Syntaxin 1A


Proteins that interact with syntaxin: Synaptotagmin

Synaptotagmin I is a synaptic vesicle protein that is thought to act as a Ca2+ sensor in neurotransmitter release. The first C2 domain of synaptotagmin I (C2A domain) contains a bipartite calcium ion-binding motif and interacts in a calcium ion-dependent manner with syntaxin, a central component of the membrane fusion complex. This interaction is mediated by the cooperative action of basic residues surrounding the calcium ion-binding sites of the C2A domain and is driven by a change in the electrostatic potential of the C2A domain induced by Ca2+ binding. A model is proposed whereby synaptotagmin acts as an electrostatic switch in calcium ion-triggered synaptic vesicle exocytosis, promoting a structural rearrangement in the fusion machinery that is effected by its interaction with syntaxin (Shao, 1997).

Syntaxin 1A plays a central role in neurotransmitter release through multiple protein-protein interactions. NMR spectroscopy was used to identify an autonomously folded N-terminal domain in syntaxin 1A and to elucidate its three-dimensional structure. This 120-residue N-terminal domain is conserved in plasma membrane syntaxins but not in other syntaxins, indicating a specific role in exocytosis. The domain contains three long alpha helices that form an up-and-down bundle with a left-handed twist. A striking residue conservation is observed throughout a long groove that is likely to provide a specific surface for protein-protein interactions. A highly acidic region binds to the C2A domain of synaptotagmin I in a Ca2+-dependent interaction that may serve as an electrostatic switch in neurotransmitter release (Fernandez, 1998).

A remarkable feature of neurotransmitter release is that it occurs very fast after Ca2+ influx. Thus, the synaptic vesicles that are "ready to fuse" appear to be in a metastable state that is hindered to proceed toward fusion in the absence of Ca2+. Based on a previous structural analyses of the C2A domain, it was predicted that the region of syntaxin involved in Ca2+-dependent binding to the C2A domain is highly negatively charged, and it was proposed that the switch in electrostatic potential caused by Ca2+ binding to the C2A domain causes the change in affinity for syntaxin. The results described in this paper demonstrate that the region of the N-terminal sequence of syntaxin that binds to the C2A domain is indeed highly acidic. Given the high density of negative charge in the Ca2+-binding region of the C2A domain before Ca2+ binding, it is most likely that the N-terminal sequence of syntaxin and the C2A domain repel each other in the absence of Ca2+. Such repulsion may be the force that prevents synaptic vesicle exocytosis to proceed before Ca2+ influx into a presynaptic terminal. Upon nerve stimulation, Ca2+ binding to the C2A domain could attract syntaxin, initiating fusion. Whether the C2A domain interaction with the N-terminal sequence of syntaxin is physiologically relevant remains to be demonstrated, since the C2A domain has also been shown to bind in a Ca2+-dependent manner to the C-terminal region of syntaxin and to negatively charged phospholipid vesicles. The picture that emerges shows that the N-terminal sequence of syntaxin is a highly conserved, independently folded domain of syntaxin with the following features: (1) it is covalently linked to the C-terminal region of syntaxin, which may be directly involved in membrane fusion; (2) it contains a synaptotagmin-binding site, which may be a part of the Ca2+ trigger; (3) it forms a highly conserved groove between two helices that may bind munc13, munc18, and/or the C-terminal region of syntaxin, with potential regulatory roles in exocytosis. These characteristics suggest that the N-terminal sequence of syntaxin may act as a multifunctional domain in neurotransmitter release. It will be interesting to study which protein(s) directly interacts with the groove and what the consequences are in vivo of mutations introduced in this groove (Fernandez, 1998).

Synaptotagmin 1 probably functions as a Ca2+ sensor in neurotransmitter release via its two C2-domains, but no common Ca2+-dependent activity that could underlie a cooperative action between them has been described. The NMR structure of the C2B-domain now reveals a ß sandwich that exhibits striking similarities and differences with the C2A-domain. Whereas the bottom face of the C2B-domain has two additional alpha helices that may be involved in specialized Ca2+-independent functions, the top face binds two Ca2+ ions and is remarkably similar to the C2A-domain. Consistent with these results, the C2B-domain binds phospholipids in a Ca2+-dependent manner similar to the C2A-domain. Evolutionary analysis has revealed that the C-terminal helix is fully conserved in the synaptotagmin 1 homologs reported from Drosophila and C. elegans, but is absent in those from squid and Aplysia, suggesting that either synaptotagmin 1 is different in molluscs or the synaptotagmins described for molluscs are not true synaptotagmin 1 homologs. These results suggest a novel view of synaptotagmin function whereby the two C2-domains cooperate in a common activity, Ca2+-dependent phospholipid binding, to trigger neurotransmitter release (Fernandez, 2001).

