Negative regulation of neurotransmitter release by calpain: a possible involvement of specific SNAP-25 cleavage

Synaptic transmission is conducted by neurotransmitters released from presynaptic nerve terminals by means of Ca2+-dependent exocytosis of synaptic vesicles. Formation of a complex of soluble N-ethylmaleimide-sensitive fusion protein receptor (SNARE) proteins, including vesicle-associated membrane protein-2 (VAMP-2) in the synaptic vesicle membrane, and syntaxin 1 and synaptosomal-associated protein of 25 kDa (SNAP-25) in the plasma membrane, is essential for exocytosis. Ionomycin treatment of cultured rat cerebellar granule cells led to cleavage of SNAP-25, but not syntaxin 1 and VAMP-2, that was dependent on extracellular Ca2+. Cleavage was also induced by N-methyl-D-aspartate (NMDA) treatment, but not by depolarization. The use of various site-specific antibodies to SNAP-25, suggested that the cleavage site was in the N-terminal domain of SNAP-25. Calpain inhibitors abolished the Ca2+-dependent cleavage of SNAP-25 and markedly facilitated Ca2+-dependent glutamate (Glu) release from cerebellar granule cells. These results suggest that calpain may play an important role in the long-lasting regulation of synaptic transmission by suppressing neurotransmitter release, possibly through the proteolytic cleavage of SNAP-25.     

Stable SNARE complex prior to evoked synaptic vesicle fusion revealed by fluorescence resonance energy transfer

Although it is clear that soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) complex plays an essential role in synaptic vesicle fusion, the dynamics of SNARE assembly during vesicle fusion remain to be determined. In this report, we employ fluorescence resonance energy transfer technique to study the formation of SNARE complexes. Donor/acceptor pair variants of green fluorescent protein (GFP), cyan fluorescent protein (CFP), and yellow fluorescent protein (YFP) are fused with the N termini of SNAP-25 and synaptobrevin, respectively. In vitro assembly of SNARE core complex in the presence of syntaxin shows strong fluorescence resonance energy transfer (FRET) between the CFP-SNAP-25 and YFP-synaptobrevin. Under the same conditions, CFP fused to the C terminus of SNAP-25, and YFP- synaptobrevin have no FRET. Adenovirus-mediated gene transfer is used to express the fusion proteins in PC12 cells and cultured rat cerebellar granule cells. Strong FRET is associated with neurite membranes and vesicular structures in PC12 cells co-expressing CFP-SNAP-25 and YFP-synaptobrevin. In cultured rat cerebellar granule cells, FRET between CFP-SNAP-25 and YFP-synaptobrevin is mostly associated with sites presumed to be synaptic junctions. Neurosecretion in PC12 cells initiated by KCl depolarization leads to an increase in the extent of FRET. These results demonstrate that significant amounts of stable SNARE complex exist prior to evoked synaptic vesicle fusion and that the assembly of SNARE complex occurs during vesicle docking/priming stage. Moreover, it demonstrates that FRET can be used as an effective tool for investigating dynamic SNARE interactions during synaptic vesicle fusion.     

The R-SNARE motif of tomosyn forms SNARE core complexes with syntaxin 1 and SNAP-25 and down-regulates exocytosis

Tomosyn is a 130-kDa syntaxin-binding protein that contains a large N-terminal domain with WD40 repeats and a C-terminal domain homologous to R-SNAREs. Here we show that tomosyn forms genuine SNARE core complexes with the SNAREs syntaxin 1 and SNAP-25. In vitro studies with recombinant proteins revealed that complex formation proceeds from unstructured monomers to a stable four-helical bundle. The assembled complex displayed features typical for SNARE core complexes, including a profound hysteresis upon unfolding-refolding transitions. No stable complexes were formed between the SNARE motif of tomosyn and either syntaxin or SNAP-25 alone. Furthermore, both native tomosyn and its isolated C-terminal domain competed with synaptobrevin for binding to endogenous syntaxin and SNAP-25 on inside-out sheets of plasma membranes. Tomosyn-SNARE complexes were effectively disassembled by the ATPase N-ethylmaleimide-sensitive factor together with its cofactor alpha-SNAP. Moreover, the C-terminal domain of tomosyn was as effective as the cytoplasmic portion of synaptobrevin in inhibiting evoked exocytosis in a cell-free preparation derived from PC12 cells. Similarly, overexpression of tomosyn in PC12 cells resulted in a massive reduction of exocytosis, but the release parameters of individual exocytotic events remained unchanged. We conclude that tomosyn is a soluble SNARE that directly competes with synaptobrevin in the formation of SNARE complexes and thus may function in down-regulating exocytosis.     

