Soluble N-ethylmaleimide-sensitive Factor Attachment Receptor Research Articles

Identification of the v-SNARE required for lytic granule fusion in cytotoxic T lymphocytes (176.27)

Abstract Cytotoxic T lymphocytes (CTLs) kill target cells by secretion of cytotoxic components such as perforin and granzymes, which are contained in lytic granules. Fusion of lytic granules occurs at the contact zone between the target cell and the CTL, the immunological synapse. Soluble NSF attachment receptor (SNARE) proteins are required for all fusion events in cells, but the SNARE proteins involved in lytic granule fusion in CTLs remain unknown. Using a targeted gene knock in strategy we identified the v-SNARE that is required for lytic granule fusion in CTLs. We first used super resolution microscopy combined with careful quantification to identify the v-SNARE that is localized exclusively to lytic granules in primary mouse CTLs. By specifically cleaving the v-SNARE with a bacterial neurotoxin and by gene knock down we show a complete loss of lytic granule fusion by two independent methods - 1) LAMP1 based degranulation assay and 2) real time imaging of exocytosis using TIRF microscopy. We therefore conclude that the v-SNARE that is localizing to the lytic granules is mediating their final fusion at the IS, thereby delivering the lethal hit needed for target cell killing.

Characterization of the functional role of VAMP8 in granule exocytosis of T lymphocytes (69.12)

Abstract One salient event in the adaptive immune response is the activation of T lymphocytes by foreign antigens. They patrol our body for providing a defense against intracellular pathogens, viruses and malignant cells. Upon recognition, T lymphocytes exert their cytotoxic activity through secretion of cytotoxic granules at a specialized cell interface, the immune synapse (IS), or engage in stimulus-coupled release of immune mediators in pre-formed granules. To induce target cell death, T lymphocytes rely on exocytosis of granules, which is the result of a cascade of events consisting of docking, priming, and finally Ca2+-dependent fusion involving soluble NSF attachment receptor (SNARE) proteins. VAMP8 is a SNARE protein that is a key component of a variety of secretion in other immune cell types and whose function in T cell killing is unknown. VAMP8 re-localizes to the immunological synapse following CTL activation suggesting it plays an important role in granule fusion. VAMP8 has also been shown to localize abundantly at the membranes of early and late endosomes. Additionally, VAMP8 plays a role in the exocytosis of organelles formed in the secretory pathway, such as exocrine granules and secretory lysosomes. Hence, this research is aimed at elucidating the involvement of VAMP8 in T lymphocytes of secretory granule release allowing us to better understand the contribution of VAMP8 to the killing process of T lymphocytes.

The voltage-dependent potassium channel subunit Kv2.1 regulates insulin secretion from rodent and human islets independently of its electrical function

It is thought that the voltage-dependent potassium channel subunit Kv2.1 (Kv2.1) regulates insulin secretion by controlling beta cell electrical excitability. However, this role of Kv2.1 in human insulin secretion has been questioned. Interestingly, Kv2.1 can also regulate exocytosis through direct interaction of its C-terminus with the soluble NSF attachment receptor (SNARE) protein, syntaxin 1A. We hypothesised that this interaction mediates insulin secretion independently of Kv2.1 electrical function. Wild-type Kv2.1 or mutants lacking electrical function and syntaxin 1A binding were studied in rodent and human beta cells, and in INS-1 cells. Small intracellular fragments of the channel were used to disrupt native Kv2.1-syntaxin 1A complexes. Single-cell exocytosis and ion channel currents were monitored by patch-clamp electrophysiology. Interaction between Kv2.1, syntaxin 1A and other SNARE proteins was probed by immunoprecipitation. Whole-islet Ca(2+)-responses were monitored by ratiometric Fura red fluorescence and insulin secretion was measured. Upregulation of Kv2.1 directly augmented beta cell exocytosis. This happened independently of channel electrical function, but was dependent on the Kv2.1 C-terminal syntaxin 1A-binding domain. Intracellular fragments of the Kv2.1 C-terminus disrupted native Kv2.1-syntaxin 1A interaction and impaired glucose-stimulated insulin secretion. This was not due to altered ion channel activity or impaired Ca(2+)-responses to glucose, but to reduced SNARE complex formation and Ca(2+)-dependent exocytosis. Direct interaction between syntaxin 1A and the Kv2.1 C-terminus is required for efficient insulin exocytosis and glucose-stimulated insulin secretion. This demonstrates that native Kv2.1-syntaxin 1A interaction plays a key role in human insulin secretion, which is separate from the channel's electrical function.