Synaptotagmin, a major intrinsic membrane protein of synaptic vesicles that binds Ca2+, was purified from bovine brain and immobilized onto Sepharose 4B. Affinity chromatography of brain membrane proteins on immobilized synaptotagmin reveals binding of alpha- and beta-neurexins to synaptotagmin in a calcium ion-independent manner. Synaptotagmin specifically interacts with the cytoplasmic domains of neurexins (see Neurexin) but not of control proteins. This interaction is dependent on a highly conserved, 40 amino acid sequence that makes up most of the cytoplasmic tails of the neurexins. These data suggest a direct interaction between the cytoplasmic domains of a plasma membrane protein (neurexin) and a protein specific for a subcellular organelle (synaptotagmin). Such an interaction could have an important role in the docking and targeting of synaptic vesicles in the nerve terminal (Hata, 1993).

C2-domains are widespread protein modules with diverse Ca2+-regulatory functions. Although multiple Ca2+ ions are known to bind at the tip of several C2-domains, the exact number of Ca2+-binding sites and their functional relevance are unknown. The first C2-domain of synaptotagmin I is believed to play a key role in neurotransmitter release via its Ca2+-dependent interactions with syntaxin and phospholipids. The Ca2+-binding mode of this C2-domain has been studied as a prototypical C2-domain using NMR spectroscopy and site-directed mutagenesis. The C2-domain is an elliptical module composed of a beta-sandwich with a long axis of 50 A. The C2-domain is found to bind three Ca2+ ions in a tight cluster spanning only 6 A at the tip of the module. The Ca2+-binding region is formed by two loops whose conformation is stabilized by Ca2+ binding. Binding involves one serine and five aspartate residues that are conserved in numerous C2-domains. All three Ca2+ ions are required for the interactions of the C2-domain with syntaxin and phospholipids. These results support an electrostatic switch model for C2-domain function whereby the beta-sheets of the domain provide a fixed scaffold for the Ca2+-binding loops, and whereby interactions with target molecules are triggered by a Ca2+-induced switch in electrostatic potential (Ubach, 1998).

Synaptotagmins constitute a large family of membrane proteins implicated in Ca2+-triggered exocytosis. Structurally similar synaptotagmins are differentially localized either to secretory vesicles or to plasma membranes, suggesting distinct functions. Using measurements of the Ca2+ affinities of synaptotagmin C2-domains in a complex with phospholipids, it has been shown that different synaptotagmins exhibit distinct Ca2+ affinities, with plasma membrane synaptotagmins binding Ca2+ with a 5- to 10-fold higher affinity than vesicular synaptotagmins. To test whether these differences in Ca2+ affinities are functionally important, the effects of synaptotagmin C2-domains on Ca2+-triggered exocytosis were examined in permeabilized PC12 cells. A precise correlation is observed between the apparent Ca2+ affinities of synaptotagmins in the presence of phospholipids and their action in PC12 cell exocytosis. This is extended to PC12 cell exocytosis triggered by Sr2+, which is also selectively affected by high-affinity C2-domains of synaptotagmins. Together, these results suggest that Ca2+ triggering of exocytosis involves tandem Ca2+ sensors provided by distinct plasma membrane and vesicular synaptotagmins. According to this hypothesis, plasma membrane synaptotagmins represent high-affinity Ca2+ sensors involved in slow Ca2+-dependent exocytosis, whereas vesicular synaptotagmins function as low-affinity Ca2+ sensors specialized for fast Ca2+-dependent exocytosis (Sugita, 2002).

The ascidian embryo, a model for the primitive mode of chordate development, rapidly forms a dorsal nervous system that consists of a small number of neurons. The transcriptional regulation of an ascidian synaptotagmin (syt) gene has been characterized to explore the molecular mechanisms underlying development of synaptic transmission. In situ hybridization has shown that syt is expressed in all neurons transiently in the embryonic epidermis. Neuronal expression of syt requires induction from the vegetal side of the embryo, whereas epidermal expression occurs autonomously in isolated ectodermal blastomeres. Introduction of green fluorescent protein reporter gene constructs into the ascidian embryos indicates that a genomic fragment of the 3.4-kb 5' upstream region contains promoter elements of syt gene. Deletion analysis of the promoter suggests that syt expression in neurons and in the embryonic epidermis depends on distinct cis-regulatory regions. The region between -1680 and -824 contains the ability to enhance neuronal expression. The construct lacking sequence between -2223 and -824 is capable of inducing neuronal gene expression in all injected larvae, indicating that the region distal to -2223 has enhancing activity of neuronal expression that can substitute for the region between -2223 and -824 (Katsuyma, 2002).

Decades ago it was proposed that exocytosis involves invagination of the target membrane, resulting in a highly localized site of contact between the bilayers destined to fuse. The vesicle protein synaptotagmin-I (syt) bends membranes in response to Ca(2+), but whether this drives localized invagination of the target membrane to accelerate fusion has not been determined. Previous studies relied on reconstituted vesicles that were already highly curved and used mutations in syt that were not selective for membrane-bending activity. This study, directly addresses this question by utilizing vesicles with different degrees of curvature. A tubulation-defective syt mutant was able to promote fusion between highly curved SNARE-bearing liposomes but exhibited a marked loss of activity when the membranes were relatively flat. Moreover, bending of flat membranes by adding an N-BAR domain rescued the function of the tubulation-deficient syt mutant. Hence, syt-mediated membrane bending is a critical step in membrane fusion (Hui, 2009).