Calmodulin and lipid binding to synaptobrevin regulates calcium-dependent exocytosis

Neurotransmitter release involves the assembly of a heterotrimeric SNARE complex composed of the vesicle protein synaptobrevin (VAMP 2) and two plasma membrane partners, syntaxin 1 and SNAP-25. Calcium influx is thought to control this process via Ca(2+)-binding proteins that associate with components of the SNARE complex. Ca(2+)/calmodulin or phospholipids bind in a mutually exclusive fashion to a C-terminal domain of VAMP (VAMP(77-90)), and residues involved were identified by plasmon resonance spectroscopy. Microinjection of wild-type VAMP(77-90), but not mutant peptides, inhibited catecholamine release from chromaffin cells monitored by carbon fibre amperometry. Pre-incubation of PC12 pheochromocytoma cells with the irreversible calmodulin antagonist ophiobolin A inhibited Ca(2+)-dependent human growth hormone release in a permeabilized cell assay. Treatment of permeabilized cells with tetanus toxin light chain (TeNT) also suppressed secretion. In the presence of TeNT, exocytosis was restored by transfection of TeNT-resistant (Q(76)V, F(77)W) VAMP, but additional targeted mutations in VAMP(77-90) abolished its ability to rescue release. The calmodulin- and phospholipid-binding domain of VAMP 2 is thus required for Ca(2+)-dependent exocytosis, possibly to regulate SNARE complex assembly.     

Inhibition of neurotransmitter release by peptides that mimic the N-terminal domain of SNAP-25

Botulinum neurotoxin serotypes A and E (BoNT/A and BoNT/E) block neurotransmitter release by cleaving the 206-amino-acid SNARE protein, SNAP-25. For each BoNT serotype, cleavage of SNAP-25 results in the loss of intact protein, the production of an N-terminal truncated protein, and the generation of a small C-terminal peptide. Peptides that mimic the C-terminal fragments of SNAP-25 following BoNT/A or BoNT/E cleavage were shown to depress transmitter release in bovine chromaffin cells and in Aplysia buccal ganglion cells. Similarly, the N-terminal–truncated SNAP-25 resulting from BoNT/A or BoNT/E cleavage has been found to inhibit transmitter exocytosis in various systems. With one exception, however, the inhibitory action of truncated SNAP-25 has not been demonstrated at a well-defined cholinergic synapse. The goal of the current study was to determine the level of inhibition of neurotransmitter release by N-terminal BoNT/A- or BoNT/E-truncated SNAP-25 in two different neuronal systems: cholinergically coupled Aplysia neurons and rat hippocampal cell cultures. Both truncated SNAP-25 products inhibited depolarization-dependent glutamate release from hippocampal cultures and depressed synaptic transmission in Aplysia buccal ganglion cells. These results suggest that truncated SNAP-25 can compete with endogenous SNAP-25 for binding with other SNARE proteins involved in transmitter release, thus inhibiting neurotransmitter exocytosis.     

SNARE complex formation is triggered by Ca2+ and drives membrane fusion

Neurotransmitter exocytosis, a process mediated by a core complex of syntaxin, SNAP-25, and VAMP (SNAREs), is inhibited by SNARE-cleaving neurotoxins. Botulinum neurotoxin E inhibition of norepinephrine release in permeabilized PC12 cells can be rescued by adding a 65 aa C-terminal fragment of SNAP-25 (S25-C). Mutations along the hydrophobic face of the S25-C helix result in SNARE complexes with different thermostabilities, and these mutants rescue exocytosis to different extents. Rescue depends on the continued presence of both S25-C and Ca2+ and correlates with complex formation. The data suggest that Ca2+ triggers S25-C binding to a low-affinity site, initiating trans-complex formation. Pairing of SNARE proteins on apposing membranes leads to bilayer fusion and results in a high-affinity cis-SNARE complex.     

