SNARE Synaptobrevin

The decisive element in exocytosis is the interaction between proteins known as SNAREs that are located on the vesicular membrane (v-SNAREs) and on the plasma membrane (t-SNAREs). In the resting state (1), the v-SNARE synaptobrevin is blocked by the vesicular protein synaptotagmin. When an action potential reaches the presynaptic membrane, voltage-gated Ca "" channels open (see p. 348). Ca "" flows in and triggers the machinery by conformational changes in proteins. Contact takes place between synaptobrevin and the t-SNARE synaptotaxin (2). Additional proteins known as SNAPs bind to the SNARE complex and allow fusion between the vesicle and the plasma membrane (3). The process is supported by the hydrolysis of GTP by the auxiliary protein Rab. 

Synaptic exocytosis involves three SNARE proteins the R-SNARE synaptobrevin/VAMP (isoforms 1 and 2) on the vesicle, and the Q-SNAREs syntaxin (isoforms 1 and 2) and SNAP-25 on the plasma membrane (Figure 4). Since SNAP-25 has two SNARE-motifs, synaptobrevin, syntaxin, and SNAP-25 together have four SNARE-motifs. Synaptobrevins and SNAP-25 are relatively simple SNARE proteins that are composed of little else besides SNARE motifs and membrane-attachment sequences (a transmembrane region for synaptobrevin, and a cysteine-rich palmitoylated sequence for SNAP-25). Syntaxins, in contrast, are complex proteins. The N-terminal two-thirds of syntaxins include a separate, autonomously folded domain (the so-called Habc-domain), while the C-terminal third is composed of a SNARE motif and transmembrane region just like synaptobrevin. 

In neurons, the SNARE complex consists of three main proteins the v-SNARE synaptobrevin or VAMP (vesicle-associated membrane protein), and two t-SNAREs, syntaxin and SNAP-25 (synaptosomal associated protein of 25 kD). Synaptobrevins traverse the synaptic vesicle membrane in an asymmetric manner a few amino acids are found inside the vesicle, but most of the molecule lies outside the vesicle, within the cytoplasm. Synaptobrevin makes contact with another protein anchored to the plasma membrane of the presynaptic neuron, syntaxin, which is associated with SNAP-25. Via these interactions, the SNARE proteins play a role in the docking and fusion of synaptic vesicles to the active zone. 

The structure and interfacial association of the full-length vesicle SNARE, synaptobrevin (I), were compared in 4 different lipid environments using NMR and ESR spectroscopy. In micelles, segments of the SNARE motif were helical and associated with the interface. However, the fraction of helix and interfacial association decreased as I was moved from micelle to bicelle to bilayer environments, indicating that the tendency toward interfacial association was sensitive to membrane curvature. In bilayers, the SNARE motif of I transiently associated with the lipid interface, and regions that were helical in micelles were in conformational and environmental exchange in bicelles and bilayers. ... 

Synaptobrevins (VAMPs) Synaptogyrin Synaptophysins PKA but diverge C-terminally. Synapsins Ia/b contain C-terminal phosphorylation sites for CaMKII and CDK 5. Interact with microfilaments, neurofilaments, microtubules, SH3 domains, calmodulin and annexin VI in vitro. Small-membrane proteins that are cleaved by tetanus toxin and by botulinum toxins B, D, F and G. Polytopic membrane protein that is tyrosine-phosphorylated. Function unknown. Polytopic membrane proteins, including synaptoporin, that are tyrosine-phosphorylated and bind to synaptobrevins. May regulate SNARE function... 

All botulin neurotoxins act in a similar way. They only differ in the amino-acid sequence of some protein parts (Prabakaran et al., 2001). Botulism symptoms are provoked both by oral ingestion and parenteral injection. Botulin toxin is not inactivated by enzymes present in the gastrointestinal tracts. Foodborne BoNT penetrates the intestinal barrier, presumably due to transcytosis. It is then transported to neuromuscular junctions within the bloodstream and blocks the secretion of the neurotransmitter acetylcholine. This results in muscle limpness and palsy caused by selective hydrolysis of soluble A-ethylmalemide-sensitive factor activating (SNARE) proteins which participate in fusion of synaptic vesicles with presynaptic plasma membrane. SNARE proteins include vesicle-associated membrane protein (VAMP), synaptobrevin, syntaxin, and synaptosomal associated protein of 25 kDa (SNAP-25). Their degradation is responsible for neuromuscular palsy due to blocks in acetylcholine transmission from synaptic terminals. In humans, palsy caused by BoNT/A lasts four to six months. 

Dissociated neuronal cultures provide a versatile system for analysis of mechanisms underlying neurotransmitter release. These cultures can be prepared from fetal or postnatal brain tissue. This preparation has been particularly instrumental in analysis of synapses deficient in key components of the release machinery. For instance, genetic deletion of synaptic SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) proteins such as synaptobrevin-2 and SNAP-25... 