Proteins that interact with syntaxin: Rop, n-sec-1, or Munc 18

Sec1 from the yeast Saccharomyces cerevisiae is a hydrophilic protein that plays an essential role in exocytosis . Two high copy suppressors of mutations in the Sec1 gene, SSO1 and SSO2, have been identified that encode proteins of the syntaxin family. Syntaxin (a T-SNARE), together with SNAP-25 and synaptobrevin/VAMP (a T- and a V-SNARE, respectively), are thought to form the core of the docking-fusion complex in synaptic vesicle exocytosis. Proteins that exhibit similarity to Sec1 were identified in the nervous system of Drosophila (Rop) and C. elegans (UNC18). Based on the amino acid sequence alignment of Sec1, Rop, and UNC18, a PCR-based approach was used to isolate a rat brain cDNA encoding a Sec1 homolog. The cDNA hybridizes to a 3.5-kb brain-specific mRNA by Northern blot analysis and encodes a protein of 593 amino acids (rbSec1). Antibodies raised against a central portion of rbSec1 recognize a 67.5-kDa protein in total homogenates of rat brain but not of nonneuronal tissues. When incubated with a Triton X-100 brain extract, rbSec1 specifically interacts with syntaxin but not with SNAP-25 or synaptobrevin/VAMP. It is concluded that the function of proteins of the Sec1 family in membrane fusion involves an interaction with a T-SNARE (Garcia, 1994).

Cyclin-dependent kinases (Cdks), which regulate the cell division cycle, have also been found in postmitotic neurons. Cdk5 (see Drosophila Cyclin-dependent kinase 5), isolated from neural tissue, has been shown to phosphorylate neurofilaments (NFs). However, instead of cyclins, other neuron-specific activators of cdk5 have been identified, including a 67-kD protein (p67) which is identical to a syntaxin-binding protein (n-sec-1, Munc 18) that is thought to play a role in synaptic vesicle trafficking and transmitter release. These functions for p67 are not mutually exclusive since regulation of cdk5 phosphorylation of cytoskeletal proteins may modulate axonal dynamics during growth, synaptogenesis and vesicle transport. To gain further insight into the role of p67 in neural tissue, an analysis of the developing rat cerebellum was carried out using antibodies to cdk5, p67, syntaxin and phosphorylated and nonphosphorylated neurofilaments. All these antigens are developmentally regulated, and increase in expression from post-natal day 2 to the adult, with p67 and cdk5 showing a close temporal correlation. p67 colocalizes with cdk5 and P-NFH in selected fiber tracts, particularly those in the deep cerebellum. For the most part, p67 also shows strong colocalization patterns with syntaxin in regions of synaptogenesis throughout development such as the molecular layer and glomeruli of the inner nuclear layer. Finally, certain fiber tracts (the afferent fibers, climbing and mossy fibers and particularly the basket cell fibers that envelop and innervate Purkinje cell somata and dendrites) display colocalization of cdk5 and P-NFH without expressing any p67 (Veeranna, 1997).

The docking/fusion of transport vesicles mediated by the soluble NSF attachment protein receptors (SNAREs) is thought to be regulated by Sec1-related proteins. Munc-18-2, a member of this family, is predominantly expressed in the epithelial cells of several tissues. Munc-18-2 colocalizes with syntaxin 3 at the apical plasma membrane of intestinal epithelium and Caco-2 cells. The presence of a physical complex of the two proteins is verified by 2-way coimmunoprecipitation. The quantity of the complex is reduced by treatment of Caco-2 cells with the alkylating agent N-ethylmaleimide, which also has an inhibitory effect on the ability of Munc-18-2 to associate with syntaxin 3 in vitro. The amount of Munc-18-2 in the complex increases upon treatment of the cells with the protein kinase C activator phorbol myristate acetate, indicating a functional connection between the complex and cell signaling. Increasing the amount of Munc-18-2 bound to syntaxin 3 by overexpression results in a marked decrease in the SNARE proteins SNAP-23 and cellubrevin, which are bound to the syntaxin. These results define a novel functional complex of Munc-18-2 and syntaxin 3 involved in the regulation of apical membrane transport (Riento, 1998).

The Munc-18/syntaxin 1A complex has been postulated to act as a negative control on the regulated exocytotic process because its formation blocks the interaction of syntaxin with vesicle SNARE proteins. However, the formation of this complex is simultaneously essential for the final stages of secretion as evidenced by the necessity of Munc-18's homologs in Saccharomyces cerevisiae (Sec1p), Drosophila (ROP), and Caenorhabditis elegans (Unc-18) for proper secretion in these organisms. As such, any event that regulates the interaction of these two proteins is important for the control of secretion. One candidate for such regulation is cyclin-dependent kinase 5 (Cdk5), a member of the Cdc2 family of cell division cycle kinases that has recently been copurified with Munc-18 from rat brain. The present study shows that Cdk5 bound to its neural specific activator p35 not only binds to Munc-18 but utilizes it as a substrate for phosphorylation. Furthermore, it is demonstrated that Munc-18, when it has been phosphorylated by Cdk5, has a significantly reduced affinity for syntaxin 1A. Cdk5 can also bind to syntaxin 1A and a complex of Cdk5, p35, Munc-18, and syntaxin 1A can be fashioned in the absence of ATP and promptly disassembled upon the addition of ATP. These results suggest a model in which p35-activated Cdk5 becomes localized to the Munc-18/syntaxin 1A complex by its affinity for both proteins so that it may phosphorylate Munc-18 and thus permit the positive interaction of syntaxin 1A with upstream protein effectors of the secretory mechanism (Shuang, 1998).

