scholarly journals Differential inhibition by botulinum neurotoxin A of cotransmitters released from autonomic vasodilator neurons

2001 ◽  
Vol 281 (5) ◽  
pp. H2124-H2132 ◽  
Author(s):  
Judy L. Morris ◽  
Phillip Jobling ◽  
Ian L. Gibbins
Keyword(s):  
Botulinum Neurotoxin ◽  
Snare Complex ◽  
Snare Proteins ◽  
Botulinum Neurotoxin A ◽  
Uterine Arteries ◽  
Synaptic Vesicle Exocytosis ◽  
Protein Receptor ◽  
Autonomic Neurons ◽  
Vesicle Exocytosis

The role of the soluble NSF attachment protein receptor (SNARE) protein complex in release of multiple cotransmitters from autonomic vasodilator neurons was examined in isolated segments of guinea pig uterine arteries treated with botulinum neurotoxin A (BoNTA; 50 nM). Western blotting of protein extracts from uterine arteries demonstrated partial cleavage of synaptosomal-associated protein of 25 kDa (SNAP-25) to a NH2-terminal fragment of ∼24 kDa by BoNTA. BoNTA reduced the amplitude (by 70–80%) of isometric contractions of arteries in response to repeated electrical stimulation of sympathetic axons at 1 or 10 Hz. The amplitude of neurogenic relaxations mediated by neuronal nitric oxide (NO) was not affected by BoNTA, whereas the duration of peptide-mediated neurogenic relaxations to stimulation at 10 Hz was reduced (67% reduction in integrated responses). In contrast, presynaptic cholinergic inhibition of neurogenic relaxations was abolished by BoNTA. These results demonstrate that the SNARE complex has differential involvement in release of cotransmitters from the same autonomic neurons: NO release is not dependant on synaptic vesicle exocytosis, acetylcholine release from small vesicles is highly dependant on the SNARE complex, and neuropeptide release from large vesicles involves SNARE proteins that may interact differently with regulatory factors such as calcium.

Botulinum neurotoxin A attenuates release of norepinephrine but not NPY from vasoconstrictor neurons

2002 ◽  
Vol 283 (6) ◽  
pp. H2627-H2635 ◽  
Author(s):  
Judy L. Morris ◽  
Phillip Jobling ◽  
Ian L. Gibbins
Keyword(s):  
Botulinum Neurotoxin ◽  
Vena Cava ◽  
Snare Proteins ◽  
Botulinum Neurotoxin A ◽  
Low Frequencies ◽  
Uterine Arteries ◽  
Venae Cavae ◽  
Protein Receptor

We examined effects of botulinum neurotoxin A (BoNTA) on sympathetic constrictions of the vena cava and uterine artery from guinea pigs to test the role of soluble NSF attachment protein receptor (SNARE) proteins in release of the cotransmitters norepinephrine (NE) and neuropeptide Y (NPY). Protein extracts of venae cavae and uterine arteries showed partial cleavage of synaptosomal associated protein of 25 kDa (SNAP-25) after treatment in vitro with BoNTA (50–100 nM). The rising phase of isometric contractions of isolated venae cavae to field stimulation at 20 Hz, mediated by NE acting on α-adrenoceptors, was reduced significantly by 100 nM BoNTA. However, sustained sympathetic contractions mediated by NPY were not affected by BoNTA. In uterine arteries, noradrenergic contractions to 1-Hz stimulation were almost abolished by BoNTA, and contractions at 10 Hz were reduced by 50–60%. We conclude that SNARE proteins are involved in exocytosis of NE from synaptic vesicles at low frequencies of stimulation but may not be essential for exocytosis of NPY and NE from large vesicles at high stimulation frequencies.

scholarly journals Botulinum Neurotoxin a Blocks Synaptic Vesicle Exocytosis but Not Endocytosis at the Nerve Terminal

1999 ◽  
Vol 147 (6) ◽  
pp. 1249-1260 ◽  
Author(s):  
Elaine A. Neale ◽  
Linda M. Bowers ◽  
Min Jia ◽  
Karen E. Bateman ◽  
Lura C. Williamson
Keyword(s):  
Synaptic Vesicle ◽  
Nerve Terminal ◽  
Neurotransmitter Release ◽  
Synaptic Vesicles ◽  
Botulinum Neurotoxin ◽  
Vesicle Membrane ◽  
Botulinum Neurotoxin A ◽  
Synaptic Vesicle Exocytosis ◽  
Vesicle Exocytosis ◽  
Botulinum Neurotoxins