SNARE protein expression and localization in human cytotoxic T lymphocytes

The major function of cytotoxic T lymphocytes (CTLs) is to eliminate pathogen-infected and tumorigenic cells. This is mediated mainly through the exocytosis of lytic granules (LGs) containing cytotoxic components, such as perforin and granzymes at the immunological synapse (IS). The soluble NSF attachment receptor (SNARE) protein isoforms are well known to be required for vesicle exocytosis in neuronal synapses, but their potential function in CTLs is only partly understood. Here, we examined the expression of SNARE proteins before and after the activation of primary human CD8(+) T cells and determined their co-localization with LGs and CD3 after IS formation with target cells. We found that several key SNARE proteins in neuronal cells were not expressed in CTLs, such as syntaxin1B2 and SNAP-25. Vti1b, Stx8 and Stx16 had the highest degrees of co-localization with LGs while Stx3, Stx4, Stx6, Stx7, Stx8, Stx13, Vti1b, VAMP3 and VAMP4 co-localized with CD3. Our data provide the first complete expression profile and localization of SNAREs in primary human CD8(+) T cells, laying the groundwork for further understanding their potential role in T-cell function.

Gelsolin Associates with the N Terminus of Syntaxin 4 to Regulate Insulin Granule Exocytosis

The plasma membrane soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) protein syntaxin (Syn)4 is required for biphasic insulin secretion, although how it regulates each phase remains unclear. In a screen to identify new Syn4-interacting factors, the calcium-activated F-actin-severing protein gelsolin was revealed. Gelsolin has been previously implicated as a positive effector of insulin secretion, although a molecular mechanism to underlie this function is lacking. Toward this, our in vitro binding studies showed the Syn4-gelsolin interaction to be direct and mediated by the N-terminal Ha domain (amino acid residues 39-70) of Syn4. Syn4-gelsolin complexes formed under basal conditions and dissociated upon acute glucose or KCl stimulation; nifedipine blocked dissociation. The dissociating action of secretagogues could be mimicked by expression of the N-terminal Ha domain of Syn4 fused to green fluorescent protein (GFP) (GFP-39-70). Furthermore, GFP-39-70 expression in isolated mouse islet and clonal MIN6 β-cells initiated insulin release in the absence of appropriate stimuli. Consistent with this, the inhibitory GFP-39-70 peptide also initiated Syn4 activation in the absence of stimuli. Moreover, although MIN6 β-cells expressing the GFP-39-70 peptide maintained normal calcium influx in response to KCl, KCl-stimulated insulin secretion and the triggering pathway of insulin secretion were significantly impaired. Taken together, these data support a mechanistic model for gelsolin's role in insulin exocytosis: gelsolin clamps unsolicited soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE)-regulated exocytosis through direct association with Syn4 in the absence of appropriate stimuli, which is relieved upon stimulus-induced calcium influx to activate gelsolin and induce its dissociation from Syn4 to facilitate insulin exocytosis.