Syntaxin 1, an essential protein in synaptic membrane fusion, contains a helical autonomously folded N-terminal domain, a C-terminal SNARE motif and a transmembrane region. The SNARE motif binds to synaptobrevin and SNAP-25 to assemble the core complex, whereas almost the entire cytoplasmic sequence participates in a complex with munc18-1, a neuronal Sec1 homolog. It is demonstrate by NMR spectroscopy that, in isolation, syntaxin adopts a 'closed' conformation. This default conformation of syntaxin is incompatible with core complex assembly, which requires an 'open' syntaxin conformation. Using site-directed mutagenesis, it has been found that disruption of the closed conformation abolishes the ability of syntaxin to bind to munc18-1 and to inhibit secretion in PC12 cells. These results indicate that syntaxin binds to munc18-1 in a closed conformation and suggest that this conformation represents an essential intermediate in exocytosis. The data suggest a model whereby, during exocytosis, syntaxin undergoes a large conformational switch that mediates the transition between the syntaxin-munc18-1 complex and the core complex (Dulubova, 1999).

The finding that the inhibition of secretion by the transfected cytoplasmic region of syntaxin 1 probably occurs by sequestering munc18-1 from the exocytotic machinery points to a critical role for munc18-1 in membrane fusion. Such a role is in agreement with the strong phenotypes observed in mutants of munc18 and munc18 homologs. At the same time, the requirement for a closed conformation of syntaxin 1 for munc18-1 binding implies that the closed conformation constitutes an obligatory intermediate in membrane fusion. All these results suggest that syntaxin switches between two conformations during exocytosis, a closed conformation that binds munc18-1 and an open conformation that forms the core complex. An N- to C-terminal interaction in syntaxin has been proposed to regulate core complex formation, perhaps by preventing reassembly after the action of alpha-SNAP/NSF disassembles the complex. Since munc18-1 binding requires a closed conformation of syntaxin 1 and prevents formation of the core complex, the question can be raised: is the primary role of the closed conformation of syntaxin 1 and its interaction with munc18-1 to ensure the proper timing of core complex formation? This possibility is difficult to reconcile with the results of transfection experiments in PC12 cells (which suggest an obligatory role for munc18-1 in exocytosis), and with the complete block of secretion caused by mutations in munc18. It is most likely that the multiprotein complex that regulates membrane fusion acts as a 'well oiled machine' that requires a minimal number of pieces to function properly. This would imply that the munc18-syntaxin complex performs a function in fusion that is as central as that of the core complex itself. As a result, at least one additional factor, perhaps with an enzymatic role, would be required to catalyze the conformational change in syntaxin 1 that mediates the transition between the munc18-syntaxin complex and the core complex. Unraveling the nature of the factor, and which of the two complexes involving syntaxin occurs first during exocytosis, are two of the main challenges faced by researchers seeking to understand the mechanism of intracellular membrane fusion (Dulubova, 1999).

Proteins that interact with syntaxin: unc13 and Munc13

unc-13 (Drosophila homolog: unc-13) mutants in Caenorhabditis elegans are characterized by a severe deficit in neurotransmitter release. Their phenotype is similar to that of the C. elegans unc-18 mutation, which is thought to affect synaptic vesicle docking to the active zone. This suggests a crucial role for the unc-13 gene product in the mediation or regulation of synaptic vesicle exocytosis. Munc13-1 is one of three closely related rat homologs of unc-13. Based on the high degree of similarity between unc-13 and Munc13 proteins, it is thought that their essential function has been conserved from C. elegans to mammals. Munc13-1 is a brain-specific peripheral membrane protein with multiple regulatory domains that may mediate diacylglycerol, phospholipid, and calcium binding. The C-terminus of Munc13-1 interacts directly with a putative coiled coil domain in the N-terminal part of syntaxin. Syntaxin is a component of the exocytotic synaptic core complex, a heterotrimeric protein complex with an essential role in transmitter release. Through this interaction, Munc13-1 binds to a subpopulation of the exocytotic core complex containing synaptobrevin, SNAP25 (synaptosomal-associated protein of 25 kDa), and syntaxin, but to no other tested syntaxin-interacting or core complex-interacting protein. The site of interaction in syntaxin is similar to the binding site for the unc-18 homolog Munc18, but different from that of all other known syntaxin interactors. These data indicate that unc-13-related proteins may indeed be involved in the mediation or regulation of synaptic vesicle exocytosis by modulating or regulating core complex formation. The similarity between the unc-13 and unc-18 phenotypes is paralleled by the coincidence of the binding sites for Munc13-1 and Munc18 in syntaxin. It is possible that the phenotype of unc-13 and unc-18 mutations is caused by the inability of the respective mutated gene products to bind to syntaxin (Betz, 1997).