The supply of synaptic vesicles in the nerve terminal is maintained by a temporally linked balance of exo- and endocytosis. Tetanus and botulinum neurotoxins block neurotransmitter release by the enzymatic cleavage of proteins identified as critical for synaptic vesicle exocytosis. We show here that botulinum neurotoxin A is unique in that the toxin-induced block in exocytosis does not arrest vesicle membrane endocytosis. In the murine spinal cord, cell cultures exposed to botulinum neurotoxin A, neither K+-evoked neurotransmitter release nor synaptic currents can be detected, twice the ordinary number of synaptic vesicles are docked at the synaptic active zone, and its protein substrate is cleaved, which is similar to observations with tetanus and other botulinal neurotoxins. In marked contrast, K+ depolarization, in the presence of Ca2+, triggers the endocytosis of the vesicle membrane in botulinum neurotoxin A–blocked cultures as evidenced by FM1-43 staining of synaptic terminals and uptake of HRP into synaptic vesicles. These experiments are the first demonstration that botulinum neurotoxin A uncouples vesicle exo- from endocytosis, and provide evidence that Ca2+ is required for synaptic vesicle membrane retrieval.

scholarly journals Reconciling the regulatory role of Munc18 proteins in SNARE-complex assembly

IUCrJ ◽  
2014 ◽  
Vol 1 (6) ◽  
pp. 505-513 ◽  
Author(s):  
Asma Rehman ◽  
Julia K. Archbold ◽  
Shu-Hong Hu ◽  
Suzanne J. Norwood ◽  
Brett M. Collins ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Transmembrane Domain ◽  
Blood Glucose Control ◽  
Vital Role ◽  
Snare Complex ◽  
Snare Proteins ◽  
Regulatory Role ◽  
Sm Proteins ◽  
Protein Receptor

Membrane fusion is essential for human health, playing a vital role in processes as diverse as neurotransmission and blood glucose control. Two protein families are key: (1) the Sec1p/Munc18 (SM) and (2) the solubleN-ethylmaleimide-sensitive attachment protein receptor (SNARE) proteins. Whilst the essential nature of these proteins is irrefutable, their exact regulatory roles in membrane fusion remain controversial. In particular, whether SM proteins promote and/or inhibit the SNARE-complex formation required for membrane fusion is not resolved. Crystal structures of SM proteins alone and in complex with their cognate SNARE proteins have provided some insight, however, these structures lack the transmembrane spanning regions of the SNARE proteins and may not accurately reflect the native state. Here, we review the literature surrounding the regulatory role of mammalian Munc18 SM proteins required for exocytosis in eukaryotes. Our analysis suggests that the conflicting roles reported for these SM proteins may reflect differences in experimental design. SNARE proteins appear to require C-terminal immobilization or anchoring, for example through a transmembrane domain, to form a functional fusion complex in the presence of Munc18 proteins.

scholarly journals Truncated SNAP-25 (1–197), Like Botulinum Neurotoxin A, Can Inhibit Insulin Secretion from HIT-T15 Insulinoma Cells

1998 ◽  
Vol 12 (7) ◽  
pp. 1060-1070 ◽  
Author(s):  
Xiaohang Huang ◽  
Michael B. Wheeler ◽  
You-hou Kang ◽  
Laura Sheu ◽  
Gergely L. Lukacs ◽  
...  
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Insulin Secretion ◽  
Botulinum Neurotoxin ◽  
Botulinum Neurotoxin A ◽  
Synaptic Vesicle Exocytosis ◽  
Protein Receptors ◽  
Vesicle Exocytosis ◽  
Insulin Secreting Cells ◽  
Insulinoma Cells

Abstract We and others have previously shown that insulin-secreting cells of the pancreas express high levels of SNAP-25 (synaptosomal-associated protein of 25 kDa), a 206-amino acid t-SNARE (target soluble N-ethylmaleimide-sensitive factor attachment protein receptors) implicated in synaptic vesicle exocytosis. In the present study, we show that SNAP-25 is required for insulin secretion by transient transfection of Botulinum Neurotoxin A (BoNT/A) into insulin-secreting HIT-T15 cells. Transient expression of BoNT/A cleaved the endogenous as well as overexpressed SNAP-25 proteins and caused significant reductions in K+ and glucose-evoked secretion of insulin. To determine whether the inhibition of release was due to the depletion of functional SNAP-25 or the accumulation of proteolytic by-products, we transfected cells with SNAP-25 proteins from which the C-terminal nine amino acids had been deleted to mimic the effects of the toxin. This modified SNAP-25 (amino acids 1–197) remained bound to the plasma membrane but was as effective as the toxin at inhibiting insulin secretion. Microfluorimetry revealed that the inhibition of secretion was due neither to changes in basal cytosolic Ca2+ levels nor in Ca2+ influx evoked by K+-mediated plasma membrane depolarization. Electron microscopy revealed that cells transfected with either BoNT/A or truncated SNAP-25 contained significantly higher numbers of insulin granules, many of which clustered close to the plasma membrane. Together, these results demonstrate that functional SNAP-25 proteins are required for insulin secretion and suggest that the inhibitory action of BoNT/A toxin on insulin secretion is in part caused by the production of the plasma membrane-bound cleavage product, which itself interferes with insulin granule docking and fusion.