SUMOylation Regulates Insulin Exocytosis Downstream of Secretory Granule Docking in Rodents and Humans

OBJECTIVEThe reversible attachment of small ubiquitin-like modifier (SUMO) proteins controls target localization and function. We examined an acute role for the SUMOylation pathway in downstream events mediating insulin secretion.RESEARCH DESIGN AND METHODSWe studied islets and β-cells from mice and human donors, as well as INS-1 832/13 cells. Insulin secretion, intracellular Ca2+, and β-cell exocytosis were monitored after manipulation of the SUMOylation machinery. Granule localization was imaged by total internal reflection fluorescence and electron microscopy; immunoprecipitation and Western blotting were used to examine the soluble NSF attachment receptor (SNARE) complex formation and SUMO1 interaction with synaptotagmin VII.RESULTSSUMO1 impairs glucose-stimulated insulin secretion by blunting the β-cell exocytotic response to Ca2+. The effect of SUMO1 to impair insulin secretion and β-cell exocytosis is rapid and does not require altered gene expression or insulin content, is downstream of granule docking at the plasma membrane, and is dependent on SUMO-conjugation because the deSUMOylating enzyme, sentrin/SUMO-specific protease (SENP)-1, rescues exocytosis. SUMO1 coimmunoprecipitates with the Ca2+ sensor synaptotagmin VII, and this is transiently lost upon glucose stimulation. SENP1 overexpression also disrupts the association of SUMO1 with synaptotagmin VII and mimics the effect of glucose to enhance exocytosis. Conversely, SENP1 knockdown impairs exocytosis at stimulatory glucose levels and blunts glucose-dependent insulin secretion from mouse and human islets.CONCLUSIONSSUMOylation acutely regulates insulin secretion by the direct and reversible inhibition of β-cell exocytosis in response to intracellular Ca2+ elevation. The SUMO protease, SENP1, is required for glucose-dependent insulin secretion.

Munc18b regulates core SNARE complex assembly and constitutive exocytosis by interacting with the N-peptide and the closed-conformation C-terminus of syntaxin 3

The interaction between SM (Sec1/Munc18) and SNARE (soluble N-ethylmaleimide-sensitive factor-attachment receptor) proteins constitutes the core eukaryotic membrane fusion machinery which manages exocytosis by mediating fusion of constitutively exocytic vesicles with the plasma membrane. However, mechanistic details on the nature and the physiological impact of SM-SNARE interactions remain largely elusive. Detailed characterization of the interaction profiles between Munc18b and its cognate SNAREs, Stx3 (syntaxin 3), SNAP-23 (soluble N-ethylmaleimide-attachment protein 23) and VAMP8 (vesicle-associated membrane protein 8), revealed that Munc18b binds Stx3, VAMP8 and the assembled core SNARE complex consisting of Stx3, SNAP-23 and VAMP8. Dissection of the Munc18b-Stx3 heterodimer suggested that Munc18b interacts with Stx3's conserved N-peptide as well as with its closed-conformation C-terminus encompassing the Habc domain, a linker and the SNARE (H3) motif. Deletion of the Habc domain or mutations interrupting the intramolecular binding of the Habc and H3 domains abrogated the Munc18b-Stx3 interaction. Although only the N-peptide deletion mutant, but not the soluble wild-type Stx3, is assembled into the core SNARE complex in the presence of Munc18b in vitro, ectopic expression of this SM protein increases constitutive exocytosis in mammalian cells. Our results suggest that Munc18b is functionally coupled to the assembly of exocytic SNARE complexes and increases exocytosis by interacting with the N-peptide and closed-conformation C-terminus of Stx3, thereby neutralizing the secretion-inhibitory effect of this SNARE.

Syntaxin 1A regulates dopamine transporter activity, phosphorylation and surface expression

We investigated the functional relationship between the soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) protein syntaxin 1A (syn 1A) and the dopamine transporter (DAT) by treating rat striatal tissue with Botulinum Neurotoxin C (BoNT/C) and co-transfecting syn 1A with DAT in non-neuronal cells, followed by analysis of DAT activity, phosphorylation, and regulation. Treatment of striatal slices with BoNT/C resulted in elevated dopamine (DA) transport Vmax and reduced DAT phosphorylation, while heterologous co-expression of syn 1A led to reduction in DAT surface expression and transport Vmax. Syn 1A was present in DAT immunoprecipitation complexes, supporting a direct or indirect interaction between the proteins. Phorbol ester regulation of DA transport activity was retained in BoNT/C-treated synaptosomes and syn 1A transfected cells, demonstrating that protein kinase C (PKC) and syn 1A effects occur through independent processes. These findings reveal a novel mechanism for regulation of DAT activity and phosphorylation, and suggest the potential for syn 1A to impact DA neurotransmission through effects on reuptake.