Presynaptic short-term plasticity is an important adaptive mechanism regulating synaptic transmitter release at varying action potential frequencies. However, the underlying molecular mechanisms are unknown. Genetically defined and functionally unique axonal subpopulations of synapses were examined in excitatory hippocampal neurons that utilize either Munc13-1 or Munc13-2 as synaptic vesicle priming factor. In contrast to Munc13-1-dependent synapses, Munc13-2-driven synapses show pronounced and transient augmentation of synaptic amplitudes following high-frequency stimulation. This augmentation is caused by a Ca2+-dependent increase in release probability and releasable vesicle pool size, and requires phospholipase C activity. Thus, differential expression of Munc13 isoforms at individual synapses represents a general mechanism that controls short-term plasticity and contributes to the heterogeneity of synaptic information coding (Rosenmund, 2002).

The efficacy of synaptic transmission between neurons can be altered transiently during neuronal network activity. This phenomenon of short-term plasticity is (1) a key determinant of network properties; (2) is involved in many physiological processes such as motor control, sound localization, or sensory adaptation, and (3) is critically dependent on cytosolic [Ca2+]. However, the underlying molecular mechanisms and the identity of the Ca2+ sensor/effector complexes involved are unclear. This study identifies a conserved calmodulin binding site in UNC-13/Munc13s, which are essential regulators of synaptic vesicle priming and synaptic efficacy. Ca2+ sensor/effector complexes consisting of calmodulin and Munc13s regulate synaptic vesicle priming and synaptic efficacy in response to a residual [Ca2+] signal and thus shape short-term plasticity characteristics during periods of sustained synaptic activity (Junge, 2004).

Neurons transfer information at chemical synapses. Interestingly, synaptic activity does not only transmit information but also regulates synaptic strength. Such activity-dependent modification of synaptic performance, or synaptic plasticity, is essential for information processing, learning, and memory (Junge, 2004).

Short-term synaptic plasticity (STP) occurs during and after repetitive synaptic activity on a timescale of milliseconds to minutes. It is a key determinant of network processes and is involved in brain functions as diverse as motor control, sensory adaptation, sound localization, and cortical gain control. STP can be expressed either as short-term enhancement (STE) or short-term depression (STD), depending on the initial release probability (Pr) of the synapses involved. High Pr is usually associated with STD, while a low Pr favors STE (Junge, 2004 and references therein).

Depletion of a readily releasable pool of fusion-competent synaptic vesicles (RRP) is a major cause for STD. The generation of this RRP is absolutely dependent on the priming action of UNC-13/Munc13s. The level of STD under steady-state conditions of RRP depletion and replenishment is controlled by a Ca2+-dependent vesicle supply process, of which the molecular mechanism and significance for STP are poorly understood. Calmodulin (CaM) may mediate this Ca2+-dependent process by acting on a subpool of the RRP with high Pr (Junge, 2004 and references therein).

A second well-known form of STP is STE. Three major forms of STE, facilitation, augmentation, and potentiation, can be distinguished based on their lifetime. During sustained activity, the efficacy of release is increased in STE, but it is unclear whether this is due to increased vesicular Pr or RRP size or both. STE is critically dependent on increased concentrations of residual Ca2+ ([Ca2+]res), which accumulates during action potential activity due to incomplete elimination. According to the original residual Ca2+ hypothesis, [Ca2+]res was thought to act on the secretory Ca2+ sensor. However, given the differences in Ca2+ requirements of fast neurotransmitter release and STE, additional, high-affinity Ca2+ sensors likely contribute to STE. The identification of such high-affinity Ca2+ sensors whose characteristics are compatible with the Ca2+ dynamics in presynaptic terminals and of molecules that transduce the residual Ca2+ signal to the secretory machinery during STE is essential for a mechanistic understanding of STE (Junge, 2004 and references therein).

The Munc13 proteins (Munc13-1, the splice isoforms bMunc13-2 and ubMunc13-2, and Munc13-3) are candidate mediators of STP. Genetic studies in mouse, fly, and nematode have established an essential role for this presynaptic protein family in synaptic vesicle priming and RRP generation. Munc13s regulate the SNARE protein Syntaxin and promote SNARE complex formation and fusion competence of synaptic vesicles (Junge, 2004 and references therein).

By determining synaptic vesicle priming, Munc13s modify synaptic strength. The domain structure of Munc13s with several binding sites for second messengers and regulatory proteins indicates that this function is tightly regulated. Indeed, Munc13s are targets of the diacylglycerol (DAG) second messenger pathway. The C1 domain function of Munc13-1 is essential for DAG and phorbol ester (PE) binding and PE potentiation of synaptic amplitudes in hippocampal neurons. Moreover, rescue experiments in Munc13-1/2 double knockout (DKO) neurons show that STE is prevalent in neurons that express only ubMunc13-2, while moderate STD is prominent in neurons expressing only Munc13-1. Thus, Munc13 isoforms can differentially control STP, but the relation of this phenomenon to the long-established role of [Ca2+]res in STP is unknown (Junge, 2004 and references therein).

