Synapsin I bundles F-actin in a phosphorylation-dependent manner

Synapsin I is a neuron-specific phosphoprotein localized to the cytoplasmic surface of synaptic vesicles. This phosphoprotein is a major substrate for cyclic AMP-dependent and calcium/calmodulin-dependent protein kinases. Its state of phosphorylation can be altered both in vivo and in vitro by a variety of physiological and pharmacological manipulations known to affect synaptic function. Recent direct evidence suggests that it may be involved in the regulation of neurotransmitter release from the nerve terminal. In the nerve terminal, synaptic vesicles are embedded in a cytoskeletal network, consisting in part of actin. We report here the ability of the dephospho-form of synapsin I to bundle F-actin. This bundling activity is reduced when synapsin I is phosphorylated by cAMP-dependent protein kinase and virtually abolished when it is phosphorylated by calcium/calmodulin-dependent protein kinase II or by both kinases. These results, demonstrating an interaction of synapsin I with actin in vitro, support the possibility that synapsin I is involved in clustering of synaptic vesicles at the presynaptic terminal and that the phosphorylation of synapsin I may be involved in regulating the translocation of synaptic vesicles to their sites of release.     

Synapsin I is a microtubule-bundling protein

Synapsin I, a synaptic vesicle protein, is thought to be involved in the regulation of neurotransmission through its phosphorylation by the cyclic AMP-dependent and Ca2+/calmodulin-dependent protein kinases which become activated upon depolarization of nerve endings. However, despite its recent characterization as a spectrin-binding protein immunologically related to erythrocyte protein 4.1, other interactions of synapsin I with structural proteins remain unknown. We report here that synapsin I can co-cycle with microtubules through three cycles of warm polymerization and cold depolymerization. Synapsin I binds saturably to microtubules stabilized by taxol, with an estimated dissociation constant (Kd) of 4.5 microM and a stoichiometry of 1.2 mol of synapsin binding sites per mol tubulin dimer. Synapsin I also increases the turbidity of tubulin solutions at 37 degrees C, but without causing detectable alterations in the critical concentration required for polymerization. Mixtures of synapsin I and tubulin observed by negative stain electron microscopy contain bundles of microtubules, accounting for the effect of synapsin I on tubulin turbidity. Synapsin I is thus a candidate to mediate or regulate the interaction of synaptic vesicles with microtubules.     

Calcium/calmodulin-dependent protein kinase II increases glutamate and noradrenaline release from synaptosomes

A variety of evidence indicates that calcium-dependent protein phosphorylation modulates the release of neurotransmitter from nerve terminals. For instance, the injection of rat calcium/calmodulin-dependent protein kinase II (Ca2+/CaM-dependent PK II) into the preterminal digit of the squid giant synapse leads to an increase in the release of a so-far unidentified neurotransmitter induced by presynaptic depolarization. But until now, it has not been demonstrated that Ca2+/CaM-dependent PK II can also regulate neurotransmitter release in the vertebrate nervous system. Here we report that the introduction of Ca2+/CaM-dependent PK II, autoactivated by thiophosphorylation, into rat brain synaptosomes (isolated nerve terminals) increases the initial rate of induced release of two neurotransmitters, glutamate and noradrenaline. We also show that introduction of a selective peptidergic inhibitor of Ca2+/CaM-dependent PK II inhibits the initial rate of induced glutamate release. These results support the hypothesis that activation of Ca2+/CaM-dependent PK II in the nerve terminal removes a constraint on neurotransmitter release.     

RIM1alpha is required for presynaptic long-term potentiation.