Acute neuroprotection by the synaptic blocker botulinum neurotoxin E in a rat model of focal cerebral ischaemia

Evidence indicates that accumulation of excitotoxic mediators, such as glutamate, contributes to neuronal damage after an ischaemic insult. It is not clear, however, whether this accumulation is due to excess synaptic release or to impaired uptake. To test a role for synaptic release, here we investigated the neuroprotective potential of the synaptic blocker botulinum neurotoxin E (BoNT/E), that prevents vesicle fusion via the cleavage of the SNARE (soluble NSF-attachment receptor) protein SNAP-25 (synaptosomal-associated protein of 25 kDa). Focal ischaemia was induced in vivo by infusing the potent vasoconstricting peptide endothelin-1 (ET-1) into the CA1 area of the hippocampus in adult rats; BoNT/E or vehicle were administered into the same site 20 min later. Injection of ET-1 was found to produce a transient and massive increase in glutamate release that was potently antagonized by BoNT/E. To assess whether blocking transmitter release translates into neuroprotection, the extent of the ischaemic damage was determined 24 h and 6 weeks after the insult. We found that BoNT/E administration consistently reduced the loss of CA1 pyramidal neurons at 24 h. The neuroprotective effect of BoNT/E, however, was no longer significant at 6 weeks. These data provide evidence that blockade of synaptic transmitter release delays neuronal cell death following focal brain ischaemia, and underline the importance of assessing long-term neuroprotection in experimental stroke studies.

Loss of synaptotagmin IV results in a reduction in synaptic vesicles and a distortion of the Golgi structure in cultured hippocampal neurons

Fusion of synaptic vesicles with the plasma membrane is mediated by the SNARE (soluble NSF attachment receptor) proteins and is regulated by synaptotagmin (syt). There are at least 17 syt isoforms that have the potential to act as modulators of membrane fusion events. Synaptotagmin IV (syt IV) is particularly interesting; it is an immediate early gene that is regulated by seizures and certain classes of drugs, and, in humans, syt IV maps to a region of chromosome 18 associated with schizophrenia and bipolar disease. Syt IV has recently been found to localize to dense core vesicles in hippocampal neurons, where it regulates neurotrophin release. Here we have examined the ultrastructure of cultured hippocampal neurons from wild-type and syt IV −/− mice using electron tomography. Perhaps surprisingly, we observed a potential synaptic vesicle transport defect in syt IV −/− neurons, with the accumulation of large numbers of small clear vesicles (putative axonal transport vesicles) near the trans-Golgi network. We also found an interaction between syt IV and KIF1A, a kinesin known to be involved in vesicle trafficking to the synapse. Finally, we found that syt IV −/− synapses exhibited reduced numbers of synaptic vesicles and a twofold reduction in the proportion of docked vesicles compared to wild-type. The proportion of docked vesicles in syt IV −/− boutons was further reduced, 5-fold, following depolarization.

Munc18/Syntaxin Interaction Kinetics Control Secretory Vesicle Dynamics

In neuronal and hormonal release, regulated exocytosis requires an essential set of proteins: the soluble N-ethylmaleimide sensitive-factor attachment receptor proteins (SNAREs) syntaxin 1, SNAP-25, VAMP, and their regulator, Munc18. Recently, it was found that Munc18-1 can interact with syntaxin 1 through distinct mechanisms: an inhibitory mode enveloping syntaxin (mode 1), sequestering it from SNARE protein interactions, and direct binding to an evolutionarily conserved N-terminal peptide of syntaxin (mode 2/3). The latter interaction has been proposed to control “priming” of the fusion reaction, defined using electrophysiology, but it is unknown how this interaction is regulated, and any dynamic effect at the molecular or vesicular level in cells remains undiscovered. We now show that a phosphorylation site in syntaxin 1 (Ser14) regulates the N-terminal interaction with Munc18-1. Probing syntaxin 1 association with Munc18-1, in real-time and in living cells, we found that modification of Ser14 modulated the dynamics of this interaction, specifically at the plasma membrane. Destabilization of this dynamic interaction enhanced vesicle immobilization at the plasma membrane with a resulting inhibition of exocytosis.