CSPα promotes SNARE-complex assembly by chaperoning SNAP-25 during synaptic activity

A neuron forms thousands of presynaptic nerve terminals on its axons, far removed from the cell body. The protein CSPα resides in presynaptic terminals, where it forms a chaperone complex with Hsc70 and SGT. Deletion of CSPα results in massive neurodegeneration that impairs survival in mice and flies. In CSPα-knockout mice, levels of presynaptic SNARE complexes and the SNARE protein SNAP-25 are reduced, suggesting that CSPα may chaperone SNARE proteins, which catalyse synaptic vesicle fusion. Here, we show that the CSPα-Hsc70-SGT complex binds directly to monomeric SNAP-25 to prevent its aggregation, enabling SNARE-complex formation. Deletion of CSPα produces an abnormal SNAP-25 conformer that inhibits SNARE-complex formation, and is subject to ubiquitylation and proteasomal degradation. Even in wild-type mouse terminals, SNAP-25 degradation is regulated by synaptic activity; this degradation is decreased by CSPα overexpression, and enhanced by CSPα deletion. Thus, SNAP-25 function is maintained during rapid SNARE cycles by equilibrium between CSPα-dependent chaperoning and ubiquitin-dependent degradation, revealing unique protein quality-control machinery within the presynaptic compartment.     

Localization and function of soluble N-ethylmaleimide-sensitive factor attachment protein-25 and vesicle-associated membrane protein-2 in functioning gastric parietal cells

The soluble N-ethylmaleimide-sensitive factor attachment protein of 25 kDa (SNAP-25) plays an important role in vesicle trafficking. Together with vesicle-associated membrane protein-2 (VAMP-2) and syntaxin, SNAP-25 forms a ternary complex implicated in docking and fusion of secretory vesicles with the plasma membrane during exocytosis. These so-called SNARE proteins are believed to regulate tubulovesicle trafficking and fusion during the secretory cycle of the gastric parietal cell. Here we examined the cellular localization and functional importance of SNAP-25 in parietal cell cultures. Adenoviral constructs were used to express SNAP-25 tagged with cyan fluorescent protein, VAMP-2 tagged with yellow fluorescent protein, and SNAP-25 in which the C-terminal 25 amino acids were deleted (SNAP-25 Delta181-206). Membrane fractionation experiments and fluorescent imaging showed that SNAP-25 is localized to the apical plasma membrane. The expression of the mutant SNAP-25 Delta181-226 inhibited the acid secretory response of parietal cells. Also, SNAP Delta181-226 bound poorly in vitro with recombinant syntaxin-1 compared with wild type SNAP-25, indicating that pairing between syntaxin-1 and SNAP-25 is required for parietal cell activation. Dual expression of SNAP-25 tagged with cyan fluorescent protein and VAMP-2 tagged with yellow fluorescent protein revealed a dynamic change in distribution associated with acid secretion. In resting cells, SNAP-25 is at the apical plasma membrane and VAMP-2 is associated with cytoplasmic H,K-ATPase-rich tubulovesicles. After stimulation, the two proteins co-localize on the apical plasma membrane. These data demonstrate the functional significance of SNAP-25 as a SNARE protein in the parietal cell and show the dynamic stimulation-associated redistribution of VAMP-2 from H,K-ATPase-rich tubulovesicles to co-localize with SNAP-25 on the apical plasma membrane.     

The last exon of SNAP-23 regulates granule exocytosis from mast cells

SNAP-25 and its ubiquitous homolog SNAP-23 are members of the SNARE family of proteins that regulate membrane fusion during exocytosis. Although SNAP-23 has been shown to participate in a variety of intracellular transport processes, the structural domains of SNAP-23 that are required for its interaction with other SNAREs have not been determined. By employing deletion mutagenesis we found that deletion of the amino-terminal 18 amino acids of SNAP-23 (encoded in the first exon) dramatically inhibited binding of SNAP-23 to both the target SNARE syntaxin and the vesicle SNARE vesicle-associated membrane protein(VAMP). By contrast, deletion of the carboxyl-terminal 23 amino acids (encoded in the last exon) of SNAP-23 does not affect SNAP-23 binding to syntaxin but profoundly inhibits its binding to VAMP. To determine the functional relevance of the modular structure of SNAP-23, we overexpressed SNAP-23 in cells possessing the capacity to undergo regulated exocytosis. Expression of human SNAP-23 in a rat mast cell line significantly enhanced exocytosis, and this effect was not observed in transfectants expressing the carboxyl-terminal VAMP-binding mutant of SNAP-23. Despite considerable amino acid identity, we found that human SNAP-23 bound to SNAREs more efficiently than did rat SNAP-23. These data demonstrate that the introduction of a "better" SNARE binder into secretory cells augments exocytosis and defines the carboxyl terminus of SNAP-23 as an essential regulator of exocytosis in mast cells.     