Two main forms of long-term potentiation (LTP)-a prominent model for the cellular mechanism of learning and memory-have been distinguished in the mammalian brain. One requires activation of postsynaptic NMDA (N-methyl d-aspartate) receptors, whereas the other, called mossy fibre LTP, has a principal presynaptic component. Mossy fibre LTP is expressed in hippocampal mossy fibre synapses, cerebellar parallel fibre synapses and corticothalamic synapses, where it apparently operates by a mechanism that requires activation of protein kinase A. Thus, presynaptic substrates of protein kinase A are probably essential in mediating this form of long-term synaptic plasticity. Studies of knockout mice have shown that the synaptic vesicle protein Rab3A is required for mossy fibre LTP, but the protein kinase A substrates rabphilin, synapsin I and synapsin II are dispensable. Here we report that mossy fibre LTP in the hippocampus and the cerebellum is abolished in mice lacking RIM1alpha, an active zone protein that binds to Rab3A and that is also a protein kinase A substrate. Our results indicate that the long-term increase in neurotransmitter release during mossy fibre LTP may be mediated by a unitary mechanism that involves the GTP-dependent interaction of Rab3A with RIM1alpha at the interface of synaptic vesicles and the active zone.     

RIM1alpha forms a protein scaffold for regulating neurotransmitter release at the active zone.

Neurotransmitters are released by synaptic vesicle fusion at the active zone. The active zone of a synapse mediates Ca2+-triggered neurotransmitter release, and integrates presynaptic signals in regulating this release. Much is known about the structure of active zones and synaptic vesicles, but the functional relation between their components is poorly understood. Here we show that RIM1alpha, an active zone protein that was identified as a putative effector for the synaptic vesicle protein Rab3A, interacts with several active zone molecules, including Munc13-1 (ref. 6) and alpha-liprins, to form a protein scaffold in the presynaptic nerve terminal. Abolishing the expression of RIM1alpha in mice shows that RIM1alpha is essential for maintaining normal probability of neurotransmitter release, and for regulating release during short-term synaptic plasticity. These data indicate that RIM1alpha has a central function in integrating active zone proteins and synaptic vesicles into a molecular scaffold that controls neurotransmitter release.     

Binding of synaptotagmin to the alpha-latrotoxin receptor implicates both in synaptic vesicle exocytosis

A vertebrate neurotoxin, alpha-latrotoxin, from black widow spider venom causes synaptic vesicle exocytosis and neurotransmitter release from presynaptic nerve terminals. Although the mechanism of action of alpha-latrotoxin is not known, it does require binding of alpha-latrotoxin to a high-affinity receptor on the presynaptic plasma membrane. The alpha-latrotoxin receptor seems to be exclusively at the presynaptic plasmamembrane. Here we report that the alpha-latrotoxin receptor specifically binds to a synaptic vesicle protein, synaptotagmin, and modulates its phosphorylation. Synaptotagmin is a synaptic vesicle-specific membrane protein that binds negatively charged phospholipids and contains two copies of a putative Ca(2+)-binding domain from protein kinase C (the C2-domain), suggesting a regulatory role in synaptic vesicle fusion. Our findings suggest that a physiological role of the alpha-latrotoxin receptor may be the docking of synaptic vesicles at the active zone. The direct interaction of the alpha-latrotoxin receptor with a synaptic vesicle protein also suggests a mechanism of action for this toxin in causing neurotransmitter release.     

Synaptotagmin I functions as a calcium regulator of release probability

In all synapses, Ca2+ triggers neurotransmitter release to initiate signal transmission. Ca2+ presumably acts by activating synaptic Ca2+ sensors, but the nature of these sensors--which are the gatekeepers to neurotransmission--remains unclear. One of the candidate Ca2+ sensors in release is the synaptic Ca2+-binding protein synaptotagmin I. Here we have studied a point mutation in synaptotagmin I that causes a twofold decrease in overall Ca2+ affinity without inducing structural or conformational changes. When introduced by homologous recombination into the endogenous synaptotagmin I gene in mice, this point mutation decreases the Ca2+ sensitivity of neurotransmitter release twofold, but does not alter spontaneous release or the size of the readily releasable pool of neurotransmitters. Therefore, Ca2+ binding to synaptotagmin I participates in triggering neurotransmitter release at the synapse.     

Long-term potentiation in the hippocampus is blocked by tyrosine kinase inhibitors.