A score for membrane fusion

Intracellular membrane fusion has been mimicked in vitro using a mix of 17 purified proteins and lipid bilayers. This technical tour de force allows the study of how cells orchestrate and perform such fusion events. Eukaryotic cells contain membrane-bound organelles that can undergo fusion with each other in a regulated manner. The widely held view is that fusion is mediated by SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) proteins. Although SNAREs are capable of fusing vesicles with high specificity, fusion reactions with native SNAREs alone are inefficient and require high concentrations of proteins. In this study, Ohya et al. reconstitute endosome fusion using the Rab machinery along with SNARE proteins. This is the first report of the reconstitution of membrane fusion efficiently and under physiological conditions and also highlights the functional relationship between Rab and SNARE proteins.

Characterization of SNARE proteins involved in granule exocytosis of cytotoxic T lymphocytes (35.25)

Abstract Cytotoxic T lymphocytes (CTLs) exert their cytotoxic activity through the polarized secretion of cytotoxic granules at the immunological synapse (IS). Because soluble NSF attachment receptor (SNARE) proteins mediate numerous intra- and intercellular fusion events and are essential effectors of exocytosis of synaptic granules in neurons, we examined the contribution of specific SNARE proteins to the formation of the IS. To determine the functional significance of specific SNAREs, our lab has examined the expression profile of more than 40 SNARE proteins by RT-PCR and quantitated various protein levels of these SNAREs in resting and activated CTLs. We have found that the expression profile of some SNARE proteins is up or down-regulated upon CTL maturation. In conjunction with these studies, we have also analyzed the sub-cellular localization and mobilization following IS formation of all SNAREs that are expressed in CD8+ T-cells for which specific antibodies are available. Some but not all SNARE proteins are translocated towards the immunological synapse and some co-localized with cytotoxic lytic granules at the immunological synapse. We found that syntaxin 4, syntaxin 6, VAMP 3, and VAMP 8, among other proteins, seem to polarize and colocalize with cytotoxic granule markers at the immune synapse suggesting they play important roles in cytotoxic granule secretion. Grant/ other support: DFG RE 1092/6-1

SNARE‐mediated homotypic fusion in Golgi apparatus ribbon formation

The ribbon‐like structure of the Golgi apparatus is thought to be the end result of homotypic fusion of cisternal membranes of ministacks, which are pre‐Golgi intermediate structures. Organization of ministack compartments for fusion is facilitated in part by the protein GRASP65 that links homotypic compartments of the ministacks and brings them into close contact. This process is called tethering. However, the Grasp65 protein alone is not sufficient to facilitate membrane fusion. Once the membranous compartments of the ministacks are in close proximity, a different set of proteins is believed to fuse the membranes into one continuous, ribbon‐like structure. The identity of these proteins is currently unknown. Soluble NSF attachment receptor (SNARE) proteins mediate membrane fusion in eukaryotic cells. A number of different SNARE proteins have been identified in Golgi membranes including Syntaxin 5, hBet1, Sec22, and Membrin. We believe that these four SNARE proteins along with GRASP 65 are sufficient to drive homotypic membrane fusion, possibly bringing about the fusion step that is required for Golgi ribbon formation. To test this hypothesis, I utilized a non‐Golgi membranous structure to model the activity of the proteins. Mitochondrial outer membranes provide an accessible model membrane to use in this case. I constructed plasmids that target the suspected Golgi SNAREs to the mitochondrial outer membrane. I then expressed these plasmids in HeLa cells along with GRASP 65 and assayed for membrane fusion using Fluorescence Recovery After Photobleaching (FRAP). As a result of my efforts, we are well on our way to determining the role of Golgi SNAREs in membrane fusion. This work was supported by the National Science Foundation's Research Opportunities for Undergraduates Program and the Mellon Institute of Carnegie Mellon University.