Two distinct effects on neurotransmission in a temperature-sensitive SNAP-25 mutant.

Vesicle fusion in eukaryotic cells is mediated by SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). In neurons, the t-SNARE SNAP-25 is essential for synaptic vesicle fusion but its exact role in this process is unknown. We have isolated a SNAP-25 temperature-sensitive paralytic mutant in Drosophila, SNAP-25(ts). The mutation causes a Gly50 to Glu change in SNAP-25's first amphipathic helix. A similar mutation in the yeast homologue SEC9 also results in temperature sensitivity, implying a conserved role for this domain in secretion. In vitro-generated 70 kDa SNARE complexes containing SNAP-25(ts) are thermally stable but the mutant SNARE multimers (of approximately 120 kDa) rapidly dissociate at 37 degrees C. The SNAP-25(ts) mutant has two effects on neurotransmitter release depending upon temperature. At 22 degrees C, evoked release of neurotransmitter in SNAP-25(ts) larvae is greatly increased, and at 37 degrees C, the release of neurotransmitter is reduced as compared with controls. Our data suggest that at 22 degrees C the mutation causes the SNARE complex to be more fusion competent but, at 37 degrees C the same mutation leads to SNARE multimer instability and fusion incompetence.     

Regulation of neuronal SNARE assembly by the membrane

In the neuron, SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) assembly acts centrally in driving membrane fusion, a required process for neurotransmitter release. In the cytoplasm, vesicular SNARE VAMP-2 (vesicle-associated membrane protein-2) engages with two plasma membrane SNAREs, syntaxin 1A and SNAP-25 (synaptosome-associated protein of 25 kDa), to form the core complex that bridges two membranes. Although various factors regulate SNARE assembly, the membrane also aids in regulation by trapping VAMP-2 in the membrane. Fluorescence and EPR analyses revealed that the insertion of seven C-terminal core-forming residues into the membrane controls complex formation of the entire core region, even though the preceding 54 core-forming residues are fully exposed and freely moving. When two interfacial tryptophan residues in this region were replaced with hydrophilic serine residues, the mutation supported rapid complex formation. The results suggest that the membrane-proximal region of VAMP-2 is a regulatory module for SNARE assembly, providing new insights into calcium-triggered membrane fusion.     

Chemomechanical Regulation of SNARE Proteins Studied with Molecular Dynamics Simulations

SNAP-25B is a neuronal protein required for neurotransmitter (NT) release and is the target of Botulinum Toxins A and E. It has two SNARE domains that form a four-helix bundle when combined with syntaxin 1A and synaptobrevin. Formation of the three-protein complex requires both SNARE domains of SNAP-25B to align parallel, stretching out a central linker. The N-terminal of the linker has four cysteines within eight amino acids. Palmitoylation of these cysteines helps target SNAP-25B to the membrane; however, these cysteines are also an obvious target for oxidation, which has been shown to decrease SNARE complex formation and NT secretion. Because the linker is only slightly longer than the SNARE complex, formation of a disulfide bond between two cysteines might shorten it sufficiently to reduce secretion by limiting complex formation. To test this idea, we have carried out molecular dynamics simulations of the SNARE complex in the oxidized and reduced states. Indeed, marked conformational differences and a reduction of helical content in SNAP-25B upon oxidation are seen. Further differences are found for hydrophobic interactions at three locations, crucial for the helix-helix association. Removal of the linker induced different conformational changes than oxidation. The simulations suggest that oxidation of the cysteines leads to a dysfunctional SNARE complex, thus downregulating NT release during oxidative stress.     

