SNARE Exocytosis

A second mechanism that impinges on the localization of transporters is through the association with proteins, the most prominent example being syntaxin. Syntaxin is a t-SNARE protein necessary for the fusion of vesicles with the plasma membrane (see the chapter on exocytosis). On the cell surface syntaxin consistently stabilizes the localization of GABA, noradrenaline, glycine, and 5HT transporters the PKCa isoform can sever the interaction with syntaxin suggesting a general mechanism for transporter internalization. 

The most ingenious exocytosis toxins, however, come from the anaerobic bacteria Clostridium botulinum and Clostridium tetani. The former produces the seven botulinum neurotoxins (BoNTs) A-G the latter produces tetanus neurotoxin (TeNT). All eight toxins consist of a heavy (H) chain and a light (L) chain that are associated by an interchain S-S bond. The L-chains enter the cytosol of axon terminals. Importantly, BoNT L-chains mainly enter peripheral cholinergic terminals, whereas the TeNT L-chain mainly enters cerebral and spinal cord GABAergic and glycinergic terminals. The L-chains are the active domains of the toxins. They are zinc-endopeptidases and specifically split the three core proteins of exocytosis, i.e. the SNAREs (Fig. 1 inset). Each ofthe eight toxins splits a... 

Vennekens R, Olausson J, Meissner M, Bloch W, Mathar I, Philipp SE, Schmitz F, Weissgerber R Nilius B, Flockerzi V, Freichel M Increased IgE-dependent mast cell activation and anaphylactic responses in mice lacking the calcium-activated nonselective cation channel XRPM4. Nat Immunol 2007 8 312-320. Guo Z, Xurner C, Castle D Relocation of the t-SNARE SNAP-23 from lamellipodia-like cell surface projections regulates compound exocytosis in mast cells. Cell 1998 94 537-548. 

Puri N. Roche PA Mast cells possess distinct secretory granule subsets whose exocytosis is regulated by different SNARE isoforms. Proc Natl Acad Sci USA 2008 105 2580-2585. 

Upon activation, neurons begin trafficking TRPVl to the membrane where the receptors become activated, desensitized and then recycled to the intracellular compartments. Translocation of TRPVl to the cell membrane occurs via SNARE (snapin and synaptotagmin IX)-mediated exocytosis [37]. Broadly speaking, activation involves phosphorylation by protein kinases (most notably, protein kinase A [PKA] and C [PKC]) and desensitization involves de-phosphorylation by phosphatases (e.g. calcineurin) [38]. Among PKC isozymes, PKCp seems to be of particular importance [39]. 

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. 

Other proteins are also needed. All cell fusion processes seem to require regulatory proteins that are essential to neurotransmission in the nematode C. elegans. Two of these are encoded by the nematode genes unc-13 and unc-18. The corresponding mammalian proteins munc-13 and munc-18 interact with syntaxin and are essential for exocytosis of synaptic vesicles.572 575 An ATPase is also needed for correct functioning of the SNARE complex574 as are other additional proteins.570... 

Most neurotransmitter release occurs by exocytosis of secretory vesicles, which involves the fusion of the secretory vesicles (synaptic vesicles and LDCVs) with the plasma membrane. All intracellular membrane fusion (except for mitochondrial fusion) is thought to operate by the same fundamental mechanism that involves a core machinery composed of four classes of proteins SNARE-proteins, SM-proteins (for... 

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. 

Gasnier B (2000) The loading of neurotransmitters into synaptic vesicle. Biochimie 82 327-37 Giraudo CG, Eng WS, Melia TJ, Rothman JE (2006) A clamping mechanism involved in SNARE-dependent exocytosis. Science 313 676-80... 

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. 4 Stages in synaptic vesicle exocytosis. Putative intermediate steps on the molecular pathway to synaptic vesicle fusion. Vesicle delivery and tethering to the presynaptic membrane most likely involves Rab-proteins and their effectors. So far, the nature of a speculative docking complex (dc) is unclear, but docking appears to be independent from SNARE proteins. In the primed state, SNAREs have assembled into a complex probably stabilized by complexin (Cpx). The fusion reaction is arrested until the intracellular calcium concentration increases. The putative calcium sensor for fast neurotransmitter release, synaptotagmin 1 (Syt), binds to intracellular calcium and in turn triggers fusion by associating with the presynaptic membrane and interacting with the SNARE complex, thereby displacing complexin (Tang et al. 2006).

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). 

Borisovska M, Zhao Y, Tsytsyura Y, Glyvuk N, Takamori S, Matti U, Rettig J, Siidhof T, Bruns D (2005) v-SNAREs control exocytosis of vesicles from priming to fusion. EMBO J. 24 2114-26 Bracher A, Weissenhorn W (2002) Structural basis for the Golgi membrane recruitment of Slylp by Sed5p. EMBO J 21 6114-24... 

---