Long-term potentiation (LTP) in the hippocampus is thought to contribute to memory formation. In the Ca1 region, LTP requires the NMDA (N-methyl-D-aspartate) receptor-dependent influx of Ca2+ and activation of serine and threonine protein kinases. Because of the high amount of protein tyrosine kinases in hippocampus and cerebellum, two regions implicated in learning and memory, we examined the possible additional requirement of tyrosine kinase activity in LTP. We first examined the specificity in brain of five inhibitors of tyrosine kinase and found that two of them, lavendustin A and genistein, showed substantially greater specificity for tyrosine kinase from hippocampus than for three serine-threonine kinases: protein kinase A, protein kinase C, and Ca2+/calmodulin kinase II. Lavendustin A and genistein selectively blocked the induction of LTP when applied in the bath or injected into the postsynaptic cell. By contrast, the inhibitors had no effect on the established LTP, on normal synaptic transmission, or on the neurotransmitter actions attributable to the actions of protein kinase A or protein kinase C. These data suggest that tyrosine kinase activity could be required postsynaptically for long-term synaptic plasticity in the hippocampus. As Ca2+ calmodulin kinase II or protein kinase C seem also to be required, the tyrosine kinases could participate postsynaptically in a kinase network together with serine and threonine kinases.     

Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C

Neurotransmitters are released at synapses by the Ca2(+)-regulated exocytosis of synaptic vesicles, which are specialized secretory organelles that store high concentrations of neurotransmitters. The rapid Ca2(+)-triggered fusion of synaptic vesicles is presumably mediated by specific proteins that must interact with Ca2+ and the phospholipid bilayer. We now report that the cytoplasmic domain of p65, a synaptic vesicle-specific protein that binds calmodulin contains an internally repeated sequence that is homologous to the regulatory C2-region of protein kinase C (PKC). The cytoplasmic domain of recombinant p65 binds acidic phospholipids with a specificity indicating an interaction of p65 with the hydrophobic core as well as the headgroups of the phospholipids. The binding specificity resembles PKC, except that p65 also binds calmodulin, placing the C2-regions in a context of potential Ca2(+)-regulation that is different from PKC. This is a novel homology between a cellular protein and the regulatory domain of protein kinase C. The structure and properties of p65 suggest that it may have a role in mediating membrane interactions during synaptic vesicle exocytosis.     

Calcium-dependent enhancement of calcium current in smooth muscle by calmodulin-dependent protein kinase II.

Calcium entry through voltage-activated Ca2+ channels is important in regulating many cellular functions. Activation of these channels in many cell types results in feedback regulation of channel activity. Mechanisms linking Ca2+ channel activity with its downregulation have been described, but little is known of the events responsible for the enhancement of Ca2+ current that in many cells follows Ca2+ channel activation and an increase in cytoplasmic Ca2+ concentration. Here we investigate how this positive feedback is achieved in single smooth muscle cells. We find that in these cells voltage-activated calcium current is persistently but reversibly enhanced after periods of activation. This persistent enhancement of the Ca2+ current is mediated by activation of calmodulin-dependent protein kinase II because it is blocked when either the rise in cytoplasmic Ca2+ is inhibited or activation of calmodulin-dependent protein kinase II is prevented by specific peptide inhibitors of calcium-calmodulin or calmodulin-dependent protein kinase II itself. This mechanism may be important in different forms of Ca2+ current potentiation, such as those that depend on prior Ca2+ channel activation or are a result of agonist-induced release of Ca2+ from internal stores.     

Synapsin I is a spectrin-binding protein immunologically related to erythrocyte protein 4.1

The membrane-associated cytoskeleton is considered to be the apparatus by which cells regulate the properties of their plasma membranes, although recent evidence has indicated additional roles for the proteins of this structure, including an involvement in intracellular transport and exocytosis (see refs 1-3 for review). Of the membrane skeletal proteins, to date only spectrin (fodrin) and ankyrin have been purified and characterized from non-erythroid sources. Protein 4.1 in the red cell is a spectrin-binding protein that enhances the binding of spectrin to actin and can apparently bind to at least one transmembrane protein Immunoreactive forms of 4.1 have been detected in several cell types, including brain. Here we report the purification of brain 4.1 on the basis of its cross-reactivity with erythrocyte 4.1 and spectrin-binding activity. We further show that brain 4.1 is identical to the synaptic vesicle protein, synapsin I, one of the brain's major substrates for cyclic AMP and Ca2+-calmodulin-dependent kinases. Spectrin and synapsin are present in brain homogenates in an approximately 1:1 molar ratio. Although synapsin I has been implicated in synaptic transmission, no activity has been previously ascribed to it.     