scholarly journals Complexin-1 regulated transition in the assembly of single neuronal SNARE complex

2021 ◽  
Author(s):  
Hao Tongrui ◽  
Feng Nan ◽  
Gong Fan ◽  
Liu Jiaquan ◽  
Lu Ma ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Molecular Mechanism ◽  
Optical Tweezers ◽  
Neurotransmitter Release ◽  
Snare Complex ◽  
Snare Proteins ◽  
Synaptic Vesicle Exocytosis ◽  
Sensitive Factor ◽  
Vesicle Exocytosis ◽  
Vesicle Pool

Neurotransmitter release is mediated by the synaptic vesicle exocytosis. Important proteins in this process have been identified including the molecular machine Synaptic-soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins, and other regulators. Complexin (Cpx) is one of the vital regulators in this process. The functions of Cpx are proposed to maintain a proper primed vesicle pool by preventing its premature depletion, which facilitates the vesicle fusion in the presence of Ca2+. However, the molecular mechanism remains unclear. Using dual-trap optical tweezers, we detected the interaction of complexin-1 (CpxI) with SNARE. We found that the CpxI stabilizes partially folded SNARE complexes by competing with C-terminal of Vamp protein and interacting with the C-terminal of t-SNARE complex.

scholarly journals A Second SNARE Role for Exocytic SNAP25 in Endosome Fusion

2006 ◽  
Vol 17 (5) ◽  
pp. 2113-2124 ◽  
Author(s):  
Yoshikatsu Aikawa ◽  
Kara L. Lynch ◽  
Kristin L. Boswell ◽  
Thomas F.J. Martin
Keyword(s):  
Plasma Membrane ◽  
Membrane Fusion ◽  
Secretory Vesicle ◽  
Neuroendocrine Cells ◽  
Snare Complex ◽  
Snare Proteins ◽  
Recycling Endosome ◽  
Protein Receptor ◽  
Interfering Rna

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins play key roles in membrane fusion, but their sorting to specific membranes is poorly understood. Moreover, individual SNARE proteins can function in multiple membrane fusion events dependent upon their trafficking itinerary. Synaptosome-associated protein of 25 kDa (SNAP25) is a plasma membrane Q (containing glutamate)-SNARE essential for Ca2+-dependent secretory vesicle–plasma membrane fusion in neuroendocrine cells. However, a substantial intracellular pool of SNAP25 is maintained by endocytosis. To assess the role of endosomal SNAP25, we expressed botulinum neurotoxin E (BoNT E) light chain in PC12 cells, which specifically cleaves SNAP25. BoNT E expression altered the intracellular distribution of SNAP25, shifting it from a perinuclear recycling endosome to sorting endosomes, which indicates that SNAP25 is required for its own endocytic trafficking. The trafficking of syntaxin 13 and endocytosed cargo was similarly disrupted by BoNT E expression as was an endosomal SNARE complex comprised of SNAP25/syntaxin 13/vesicle-associated membrane protein 2. The small-interfering RNA-mediated down-regulation of SNAP25 exerted effects similar to those of BoNT E expression. Our results indicate that SNAP25 has a second function as an endosomal Q-SNARE in trafficking from the sorting endosome to the recycling endosome and that BoNT E has effects linked to disruption of the endosome recycling pathway.

What is the role of SNARE proteins in membrane fusion?

2003 ◽  
Vol 285 (2) ◽  
pp. C237-C249 ◽  
Author(s):  
Joseph G. Duman ◽  
John G. Forte
Keyword(s):  
Subcellular Localization ◽  
Membrane Fusion ◽  
Biological Membrane ◽  
Snare Complex ◽  
Model Systems ◽  
Snare Proteins ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Membrane Contact

Soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) proteins have been at the fore-front of research on biological membrane fusion for some time. The subcellular localization of SNAREs and their ability to form the so-called SNARE complex may be integral to determining the specificity of intracellular fusion (the SNARE hypothesis) and/or serving as the minimal fusion machinery. Both the SNARE hypothesis and the idea of the minimal fusion machinery have been challenged by a number of experimental observations in various model systems, suggesting that SNAREs may have other functions. Considering recent advances in the SNARE literature, it appears that SNAREs may actually function as part of a complex fusion “machine.” Their role in the machinery could be any one or a combination of roles, including establishing tight membrane contact, formation of a scaffolding on which to build the machine, binding of lipid surfaces, and many others. It is also possible that complexations other than the classic SNARE complex participate in membrane fusion.

scholarly journals Exploring the Two Coupled Conformational Changes That Activate the Munc18-1/Syntaxin-1 Complex