Novel Interactions of CAPS (Ca2+-dependent Activator Protein for Secretion) with the Three Neuronal SNARE Proteins Required for Vesicle Fusion

CAPS (aka CADPS) is required for optimal vesicle exocytosis in neurons and endocrine cells where it functions to prime the exocytic machinery for Ca²⁺-triggered fusion. Fusion is mediated by trans complexes of the SNARE proteins VAMP-2, syntaxin-1, and SNAP-25 that bridge vesicle and plasma membrane. CAPS promotes SNARE complex formation on liposomes, but the SNARE binding properties of CAPS are unknown. The current work revealed that CAPS exhibits high affinity binding to syntaxin-1 and SNAP-25 and moderate affinity binding to VAMP-2. CAPS binding is specific for a subset of exocytic SNARE protein isoforms and requires membrane integration of the SNARE proteins. SNARE protein binding by CAPS is novel and mediated by interactions with the SNARE motifs in the three proteins. The C-terminal site for CAPS binding on syntaxin-1 does not overlap the Munc18-1 binding site and both proteins can co-reside on membrane-integrated syntaxin-1. As expected for a C-terminal binding site on syntaxin-1, CAPS stimulates SNARE-dependent liposome fusion with N-terminal truncated syntaxin-1 but exhibits impaired activity with C-terminal syntaxin-1 mutants. Overall the results suggest that SNARE complex formation promoted by CAPS may be mediated by direct interactions of CAPS with each of the three SNARE proteins required for vesicle exocytosis.     

In vivo analysis of conserved C. elegans tomosyn domains

Neurosecretion is critically dependent on the assembly of a macromolecular complex between the SNARE proteins syntaxin, SNAP-25 and synaptobrevin. Evidence indicates that the binding of tomosyn to syntaxin and SNAP-25 interferes with this assembly, thereby negatively regulating both synaptic transmission and peptide release. Tomosyn has two conserved domains: an N-terminal encompassing multiple WD40 repeats predicted to form two β-propeller structures and a C-terminal SNARE-binding motif. To assess the function of each domain, we performed an in vivo analysis of the N- and C- terminal domains of C. elegans tomosyn (TOM-1) in a tom-1 mutant background. We verified that both truncated TOM-1 constructs were transcribed at levels comparable to rescuing full-length TOM-1, were of the predicted size, and localized to synapses. Unlike full-length TOM-1, expression of the N- or C-terminal domains alone was unable to restore inhibitory control of synaptic transmission in tom-1 mutants. Similarly, co-expression of both domains failed to restore TOM-1 function. In addition, neither the N- nor C-terminal domain inhibited release when expressed in a wild-type background. Based on these results, we conclude that the ability of tomosyn to regulate neurotransmitter release in vivo depends on the physical integrity of the protein, indicating that both N- and C-terminal domains are necessary but not sufficient for effective inhibition of release in vivo.     

Rabphilin regulates SNARE-dependent re-priming of synaptic vesicles for fusion

Synaptic vesicle fusion is catalyzed by assembly of synaptic SNARE complexes, and is regulated by the synaptic vesicle GTP-binding protein Rab3 that binds to RIM and to rabphilin. RIM is a known physiological regulator of fusion, but the role of rabphilin remains obscure. We now show that rabphilin regulates recovery of synaptic vesicles from use-dependent depression, probably by a direct interaction with the SNARE protein SNAP-25. Deletion of rabphilin dramatically accelerates recovery of depressed synaptic responses; this phenotype is rescued by viral expression of wild-type rabphilin, but not of mutant rabphilin lacking the second rabphilin C2 domain that binds to SNAP-25. Moreover, deletion of rabphilin also increases the size of synaptic responses in synapses lacking the vesicular SNARE protein synaptobrevin in which synaptic responses are severely depressed. Our data suggest that binding of rabphilin to SNAP-25 regulates exocytosis of synaptic vesicles after the readily releasable pool has either been physiologically exhausted by use-dependent depression, or has been artificially depleted by deletion of synaptobrevin.     

Accessory α-Helix of Complexin I Can Displace VAMP2 Locally in the Complexin-SNARE Quaternary Complex

The calcium-triggered neurotransmitter release requires three SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins: synaptobrevin 2 (or vesicle-associated membrane protein 2) on the synaptic vesicle and syntaxin 1 and SNAP-25 (synaptosome-associated protein of 25 kDa) at the presynaptic plasma membrane. This minimal fusion machinery is believed to drive fusion of the vesicle to the presynaptic membrane. Complexin, also known as synaphin, is a neuronal cytosolic protein that acts as a major regulator of synaptic vesicle exocytosis. Stimulatory and inhibitory effects of complexin have both been reported, suggesting the duality of its function. To shed light on the molecular basis of the complexin's dual function, we have performed an EPR investigation of the complexin-SNARE quaternary complex. We found that the accessory α-helix (amino acids 27-48) by itself has the capacity to replace the C-terminus of the SNARE motif of vesicle-associated membrane protein 2 in the four-helix bundle and makes the SNARE complex weaker when the N-terminal region of complexin I (amino acids 1-26) is removed. However, the accessory α-helix remains detached from the SNARE core when the N-terminal region of complexin I is present. Thus, our data show the possibility that the balance between the activities of the accessory α-helix and the N-terminal domain might determine the final outcome of the complexin function, either stimulatory or inhibitory.     