The SNARE protein syntaxin 7 is required for immunological synapse formation in cytotoxic T lymphocytes (35.24)

Abstract Cytotoxic T lymphocytes (CTLs) kill target cells by secretion of cytotoxic components such as perforin and granzymes which are contained in lytic granules. Fusion of lytic granules occurs at the contact zone between the target cell and the CTL, the immunological synapse (IS). T cell receptor (TCR) enrichment at the IS is one of the key early events of IS formation. Soluble NSF attachment receptor (SNARE) proteins are required for all fusion events in cells, but the individual SNARE proteins that are important for CTL function are not yet known. We identified syntaxin 7 in CTLs by reverse transcriptase PCR and immunocytochemistry. We found that syntaxin 7 is localised to the IS with a similar distribution to the lytic granule marker - perforin. We then blocked the function of syntaxin 7 by deleting its transmembrane domain and observed a complete loss of TCR accumulation at the IS, indicating that no IS is formed. These results imply that syntaxin 7 is required for IS formation in CTLs. Further studies using specific endosome markers will help us determine the mechanism of syntaxin 7 function in CTLs. Source of research support - GRK1326

Phylogeny of the SNARE vesicle fusion machinery yields insights into the conservation of the secretory pathway in fungi

BackgroundIn eukaryotic cells, directional transport between different compartments of the endomembrane system is mediated by vesicles that bud from a donor organelle and then fuse with an acceptor organelle. A family of integral membrane proteins, termed soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins, constitute the key machineries of these different membrane fusion events. Over the past 30 years, the yeast Saccharomyces cerevisiae has served as a powerful model organism for studying the organization of the secretory and endocytic pathways, and a few years ago, its entire set of SNAREs was compiled.ResultsHere, we make use of the increasing amount of genomic data to investigate the history of the SNARE family during fungi evolution. Moreover, since different SNARE family members are thought to demarcate different organelles and vesicles, this approach allowed us to compare the organization of the endomembrane systems of yeast and animal cells. Our data corroborate the notion that fungi generally encompass a relatively simple set of SNARE proteins, mostly comprising the SNAREs of the proto-eukaryotic cell. However, all fungi contain a novel soluble SNARE protein, Vam7, which carries an N-terminal PX-domain that acts as a phosphoinositide binding module. In addition, the points in fungal evolution, at which lineage-specific duplications and diversifications occurred, could be determined. For instance, the endosomal syntaxins Pep12 and Vam3 arose from a gene duplication that occurred within the Saccharomycotina clade.ConclusionAlthough the SNARE repertoire of baker's yeast is highly conserved, our analysis reveals that it is more deviated than the ones of basal fungi. This highlights that the trafficking pathways of baker's yeast are not only different to those in animal cells but also are somewhat different to those of many other fungi.

Synaptotagmin C2B Domain Regulates Ca2+-triggered Fusion in Vitro: CRITICAL RESIDUES REVEALED BY SCANNING ALANINE MUTAGENESIS

Synaptotagmin (syt) 1 is localized to synaptic vesicles, binds Ca2+, and regulates neuronal exocytosis. Syt 1 harbors two Ca2+-binding motifs referred to as C2A and C2B. In this study we examine the function of the isolated C2 domains of Syt 1 using a reconstituted, SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor)-mediated, fusion assay. We report that inclusion of phosphatidylethanolamine into reconstituted SNARE vesicles enabled isolated C2B, but not C2A, to regulate Ca2+-triggered fusion. The isolated C2B domain had a 6-fold lower EC for Ca2+ 50-activated fusion than the intact cytosolic domain of Syt 1 (C2AB). Phosphatidylethanolamine increased both the rate and efficiency of C2AB- and C2B-regulated fusion without affecting their abilities to bind membrane-embedded syntaxin-SNAP-25 (t-SNARE) complexes. At equimolar concentrations, the isolated C2A domain was an effective inhibitor of C2B-, but not C2AB-regulated fusion; hence, C2A has markedly different effects in the fusion assay depending on whether it is tethered to C2B. Finally, scanning alanine mutagenesis of C2AB revealed four distinct groups of mutations within the C2B domain that play roles in the regulation of SNARE-mediated fusion. Surprisingly, substitution of Arg-398 with alanine, which lies on the opposite end of C2B from the Ca2+/membrane-binding loops, decreases C2AB t-SNARE binding and Ca2+-triggered fusion in vitro without affecting Ca2+-triggered interactions with phosphatidylserine or vesicle aggregation. In addition, some mutations uncouple the clamping and stimulatory functions of syt 1, suggesting that these two activities are mediated by distinct structural determinants in C2B.