2021 ◽  
Vol 14 ◽  
Author(s):  
Jihong Gong ◽  
Xianping Wang ◽  
Chaoyang Cui ◽  
Yuyang Qin ◽  
Ziqi Jin ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Current Data ◽  
Snare Complex ◽  
Linker Region ◽  
Calcium Dependent ◽  
Synaptic Vesicle Exocytosis ◽  
Vesicle Exocytosis ◽  
The Relationship ◽  
Snare Complex Formation

Calcium-dependent synaptic vesicle exocytosis is mediated by SNARE complex formation. The transition from the Munc18-1/syntaxin-1 complex to the SNARE complex is catalyzed by the Munc13-1 MUN domain and involves at least two conformational changes: opening of the syntaxin-1 linker region and extension of Munc18-1 domain 3a. However, the relationship and the action order of the two conformational changes remain not fully understood. Here, our data show that an open conformation in the syntaxin-1 linker region can bypass the requirement of the MUN NF sequence. In addition, an extended state of Munc18-1 domain 3a can compensate the role of the syntaxin-1 RI sequence. Altogether, the current data strongly support our previous notion that opening of the syntaxin-1 linker region by Munc13-1 is a key step to initiate SNARE complex assembly, and consequently, Munc18-1 domain 3a can extend its conformation to serve as a template for association of synaptobrevin-2 and syntaxin-1.

scholarly journals Epileptic Phenotypes Associated With SNAREs and Related Synaptic Vesicle Exocytosis Machinery

2022 ◽  
Vol 12 ◽  
Author(s):  
Elisa Cali ◽  
Clarissa Rocca ◽  
Vincenzo Salpietro ◽  
Henry Houlden
Keyword(s):  
Synaptic Vesicle ◽  
Neurodevelopmental Disorder ◽  
Febrile Seizures ◽  
Autism Spectrum ◽  
Snare Complex ◽  
Synaptic Vesicle Exocytosis ◽  
Protein Receptor ◽  
Genes Encoding ◽  
Associated Proteins ◽  
Vesicle Exocytosis

SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptor) are an heterogeneous family of proteins that, together with their key regulators, are implicated in synaptic vesicle exocytosis and synaptic transmission. SNAREs represent the core component of this protein complex. Although the specific mechanisms of the SNARE machinery is still not completely uncovered, studies in recent years have provided a clearer understanding of the interactions regulating the essential fusion machinery for neurotransmitter release. Mutations in genes encoding SNARE proteins or SNARE complex associated proteins have been associated with a variable spectrum of neurological conditions that have been recently defined as “SNAREopathies.” These include neurodevelopmental disorder, autism spectrum disorder (ASD), movement disorders, seizures and epileptiform abnormalities. The SNARE phenotypic spectrum associated with seizures ranges from simple febrile seizures and infantile spasms, to severe early-onset epileptic encephalopathies. Our study aims to review and delineate the epileptic phenotypes associated with dysregulation of synaptic vesicle exocytosis and transmission, focusing on the main proteins of the SNARE core complex (STX1B, VAMP2, SNAP25), tethering complex (STXBP1), and related downstream regulators.

scholarly journals The septin Sept5/CDCrel-1 competes with α-SNAP for binding to the SNARE complex

2005 ◽  
Vol 385 (2) ◽  
pp. 347-353 ◽  
Author(s):  
Crestina L. BEITES ◽  
Kristen A. CAMPBELL ◽  
William S. TRIMBLE
Keyword(s):  
Protein Complexes ◽  
Snare Complex ◽  
Snare Proteins ◽  
Interacting Protein ◽  
Protein Receptor ◽  
Snare Regulators ◽  
Protein Attachment ◽  
The Brain ◽  
Insight Into

SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) proteins are supposed to mediate the docking and/or fusion of the vesicle with the plasma membrane. However, it is not clearly understood how this process is regulated. In a search for potential SNARE regulators, we recently identified septin 5 (Sept5) as a novel SNARE interacting protein. Septins were first identified as filamentous proteins required for cytokinesis in yeast. Several septins have now been identified in mammals but little is known about their functions. We have previously shown that Sept5 is predominantly expressed in the brain, where it associates with vesicles and membranes through its interaction with the SNARE domain of syntaxin 1A. Furthermore, Sept5 appears to inhibit exocytosis, possibly by regulating vesicle targeting and/or fusion events. To gain insight into the role of Sept5, we have mapped the Sept5 domains important for syntaxin binding. We also investigated the ability of Sept5 to bind to syntaxin when in various protein complexes. Although Sept5 cannot bind an nSec1–syntaxin complex, it can bind syntaxin in a SNARE complex. This interaction is occluded by the binding of α-SNAP, suggesting that Sept5 may regulate the availability of SNARE proteins through its interaction with syntaxin and the 7 S complex.

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