lack synapsins. This apparatus appears to utilize the conserved N-terminal domains that are shared between all synapsins.

Animals↗
PubMed2003

Interaction of synapsin I with membranes.

The synapsins (I, II, and III) comprise a family of peripheral membrane proteins that are involved in both regulation of neurotransmitter release and synaptogenesis. Synapsins are concentrated at presynaptic nerve terminals and are associated with the cytoplasmic surface of synaptic vesicles. Membrane-binding of synapsins involves interaction with both protein and lipid components of synaptic vesicles. Synapsin I binds rapidly and with high affinity to liposomes containing anionic lipids. The binding of bovine synapsin I to liposomes was studied using fluoresceinphosphatidyl-ethanolamine (FPE) to measure membrane electrostatic potential. Synapsin binding to liposomes caused a rapid increase in FPE fluorescence, indicating an increase in positive charge at the membrane surface. Synapsin I binding to monolayers resulted in a substantial increase in monolayer surface pressure. At higher initial surface pressures, the synapsin-induced increase in monolayer surface pressure is dependent on the presence of anionic lipids in the monolayer. Synapsin I also induced rapid aggregation of liposomes, but did not induce leakage of entrapped carboxyfluorescein, while other aggregation-inducing agents promoted extensive leakage. These results are in agreement with the presence of amphipathic stretches of amino acids in synapsin I that exhibit both electrostatic and hydrophobic interactions with membranes, and offer a molecular explanation for the high affinity binding of synapsin I to liposomes and for stabilization of membranes by synapsin I.

Cell Fusion↗
PubMed1997

Anti-synapsin monoclonal antibodies: epitope mapping and inhibitory effects on phosphorylation and Grb2 binding.

The synapsins are a family of major neuron-specific synaptic vesicle-associated phosphoproteins which play important roles in synaptic function. In an effort to identify molecular tools which can be used to perturb the activity of the synapsins in in vitro as well as in vivo experiments, we have localized the epitopes of a panel of monoclonal antibodies (mAbs) raised against synapsins I and II and have characterized their ability to interfere with the interactions of the synapsins with protein kinases, actin and Src homology-3 (SH3) domains. The epitopes of the six mAbs were found to be concentrated in the N-terminal region within domains A and B for the synapsin II-reactive mAbs 19.4, 19.11, 19.51 and 19.21, and in two C-terminal clusters in the proline-rich domains D for synapsin I (mAbs 10.22, 19.51, 19.11 and 19.8) and G for synapsin II (mAb 19.8). The synapsin II-specific mAbs 19.4 and 19.21, whose overlapping epitopes are adjacent to phosphorylation site 1, specifically inhibited synapsin II phosphorylation by endogenous or exogenous cAMP-dependent protein kinase. While all the anti-synapsin I mAbs were unable to affect the interactions of synapsin I both with Ca2+/calmodulin-dependent protein kinase II and with actin monomers and filaments, mAbs 19.8 and 19.51 were found to inhibit the binding of Grb2 SH3 domains to the proline-rich C-terminal region of synapsin I.

Adaptor Proteins, Signal Transducing↗
PubMed1991

Actin and tubulin binding domains of synapsins Ia and Ib.

Synapsins Ia and Ib are neuronal phosphoproteins involved with the regulated clustering of small synaptic vesicles at the presynaptic terminus. In vitro they bind and bundle filaments of both actin and tubulin. Previously, we identified an actin binding domain in the NH2-terminal 25-kDa fragment (N25) generated by 2-nitro-5-thiocyanobenzoic acid (NTCB) cleavage of synapsin I and found that a complementary COOH-terminal 52-kDa portion of the molecule (N52) contained either a second actin binding site or a site of self-association [Petrucci, T. P., & Morrow, J. S. (1987) J. Cell. Biol. 105, 1355]. Using direct binding assays between actin, tubulin, and specific synapsin NTCB-derived peptides, we confirm the ability of purified N25 to bind but not bundle actin and demonstrate that the complementary N52 (or N50) fragments from synapsins Ia and Ib and a 14-kDa fragment derived from the middle of the molecule also associate directly with actin. An antibody specific for N25 inhibits the actin binding activity of N25 and the actin bundling but not the actin binding activity of intact synapsin I. Similar studies conducted with purified tubulin and tubulin immobilized on Sepharose demonstrate that both tubulin and actin bind at approximately the same sites in the NH2-terminal half of synapsin I. Although the fragments derived from the COOH terminus of both synapsin Ia and synapsin Ib (N40b/N34) were devoid of measurable actin binding activity after NTCB cleavage, they were specifically labeled in the intact molecule by a photoactivated cross-linker bound to F-actin. Collectively, these results indicate that synapsins Ia and Ib possess two actin and tubulin binding domains located in the NH2-terminal half of the molecule and suggest that a third actin binding domain is located in the COOH-terminal region. The NH2-terminal sites are found in NTCB peptides N25 and N14, while the third site, apparently of lower affinity, resides in N40b/N34. It is hypothesized that, in the intact molecule, the two NH2-terminal domains contribute to a single high-affinity actin and/or tubulin binding site in the "globular" head region of synapsin I, while the third actin binding domain constitutes the topographically distinct site required for the actin bundling activity of the native molecule. The 45-residue COOH extension that distinguishes synapsin Ia from synapsin Ib appears not to be involved with actin binding, since no differences were found in the ability of N40b and N34 to be photo-cross-linked to actin.(ABSTRACT TRUNCATED AT 400 WORDS)

Actins↗
PubMed1999

Characterization of transcripts from the synapsin III gene locus.