This study reports that Munc13-1 and ubMunc13-2 bind CaM in a Ca2+-dependent manner via an evolutionarily conserved CaM recognition motif. Using synaptic depression, frequency facilitation, and augmentation protocols in autaptic hippocampal neurons (a special type of neuron that incorporates synaptical positive feedback through recurrent collaterals of its own axons) as a model of STP, it is shown that CaM binding to Munc13 proteins causes increased priming activity and RRP sizes. It is concluded that activation of the CaM/Munc13 complex by [Ca2+]res represents a molecular correlate for the phenomenon of Ca2+-dependent vesicle pool refilling. This mechanism controls STP characteristics and is likely to be evolutionarily conserved (Junge, 2004).

Other proteins that interact with syntaxin

The membrane proteins synaptobrevin, syntaxin, and SNAP-25 form the core of a ubiquitous fusion machine that interacts with the soluble proteins NSF and alpha-SNAP. During regulated exocytosis, membrane fusion is usually strictly controlled by Ca2+ ions. However, the mechanism by which Ca2+ regulates exocytosis is still unclear. The membranes of exocrine secretory granules contain an 18-kDa protein, syncollin, that binds to syntaxin at low Ca2+ concentrations and dissociates at concentrations known to stimulate exocytosis. Syncollin binds to the cytoplasmic domain of syntaxin and also to the C-terminal region (albeit less well), but fails to bind the N-terminal region. alpha-SNAP displaces syncollin from immobilized syntaxin in a concentration dependent manner. Syncollin has a single hydrophobic domain at its N-terminus and shows no significant homology with any known protein. Recombinant syncollin inhibits fusion in vitro between zymogen granules and pancreatic plasma membranes; its potency falls as Ca2+ concentration rises. It has been suggested that syncollin acts as a Ca2(+)-sensitive regulator of exocytosis in exocrine tissues (Edwardson, 1997).

Syntaxin-1 is a component of the synaptic vesicle docking and/or membrane fusion soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) complex (7S and 20S complexes) in nerve terminals. Syntaxin-1 also forms a heterodimer with Munc18/n-Sec1/rbSec1 in a complex that is distinct from the 7S and 20S complexes. A novel syntaxin-1-binding protein, tomosyn (related to Drosophila lethal (2) giant larvae), has been identified that is capable of dissociating Munc18 from syntaxin-1 and forming a novel 10S complex with syntaxin-1, soluble N-etyhlmaleimide-sensitive factor attachment (SNAP) 25, and synaptotagmin. The 130 kDa isoform of tomosyn is specifically expressed in brain, where its distribution partly overlaps with that of syntaxin-1 in nerve terminals. High level expression of either syntaxin-1 or tomosyn results in a specific reduction in Ca2+-dependent exocytosis from PC12 cells. These results suggest that tomosyn is an important component in the neurotransmitter release process where it may stimulate SNARE complex formation (Fujita, 1998).

Syntaxin-1 is a key component of the synaptic vesicle docking/fusion machinery that forms the SNARE complex with VAMP/synaptobrevin and SNAP-25. Identifying proteins that modulate SNARE complex formation is critical for understanding the molecular mechanisms underlying neurotransmitter release and its modulation. A protein called syntaphilin has been cloned and characterized that is selectively expressed in brain. Syntaphilin competes with SNAP-25 for binding to syntaxin-1 and inhibits SNARE complex formation by absorbing free syntaxin-1. Transient overexpression of syntaphilin in cultured hippocampal neurons significantly reduces neurotransmitter release. Furthermore, introduction of syntaphilin into presynaptic superior cervical ganglion neurons in culture inhibits synaptic transmission. These findings suggest that syntaphilin may function as a molecular clamp that controls free syntaxin-1 availability for the assembly of the SNARE complex, and thereby regulates synaptic vesicle exocytosis (Lao, 2000).

Modification of Syntaxin by phosphorylation

Synaptic membrane trafficking proteins are substrates for three kinases implicated in the modulation of synaptic transmission: casein kinase II, calcium/calmodulin-dependent protein kinase II (see Drosophila CaM kinase II), and cAMP-dependent protein kinase (see Drosophila PKA). Each kinase phosphorylates a specific set of the vesicle proteins syntaxin 1A, N-ethylmaleimide-sensitive factor (NSF), vesicle-associated membrane protein (VAMP), synaptosome-associated 25-kDa protein (SNAP-25), n-sec1, alpha soluble NSF attachment protein (alpha SNAP), and synaptotagmin. VAMP is phosphorylated by calcium/calmodulin-dependent protein kinase II on serine 61. alpha SNAP is phosphorylated by PKA; however, the beta SNAP isoform is phosphorylated only 20% as efficiently. alpha SNAP phosphorylated by PKA binds to the core docking and fusion complex with a strength only one-tenth that of the dephosphorylated form. These studies provide a first glimpse at regulatory events that may be important in modulating neurotransmitter release during learning and memory (Hirling, 1996).