Differential control of the releasable vesicle pools by SNAP-25 splice variants and SNAP-23

The SNARE complex, consisting of synaptobrevin, syntaxin, and SNAP-25, is essential for calcium-triggered exocytosis in neurosecretory cells. Little is known, however, about how developmentally regulated isoforms and other cognate SNARE components regulate vesicular fusion. To address this question, we examined neuroexocytosis from chromaffin cells of Snap25 null mice rescued by the two splice variants SNAP-25a and SNAP-25b and the ubiquitously expressed homolog SNAP-23. In the absence of SNAP-25, vesicle docking persisted, but primed vesicle pools were empty and fast calcium-triggered release abolished. Single vesicular fusion events showed normal characteristics, except for a shorter duration of the fusion pore. Overexpression of SNAP-25a, SNAP-25b, and SNAP-23 resulted in three distinct phenotypes; SNAP-25b induced larger primed vesicle pools than SNAP-25a, whereas SNAP-23 did not support a standing pool of primed vesicles. We conclude that three alternative SNARE components support exocytosis, but they differ in their ability to stabilize vesicles in the primed state.     

Homo- and heterooligomeric SNARE complexes studied by site-directed spin labeling

SNARE (soluble NSF acceptor protein receptor) proteins are thought to mediate membrane fusion by assembling into heterooligomeric complexes that connect the fusing membranes and initiate the fusion reaction. Here we used site-directed spin labeling to map conformational changes that occur upon homo- and heterooligomeric complex formation of neuronal SNARE proteins. We found that the soluble domains of synaptobrevin, SNAP-25, and syntaxin 1 are unstructured. At higher concentrations, the SNARE motif of syntaxin 1 forms homooligomeric helical bundles with at least some of the alpha-helices aligned in parallel. In the assembled SNARE complex, mapping of thirty side chain positions yielded spectra which are in good agreement with the recently published crystal structure. The loop region of SNAP-25 that connects the two SNARE motifs is largely unstructured. C-terminal truncation of synaptobrevin resulted in complexes that are completely folded N-terminal of the truncation but become unstructured at the C-terminal end. The binary complex of syntaxin and SNAP-25 consists of a parallel four helix-bundle with properties resembling that of the ternary complex.     

Formation of the full SNARE complex eliminates interactions of its individual protein components with the Kv2.1 channel

Previously, we have demonstrated physical and functional interactions of the voltage-gated potassium channel Kv2.1 with the plasma membrane protein components of the exocytotic SNARE complex, syntaxin 1A, and the t-SNARE, syntaxin 1A/SNAP-25, complex. Importantly, the physical interaction of Kv2.1 with syntaxin was shown to be involved in the facilitation of secretion from PC12 cells, which was independent of potassium currents. Recently, we showed that also VAMP2, the vesicular SNARE, interacts physically and functionally with Kv2.1. Here, we first set out to test the interaction of the full SNARE, syntaxin/SNAP-25/VAMP2, complex with the channel. Using the interaction of VAMP2 with Kv2.1 in Xenopus oocytes as a probe, we showed that coexpression of the t-SNARE complex with VAMP2 abolished the VAMP2 effect on channel inactivation and reduced the amount of VAMP2 that coprecipitated with Kv2.1. Further, in vitro pull down assays showed that the full SNARE complex failed to interact with Kv2.1 N- and C-termini in tandem, in contrast to the individual SNARE components. This suggests that the interactions of the SNARE components with Kv2.1 are abolished upon their recruitment into a full SNARE complex, which does not interact with the channel. Other important findings arising from the in vitro study are that the t-SNARE complex, in addition to syntaxin, interacts with a specific C-terminal channel domain, C1a, shown to mediate the facilitation of release by Kv2.1 and that the presence of Kv2.1 N-terminus has crucial contribution to these interactions. These findings provide important insights into the understanding of the complex molecular events involved in the novel phenomenon of secretion facilitation in neuroendocrine cells by Kv2.1.