Synapsin III, the most recently described member of the synapsin gene family, displays a gene structure and protein domain structure similar to those of synapsins I and II. In this report, however, we describe major differences in the temporal- and tissue-specific expressions of synapsin III. Whereas synapsins I and II each give rise to two isoforms that are expressed predominantly in adult brain, there are at least six synapsin III transcripts (synapsin IIIa-IIIf) that differ with respect to tissue- and developmental stage-specific expression. Three of the neuronal transcripts are detected in fetal and to a lesser extent in adult brain (IIa-IIIc), whereas one (IIId) is detected only in fetal brain. Two additional transcripts (IIIe and IIIf) are detected only in nonneuronal tissues. A putative second promoter, which is contained within an intron in the synapsin III gene locus, appears to generate the nonneuronal synapsin IIIe and IIIf transcripts. This level of genome complexity is far greater than that described previously for the synapsin I and II genes and suggests that synapsin III may have functions distinct from those described for synapsins I and II.

Animals↗
PubMed1994

Evidence that two non-overlapping high-affinity calmodulin-binding sites are present in the head region of synapsin I.

Calmodulin is an important element in the regulation of nerve terminal exocytosis by Ca2+. Calmodulin has been shown to interact with the synaptic vesicle phosphoproteins synapsins Ia and Ib [Okabe, T. & Sobue, K. (1987) FEBS Lett. 213, 184-188; Hayes, N. V. L., Bennett, A. F. & Baines, A. J. (1991) Biochem. J. 275, 93-97]. These proteins are thought to provide regulated linkages between synaptic vesicles and cytoskeletal elements. It is well established that calmodulin modulates synapsin I activities via calmodulin-dependent protein-kinase-II-catalysed phosphorylation. The direct binding of calmodulin to synapsin I suggests a second mode of regulation in addition to phosphorylation. In this study, we present evidence indicating that two sites for calmodulin binding exist in the N-terminal head region of synapsins Ia and Ib. In unphosphorylated synapsin I, these sites had a Kd value of = 36 +/- 14 nM for binding to calmodulin labelled with acetyl-N'-(5-sulpho-1-naphthyl)ethylene diamine. The Kd values for synapsin I phosphorylated at various sites were as follows: site I 18 +/- 11 nM; sites II and III 35 +/- 14 nM; sites I-III 16 +/- 9 nM. The fluorescence data indicated a stoichiometry of not less than 2 mol calmodulin bound to 1 mol synapsin I at saturation in each case. Consistent with this stoichiometry, two chemically cross-linked species (96 kDa and 116 kDa) containing calmodulin and synapsin I were generated in vitro, corresponding to one and two calmodulin molecules bound/synapsin I. Defined fragments of synapsin I were generated with the reagent 2-nitro-5-thiocyanobenzoic acid, which cleaves at cysteine residues. Cysteine-specific cleavage of whole synapsin I after cross-linking to biotinylated calmodulin generated a pair of polypeptide complexes (approximately 46 kDa and 38 kDa), the masses of which indicated cross-linking of calmodulin to the N-terminal and middle regions of synapsin I. Purified N-terminal and middle fragments each showed a Ca(2+)-dependent interaction with calmodulin affinity columns. Two calmodulin-binding fragments (7.4 kDa and 6.5 kDa) were generated using Staphylococcus aureus V8 protease digestion of synapsin I. These fragments were isolated by calmodulin affinity chromatography and reverse-phase HPLC. N-terminal sequence analysis indicated that each was contained within one of the 2-nitro-5-thiocyanobenzoic-acid-derived calmodulin-binding fragments.(ABSTRACT TRUNCATED AT 400 WORDS)

Actins↗
PubMed1997

Kinetic analysis of the phosphorylation-dependent interactions of synapsin I with rat brain synaptic vesicles.

1. Synapsin I, a major synaptic vesicle (SV)-associated phosphoprotein, is involved in the regulation of neurotransmitter release and synapse formation. By binding to both phospholipid and protein components of SV with high affinity and in a phosphorylation-dependent fashion, synapsin I is believed to cluster SV and to attach them to the actin-based cytoskeleton of the nerve terminal. 2. In the present study we have investigated the kinetic aspects of synapsin I-SV interactions and the mechanisms of their modulation by ionic strength and site-specific phosphorylation, using fluorescence resonance energy transfer between suitable fluorophores linked to synapsin I and to the membrane bilayer. 3. The binding of synapsin I to the phospholipid and protein components of SV has fast kinetics: mean time constants ranged between 1 and 4 s for association and 9 and 11's for ionic strength-induced dissociation at 20 degrees C. The interaction with the phospholipid component consists predominantly of a hydrophobic binding with the core of the membrane which may account for the membrane stabilizing effect of synapsin I. 4. Phosphorylation of synapsin I by either SV-associated or purified exogenous Ca2+/calmodulin-dependent protein kinase II (CaMPKII) inhibited the association rate and the binding to SV at steady state by acting on the ionic strength-sensitive component of the binding. When dephosphorylated synapsin I was previously bound to SV, exposure of SV to Ca2+/calmodulin in the presence of ATP triggered a prompt dissociation of synapsin I with a time constant similar to that of ionic strength-induced dissociation. 5. In conclusion, the reversible interactions between synapsin I and SV are highly regulated by site-specific phosphorylation and have kinetics of the same order of magnitude as the kinetics of SV recycling determined in mammalian neurons under comparable temperature conditions. These findings are consistent with the hypothesis that synapsin I associates with, and dissociates from, SV during the exo-endocytotic cycle. The on-vesicle phosphorylation of synapsin I by the SV-associated CaMPKII, and the subsequent dissociation of the protein from the vesicle membrane, though not involved in mediating exocytosis of primed vesicles evoked by a single stimulus, may represent a prompt and efficient mechanism for the modulation of neurotransmitter release and presynaptic plasticity.

Animals↗