Syntaxins and Ca2+ channels

N-type Ca2+ channels bind directly to the synaptic core complex of VAMP/synaptobrevin, syntaxin, and SNAP-25. Peptides containing the synaptic protein interaction ("synprint") site cause dissociation of N-type Ca2+ channels from the synaptic core complex. Introduction of synprint peptides into presynaptic superior cervical ganglion neurons reversibly inhibited synaptic transmission. Fast EPSPs due to synchronous transmitter release are inhibited, while late EPSPs arising from asynchronous release following a train of action potentials are increased and paired-pulse facilitation is increased. The corresponding peptides from L-type Ca2+ channels have no effect, and the N-type peptides have no effect on Ca2+ currents through N-type Ca2+ channels. These results are consistent with the hypothesis that binding of the synaptic core complex to presynaptic N-type Ca2+ channels is required for Ca2+ influx to elicit rapid, synchronous neurotransmitter release (Mochida, 1996).

An electrophysiological assay was used to investigate the functional interaction of syntaxin 1A and SNAP-25 with the class C, L-type, and the class B, N-type, voltage-sensitive calcium channels. Co-expression of syntaxin 1A with the pore-forming subunits of the L- and N-type channels in Xenopus oocytes generates a dramatic inhibition of inward currents (>60%) and modifies the rate of inactivation (tau) and the steady-state voltage-dependence of inactivation. Syntaxin 1-267, which lacks the transmembrane region (TMR), and syntaxin 2 do not modify channel properties, suggesting that the syntaxin 1A interaction site resides predominantly in the TMR. Co-expression of SNAP-25 significantly modifies the gating properties of L- and N-type channels and displays modest inhibition of current amplitude. Syntaxin 1A and SNAP-25 combined restore the syntaxin-inhibited N-type inward current but not the reduced rate of inactivation. Hence, a distinct interaction of a putative syntaxin 1A-SNAP-25 complex with the channel is apparent, consistent with the formation of a synaptosomal SNAP receptors (SNAREs) complex. Three conclusions may be drawn regarding the in vivo functional reconstitution: (1) it establishes the proximity of the SNAREs to calcium channels, (2) provides new insight into a potential regulatory role for the two SNAREs in controlling calcium influx through N- and L-type channels, and (3) it may suggest a pivotal role for calcium channels in the secretion process (Wiser, 1996).

Neurotransmitter release into the synapse is stimulated by calcium influx through ion channels that are closely associated with the transmitter release sites. This link may involve the membrane protein syntaxin, which is known to be associated with the release sites and to bind to the calcium channels. There is evidence that presynaptic calcium channels are downregulated by second messenger pathways involving G proteins. The patch-clamp technique was used to test whether calcium current is regulated by G proteins in a vertebrate presynaptic nerve terminal, and whether this regulation is affected by the linkage to syntaxin. The calcium current in the nerve terminal shows typical G-protein-mediated changes in amplitude and activation kinetics, reversed by a preceding depolarization. These effects of the G protein are virtually eliminated if syntaxin is first cleaved with botulinum toxin C1. These findings indicate that this sensitivity of the current to modulation by G proteins requires the association of the presynaptic calcium channel with elements of the transmitter release site, which may ensure that channels tethered at release sites are preferentially regulated by the G-protein second messenger pathway (Stanley, 1997).

Fast neurotransmission requires that docked synaptic vesicles be located near the presynaptic N-type or P/Q-type calcium channels. Specific protein-protein interactions between a synaptic protein interaction (synprint) site on N-type and P/Q-type channels and the presynaptic SNARE proteins syntaxin, SNAP-25, and synaptotagmin are required for efficient, synchronous neurotransmitter release. Interaction of the synprint site of N-type calcium channels with syntaxin and SNAP-25 shows a biphasic calcium dependence with maximal binding at 10-20 microM. The synprint sites of the BI and rbA isoforms of the alpha1A subunit of P/Q-type Ca2+ channels have different patterns of interactions with synaptic proteins. The BI isoform of alpha1A interacts specifically with syntaxin, SNAP-25, and synaptotagmin, all independent of Ca2+ concentrations and binds with high affinity to the C2B domain of synaptotagmin but not the C2A domain. The rbA isoform of alpha1A interacts specifically with synaptotagmin and SNAP-25 but not with syntaxin. Binding of synaptotagmin to the rbA isoform of alpha1A is Ca2+-dependent, with maximum affinity at 10-20 microM Ca2+. Although the rbA isoform of alpha1A binds well to both the C2A and C2B domains of synaptotagmin, only the interaction with the C2A domain is Ca2+-dependent. These differential, Ca2+-dependent interactions of Ca2+ channel synprint sites with SNARE proteins may modulate the efficiency of transmitter release triggered by Ca2+ influx through these channels (Kim, 1997).