Interaction with the NMDA receptor locks CaMKII in an active conformation.

Calcium- and calmodulin-dependent protein kinase II (CaMKII) and glutamate receptors are integrally involved in forms of synaptic plasticity that may underlie learning and memory. In the simplest model for long-term potentiation, CaMKII is activated by Ca2+ influx through NMDA (N-methyl-D-aspartate) receptors and then potentiates synaptic efficacy by inducing synaptic insertion and increased single-channel conductance of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors. Here we show that regulated CaMKII interaction with two sites on the NMDA receptor subunit NR2B provides a mechanism for the glutamate-induced translocation of the kinase to the synapse in hippocampal neurons. This interaction can lead to additional forms of potentiation by: facilitated CaMKII response to synaptic Ca2+; suppression of inhibitory autophosphorylation of CaMKII; and, most notably, direct generation of sustained Ca2+/calmodulin (CaM)-independent (autonomous) kinase activity by a mechanism that is independent of the phosphorylation state. Furthermore, the interaction leads to trapping of CaM that may reduce down-regulation of NMDA receptor activity. CaMKII-NR2B interaction may be prototypical for direct activation of a kinase by its targeting protein.     

Intracellular calcium dependence of transmitter release rates at a fast central synapse

Calcium-triggered fusion of synaptic vesicles and neurotransmitter release are fundamental signalling steps in the central nervous system. It is generally assumed that fast transmitter release is triggered by elevations in intracellular calcium concentration ([Ca2+]i) to at least 100 microM near the sites of vesicle fusion. For synapses in the central nervous system, however, there are no experimental estimates of this local [Ca2+]i signal. Here we show, by using calcium ion uncaging in the large synaptic terminals of the calyx of Held, that step-like elevations to only 10 microM [Ca2+]i induce fast transmitter release, which depletes around 80% of a pool of available vesicles in less than 3 ms. Kinetic analysis of transmitter release rates after [Ca2+]i steps revealed the rate constants for calcium binding and vesicle fusion. These show that transient (around 0.5 ms) local elevations of [Ca2+]i to peak values as low as 25 microM can account for transmitter release during single presynaptic action potentials. The calcium sensors for vesicle fusion are far from saturation at normal release probability. This non-saturation, and the high intracellular calcium cooperativity in triggering vesicle fusion, make fast synaptic transmission very sensitive to modulation by changes in local [Ca2+]i.     

Activation of chloride channels in normal and cystic fibrosis airway epithelial cells by multifunctional calcium/calmodulin-dependent protein kinase.

Cystic fibrosis is associated with defective regulation of apical membrane chloride channels in airway epithelial cells. These channels in normal cells are activated by cyclic AMP-dependent protein kinase and protein kinase C. In cystic fibrosis these kinases fail to activate otherwise normal Cl- channels. But Cl- flux in cystic fibrosis cells, as in normal cells, can be activated by raising intracellular Ca2+ (refs 5-10). We report here whole-cell patch clamp studies of normal and cystic fibrosis-derived airway epithelial cells showing that Cl- channel activation by Ca2+ is mediated by multifunctional Ca2+/calmodulin-dependent protein kinase. We find that intracellular application of activated kinase and ATP activates a Cl- current similar to that activated by a Ca2+ ionophore, that peptide inhibitors of either the kinase or calmodulin block Ca2(+)-dependent activation of Cl- channels, and that a peptide inhibitor of protein kinase C does not block Ca2(+)-dependent activation. Ca2+/calmodulin activation of Cl- channels presents a pathway with therapeutic potential for circumventing defective regulation of Cl- channels in cystic fibrosis.     