Botulinum Neurotoxin as a Therapeutic Modality in Orthopaedic Surgery: More Than Twenty Years of Experience

Seyler, Thorsten M. MD1; Smith, Beth P. PhD1; Marker, David R. BS2; Ma, Jianjun MD, PhD1; Shen, Jian MD, PhD1; Smith, Tom L. PhD1; Mont, Michael A. MD2; Kolaski, Kat MD1; Koman, L. Andrew MD1 Author Information

Thymic alterations in mice deficient for the SNARE protein VAMP8/endobrevin

SNARE (soluble-N-ethylmaleimide-sensitive factor attachment receptor) proteins mediate the recognition and fusion of transport vesicles in eukaryotic cells. The SNARE protein VAMP8 (also called endobrevin) is involved in the fusion of late endosomes and in some pathways of regulated exocytosis. In a subset of mice deficient for the SNARE protein VAMP8, a severe alteration of the thymus and in T lymphocyte development was observed and characterized. The size of the thymus and the number of thymocytes were dramatically reduced compared with those in heterozygous littermates. Further, the compartmentalization into cortex and medulla and the organization of the thymus epithelium were disturbed. The numbers of all thymocyte subpopulations were reduced, with the CD4 and CD8 double-positive thymocytes being most severely affected. The proportion of proliferating thymocytes was reduced, and the staining of apoptotic cells in situ and ex vivo indicated an increased number of apoptotic cells. Isolated thymocytes of Vamp8−/− mice were more susceptible to various apoptotic stimuli including glucocorticoids, FAS receptor, and CD3/CD28-mediated signaling in vitro, even before an increased number of apoptotic cells was detectable in situ. However, bone marrow of phenotypically affected Vamp8−/− mice was readily able to repopulate immunodeficient hosts suggesting that the SNARE protein VAMP8 has a specific function in the thymic stroma affecting the proliferation and apoptosis of T lymphocytes during maturation in the thymus.

VAMP2 interacts directly with the N terminus of Kv2.1 to enhance channel inactivation

Recently, we demonstrated that the Kv2.1 channel plays a role in regulated exocytosis of dense-core vesicles (DCVs) through direct interaction of its C terminus with syntaxin 1A, a plasma membrane soluble NSF attachment receptor (SNARE) component. We report here that Kv2.1 interacts with VAMP2, the vesicular SNARE partner that is also present at high concentration in neuronal plasma membrane. This is the first report of VAMP2 interaction with an ion channel. The interaction was demonstrated in brain membranes and characterized using electrophysiological and biochemical analyses in Xenopus oocytes combined with an in vitro binding analysis and protein modeling. Comparative study performed with wild-type and mutant Kv2.1, wild-type Kv1.5, and chimeric Kv1.5N/Kv2.1 channels revealed that VAMP2 enhanced the inactivation of Kv2.1, but not of Kv1.5, via direct interaction with the T1 domain of the N terminus of Kv2.1. Given the proposed role for surface VAMP2 in the regulation of the vesicle cycle and the important role for the sustained Kv2.1 current in the regulation of dendritic calcium entry during high-frequency stimulation, the interaction of VAMP2 with Kv2.1 N terminus may contribute, alongside with the interaction of syntaxin with Kv2.1 C terminus, to the activity dependence of DCV release.