Syntaxins and the sequential assembly of the SNARE assembly in exocytosis

Changes in SNARE conformations during MgATP-dependent priming of cracked PC12 cells were probed by their altered accessibility to various inhibitors. Dominant negative soluble syntaxin and, to a much lesser extent, VAMP coil domains inhibit exocytosis more efficiently after priming. Neurotoxins and an anti-SNAP25 antibody inhibit exocytosis less effectively after priming. It is proposed that SNAREs partially and reversibly assemble during priming, and that the syntaxin H3 domain is prevented from fully joining the complex until the arrival of the Ca2+ trigger. Furthermore, mutation of hydrophobic residues of the SNAP25 C-terminal coil that contribute to SNARE core interactions affects the maximal rate of exocytosis, while mutation of charged residues on the surface of the complex affects the apparent affinity of the coil domain for the partially assembled complex (Chen, 2001).

In neurons and neuroendocrine cells, exocytosis is highly regulated, and therefore SNARE-complex formation must also be highly regulated, since this complex formation catalyzes a late step or perhaps the final step of the membrane fusion reaction. How is the formation of the SNARE complex regulated in Ca2+-triggered exocytosis? Based on functional data obtained in the cracked PC12 cell system, the following is proposed: (1) SNAREs partially and reversibly assemble during priming, so that full assembly can occur very rapidly once the cell is triggered; (2) the syntaxin coil is likely to be less tightly associated than the VAMP and SNAP25 coils and it is proposed that the syntaxin coil is held back by a calcium sensor until the arrival of the Ca2+ trigger; (3) surface and core hydrophobic residues of the SNARE complex play different roles in the assembly process, with only the hydrophobic ones being important for the maximal membrane fusion rates, while the surface residues are important in the initial formation of partially assembled complexes (Chen, 2001).

The first two proposals are supported by the following observations: (1) VAMP-cleaving neurotoxins and an anti-SNAP25 antibody show decreased inhibition efficiency after priming, suggesting reduced accessibility to these two SNAREs. Note that these inhibitors are proteins of relatively large size (>50 kDa) compared to the H3 and V2 coils (8-9 kDa). The fact that the toxins are able to inhibit in primed cells also suggests that trans-SNARE complexes are reversibly (and therefore likely only partially) zippered after priming, since fully zippered complexes resist toxin cleavage. (2) Primed cells are more sensitive to inhibition by the syntaxin H3 coil, and, to a lesser extent, the VAMP2 coil than unprimed cells, suggesting that the SNAREs are more free to bind each other or are loosely associated after priming. However, the inhibition by H3 was increased much more dramatically than that by V2 after priming, suggesting that endogenous VAMP and SNAP25 preferentially associate during priming in vivo. Because the inhibition results obtained with toxins and antibody show effects opposite to those of V2 and H3, it is unlikely that probe accessibility, such as loss of a diffusion barrier, or an artifact of data normalization is the explanation for the enhanced H3 or V2 inhibition. (3) The experiments using soluble V2 + S25 or H3 + S25 as the inhibitors suggest that, whereas endogenous syntaxin is not readily available for exogenous SNARE binding regardless of priming, endogenous VAMP becomes less available for exogenous SNARE binding after priming. Perhaps in unprimed cells, n-sec1 binding to syntaxin prevents it from forming a core complex with V2 + S25, while VAMP is available to bind H3 + S25. In primed cells, however, VAMP and syntaxin both become reversibly complexed with other SNAREs, making both V2 + S25 and H3 + S25 ineffective inhibitors. It is also possible that the binding of a calcium sensor to syntaxin after priming prevents strong binding of V2 + S25 to syntaxin (Chen, 2001).

Taken together, these results are most consistent with the existence of a dynamic, partially assembled SNARE complex lacking the full syntaxin H3 domain. This is consistent with earlier electrophysiological data proposing the existence of loose or partially zippered SNARE complexes. It is proposed that, in vivo, syntaxin might be regulated to fully join the SNARE complex only after VAMP and SNAP25 partially and reversibly assemble. Previous biochemical studies demonstrate a moderate affinity (1-1.4 µM) between SNAP25 and VAMP in vitro, and circular dichroism experiments show that their binding causes an increase in the observed alpha-helicity, suggesting that the VAMP and SNAP25 interaction may occur in vivo, particularly if stabilized by other proteins. Moreover, since syntaxin is the only neuronal SNARE that has a long N-terminal region beyond the coiled-coil-forming domain, it may be the most suitable for regulation by other proteins, such as a Ca2+ sensor. Unfortunately, due to the high propensity of the SNAREs to form unproductive cis complexes in vitro, it is not possible to directly study the nature of the partial or trans-SNARE complexes involved in the priming and triggering steps of exocytosis by common biochemical methods such as immunoprecipitation or SDS-PAGE analysis (Chen, 2001).

Vertebrate syntaxins, the Golgi apparatus, and vesicular trafficking

Continued: see Syntaxin 1A Evolutionary Homologs part 3/3 | back to part 1/3

Syntaxin 1A: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.