The SNAREs involved in the fusion of synaptic vesicles and of secretory granules in neuroendocrine cells, referred to as neuronal SNAREs, have been intensely studied and serve as a paradigm for all SNAREs. They include syntaxin 1A and SNAP-25 at the presynaptic membrane and synaptobrevin 2 (also referred to as VAMP 2) at the vesicle membrane. Their importance for synaptic neurotransmission is documented by the fact that the block in neurotransmitter release caused by botulinum and tetanus neurotoxins is due to proteolysis of the neuronal SNAREs (Schiavo et al. 2000). Genetic deletion of these SNAREs confirmed their essential role in the last steps of neurotransmitter release. Intriguingly, analysis of chromaffin cells from KO mice lacking synaptobrevin or SNAP-25 showed that these proteins can be at least partially substituted by SNAP-23 and cellubrevin, respectively (Sorensen et al. 2003 Borisovska et al. 2005), i.e., the corresponding SNAREs involved in constitutive exocytosis. 

Fig. 1 Structure of the neuronal SNAREs. Upper panel domain structure of the three neuronal SNARE proteins involved in synaptic vesicle fusion. Syntaxin 1A and SNAP-25 (contains two SNARE motifs) are associated with the presynaptic membrane, whereas synaptobrevin 2 is synaptic vesicle associated. The SNARE motifs form a stable complex (core complex) whose crystal structure has been analyzed (lower panel). In the complex, each of the SNARE motifs adopts an alpha-helical structure, and the four alpha-helices are aligned in parallel forming a twisted bundle (modified from Sutton et al. 1998). Stability of the complex is mediated by layers of interaction (—7 to +8) in which amino acids from each of the four alpha-helices participate (see text).

Synaptobrevin 2 is a small protein composed of 118 amino acids. It contains a SNARE motif with a short N-terminal proline-rich extension but lacks an independently folded N-terminal domain. Like syntaxin 1, the protein possesses a C-terminal transmembrane domain that is connected to the SNARE motif by a short linker (Figure 1). Synaptobrevin is palmitoylated at cysteine residues close to its transmembrane domain. Synaptobrevin 2 is highly expressed in neurons and neuroendocrine cells, but unlike syntaxin 1 it is also present in many non-neuronal tissues albeit at low levels. 

Qb-, Qc- and R-SNAREs (Bock and Scheller 2001). Following this classification, syntaxin 1A, SNAP-25, and synaptobrevin 2 represent the Qa-, Qb- and Qc-, and R-SNAREs, respectively (Fasshauer et al. 1998). It turned out later that actually all functional SNARE complexes assigned to trafficking steps in yeast and mammals have a QaQbQcR-composition (Hong 2005 Jahn and Scheller 2006). 

In addition to the proteins discussed above, neuronal SNAREs were reported to interact with numerous other proteins in a specific manner, but in most cases both the structural basis and the biological function of these interactions need to be defined. For instance, synaptophysin, a membrane protein of synaptic vesicles, forms a complex with synaptobrevin in which synaptobrevin is not available for interactions with its partner SNAREs syntaxin 1A and SNAP-25, suggesting that this complex represents a reserve pool of recruitable synaptobrevin (Becher et al. 1999) or regulates interactions between the vesicle-associated synaptobrevin and the plasmalem-mal SNAREs. Alternatively, it has been suggested that this complex is involved in synaptobrevin sorting to synaptic vesicles. 

Tomosyn is a soluble protein of 130 kDa with a C-terminal R-SNARE motif that is capable of replacing synaptobrevin in the neuronal SNARE complex. Most available data indicate that tomosyn negatively regulates exocytosis by competing with synaptobrevin in the formation of SNARE complexes (Brunger 2005), thereby leading to the inhibition of synaptic vesicle priming (McEwen et al. 2006). 

Fig. 5 GPCR regulation of exocytosis downstream of Ca2+-entry. (a) Sequence of steps leading from recruitment to maturation of synaptic vesicles from a reserve pool (RP) to a readily-releasable pool (RRP) displaying slow (asynchronous) and fast (synchronous highly Ca2+-sensitive pool, HCSP synaptotagmin 1 (SYT 1) supported) components, (b) Protein-protein interactions of SNARES (SYX, syntaxin SYB, synaptobrevin and SNAP-2s-7S complex) and major putative regulatory proteins. Phosphoproteins are shown in shaded boxes (phosphorylation sites for PKA and PKC are indicated where known) with phosphorylation-dependent interactions depicted by arrows (increase indicated by filled arrows decrease indicated by open arrows). Circle-end connectors indicate a phosphorylation-independent or as yet unspecified interaction. Potential effects of interactions at various points of the sequence in A are discussed in the text.

Glutamate synapses are specialized for rapid vesicle fusion/neurotransmitter release. Fusion is initiated by SNARE proteins such as syntaxin-1, synaptobrevin-2, and SNAP-25 (see Chapter 2.4 this volume). Presynaptic gene expression, in general, is widely disturbed in schizophrenia (Mimics et al., 2000 Mimics et al., 2001 Hemby et al., 2002), including reduced expression of SNAP-25 RNA(Hemby et al., 2002) and... 

The changes in SNARE protein expression do not appear to be due to the effects of antipsychotic treatment. In rats, haloperidol and chlorpromazine increased SNAP-25 protein levels in the hippocampus (Barr et al., 2006), while in the postmortem brains of individuals with schizophrenia, SNAP-25 levels are lower in the hippocampus (Young et al., 1998 Fatemi et al., 2001 Thompson et al., 2003a). Likewise, no changes in the level of mRNA encoding SNAP-25, syntaxin or synaptobrevin were observed in the prefrontal cortex of rats chronically treated with haloperidol (Nakahara et al., 1998). 

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