Induction of formation of presynaptic terminals in neuroblastoma cells by synapsin IIb

The synapsins are a family of closely related phosphoproteins (termed synapsins Ia, Ib, IIa and IIb) associated with synaptic vesicles and implicated in the short-term regulation of neurotransmitter release from nerve endings. During development, expression of the synapsins correlates temporally with synapse formation, but there has been no direct evidence that they are involved in synaptogenesis. Here we report that overexpression of synapsin IIb in the neuroblastoma x glioma hybrid clonal cell line NG108-15 leads, during cell differentiation, to marked increases in the number of neuritic varicosities and in the numbers of small clear vesicles and large dense core vesicles per varicosity, as well as to the appearance of synapse-like cell-cell contacts. Thus, synapsin IIb may be involved in the regulation of synapse formation and, as a result, in long-term neuronal signalling.     

cGMP-dependent protein kinase enhances Ca2+ current and potentiates the serotonin-induced Ca2+ current increase in snail neurones

Protein phosphorylation catalysed by cyclic AMP-dependent, Ca2+/calmodulin-dependent and Ca2+/diacylglycerol-dependent protein kinases is important both in the modulation of synaptic transmission and in the regulation of neuronal membrane permeability (for reviews see refs 5-7). However, there has previously been no evidence for the involvement of cyclic GMP-dependent protein kinase (cGMP-PK) in the regulation of neuronal function. Serotonin induces an increase of Ca2+ current in a group of identified ventral neurones of the snail Helix aspersa. This effect is probably mediated by cGMP because it is mimicked by the intracellular injection of cGMP or the application of zaprinast, an inhibitor of cGMP-dependent phosphodiesterase. We have now found that the effect of either serotonin or zaprinast on the Ca2+ current is potentiated by the intracellular injection of cGMP-PK. Moreover, the intracellular injection of activated cGMP-PK (cGMP-PK + 1 microM cGMP) greatly enhances the Ca2+ current of the identified ventral neurones seen in the absence of serotonin. These results indicate that cGMP-PK has a physiological role in the control of the membrane permeability of these neurones.     

Allosteric modulation of the presynaptic Ca2+ sensor for vesicle fusion

Neurotransmitter release is triggered by an increase in the cytosolic Ca2+ concentration ([Ca2+]i), but it is unknown whether the Ca2+-sensitivity of vesicle fusion is modulated during synaptic plasticity. We investigated whether the potentiation of neurotransmitter release by phorbol esters, which target presynaptic protein kinase C (PKC)/munc-13 signalling cascades, exerts a direct effect on the Ca2+-sensitivity of vesicle fusion. Using direct presynaptic Ca2+-manipulation and Ca2+ uncaging at a giant presynaptic terminal, the calyx of Held, we show that phorbol esters potentiate transmitter release by increasing the apparent Ca2+-sensitivity of vesicle fusion. Phorbol esters potentiate Ca2+-evoked release as well as the spontaneous release rate. We explain both effects by an increased fusion 'willingness' in a new allosteric model of Ca2+-activation of vesicle fusion. In agreement with an allosteric mechanism, we observe that the classically high Ca2+ cooperativity in triggering vesicle fusion (approximately 4) is gradually reduced below 3 microM [Ca2+]i, reaching a value of <1 at basal [Ca2+]i. Our data indicate that spontaneous transmitter release close to resting [Ca2+]i is a consequence of an intrinsic property of the molecular machinery that mediates synaptic vesicle fusion.     

Alpha-neurexins couple Ca2+ channels to synaptic vesicle exocytosis

Synapses are specialized intercellular junctions in which cell adhesion molecules connect the presynaptic machinery for neurotransmitter release to the postsynaptic machinery for receptor signalling. Neurotransmitter release requires the presynaptic co-assembly of Ca2+ channels with the secretory apparatus, but little is known about how synaptic components are organized. Alpha-neurexins, a family of >1,000 presynaptic cell-surface proteins encoded by three genes, link the pre- and postsynaptic compartments of synapses by binding extracellularly to postsynaptic cell adhesion molecules and intracellularly to presynaptic PDZ domain proteins. Using triple-knockout mice, we show that alpha-neurexins are not required for synapse formation, but are essential for Ca2+-triggered neurotransmitter release. Neurotransmitter release is impaired because synaptic Ca2+ channel function is markedly reduced, although the number of cell-surface Ca2+ channels appears normal. These data suggest that alpha-neurexins organize presynaptic terminals by functionally coupling Ca2+ channels to the presynaptic machinery.