scholarly journals Role of α-SNAP in Promoting Efficient Neurotransmission at the Crayfish Neuromuscular Junction

1999 ◽  
Vol 82 (6) ◽  
pp. 3406-3416 ◽  
Author(s):  
Ping He ◽  
R. Chase Southard ◽  
Dong Chen ◽  
S. W. Whiteheart ◽  
R. L. Cooper
Keyword(s):  
Neuromuscular Junction ◽  
Neurotransmitter Release ◽  
Synaptic Vesicles ◽  
Synaptic Current ◽  
Attachment Protein ◽  
Quantal Analysis ◽  
Sensitive Factor ◽  
Electrophysiological Studies ◽  
Crayfish Neuromuscular Junction

In this manuscript, we address the role of the soluble N-ethylmaleimide sensitive factor attachment protein (α-SNAP) in synaptic transmission at the neuromuscular junction of the crayfish opener muscle. Immunochemcial methods confirm the presence of α-SNAP in these preparations and show that it is concentrated in the synaptic areas. Microinjection and electrophysiological studies show that α-SNAP causes an increase in neurotransmitter release yet does not significantly affect the kinetics. More specific quantal analysis, using focal, macropatch, synaptic current recordings, shows that α-SNAP increases transmitter release by increasing the probability of exocytosis but not the number of potential release sites. These data demonstrate that the role of α-SNAP is to increase the efficiency of neurotransmission by increasing the probability that a stimulus will result in neurotransmitter release. What this suggests is that α-SNAP is critical for the formation and maintenance of a “ready release” pool of synaptic vesicles.

scholarly journals Quantitative analysis of the native presynaptic cytomatrix by cryoelectron tomography

2010 ◽  
Vol 188 (1) ◽  
pp. 145-156 ◽  
Author(s):  
Rubén Fernández-Busnadiego ◽  
Benoît Zuber ◽  
Ulrike Elisabeth Maurer ◽  
Marek Cyrklaff ◽  
Wolfgang Baumeister ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Neurotransmitter Release ◽  
Synaptic Vesicles ◽  
Attachment Protein ◽  
Readily Releasable Pool ◽  
Protein Receptor ◽  
Membrane Contact ◽  
Synaptic Vesicle Release ◽  
Protein Attachment

The presynaptic terminal contains a complex network of filaments whose precise organization and functions are not yet understood. The cryoelectron tomography experiments reported in this study indicate that these structures play a prominent role in synaptic vesicle release. Docked synaptic vesicles did not make membrane to membrane contact with the active zone but were instead linked to it by tethers of different length. Our observations are consistent with an exocytosis model in which vesicles are first anchored by long (>5 nm) tethers that give way to multiple short tethers once vesicles enter the readily releasable pool. The formation of short tethers was inhibited by tetanus toxin, indicating that it depends on soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor complex assembly. Vesicles were extensively interlinked via a set of connectors that underwent profound rearrangements upon synaptic stimulation and okadaic acid treatment, suggesting a role of these connectors in synaptic vesicle mobilization and neurotransmitter release.

Different VAMP/Synaptobrevin Complexes for Spontaneous and Evoked Transmitter Release at the Crayfish Neuromuscular Junction

1998 ◽  
Vol 80 (6) ◽  
pp. 3233-3246 ◽  
Author(s):  
Shao-Ying Hua ◽  
Dorota A. Raciborska ◽  
William S. Trimble ◽  
Milton P. Charlton
Keyword(s):  
Neuromuscular Junction ◽  
Transmitter Release ◽  
Neurotransmitter Release ◽  
Action Potentials ◽  
Neuromuscular Junctions ◽  
Spontaneous Release ◽  
Intracellular Ca ◽  
Crayfish Neuromuscular Junction ◽  
Evoked Transmitter Release

Hua, Shao-Ying, Dorota A. Raciborska, William S. Trimble, and Milton P. Charlton. Different VAMP/synaptobrevin complexes for spontaneous and evoked transmitter release at the crayfish neuromuscular junction. J. Neurophysiol. 80: 3233–3246, 1998. Although vesicle-associated membrane protein (VAMP/synaptobrevin) is essential for evoked neurotransmitter release, its role in spontaneous transmitter release remains uncertain. For instance, many studies show that tetanus toxin (TeNT), which cleaves VAMP, blocks evoked transmitter release but leaves some spontaneous transmitter release. We used recombinant tetanus and botulinum neurotoxin catalytic light chains (TeNT-LC, BoNT/B-LC, and BoNT/D-LC) to examine the role of VAMP in spontaneous transmitter release at neuromuscular junctions (nmj) of crayfish. Injection of TeNT-LC into presynaptic axons removed most of the VAMP immunoreactivity and blocked evoked transmitter release without affecting nerve action potentials or Ca2+ influx. The frequency of spontaneous transmitter release was little affected by the TeNT-LC when the evoked transmitter release had been blocked by >95%. The spontaneous transmitter release left after TeNT-LC treatment was insensitive to increases in intracellular Ca2+. BoNT/B-LC, which cleaves VAMP at the same site as TeNT-LC but uses a different binding site, also blocked evoked release but had minimal effect on spontaneous release. However, BoNT/D-LC, which cleaves VAMP at a different site from the other two toxins but binds to the same position on VAMP as TeNT, blocked both evoked and spontaneous transmitter release at similar rates. The data indicate that different VAMP complexes are employed for evoked and spontaneous transmitter release; the VAMP used in spontaneous release is not readily cleaved by TeNT or BoNT/B. Because the exocytosis that occurs after the action of TeNT cannot be increased by increased intracellular Ca2+, the final steps in neurotransmitter release are Ca2+ independent.

Role of NSF in Neurotransmitter Release: A Peptide Microinjection Study at the Crayfish Neuromuscular Junction

2006 ◽  
Vol 96 (3) ◽  
pp. 1053-1060 ◽  
Author(s):  
I. Parnas ◽  
G. Rashkovan ◽  
V. O'Connor ◽  
O. El-Far ◽  
H. Betz ◽  
...  
Keyword(s):  
Amino Acid ◽  
Neuromuscular Junction ◽  
Neurotransmitter Release ◽  
Action Potentials ◽  
Time Course ◽  
Synaptic Delay ◽  
Quantal Content ◽  
Presynaptic Mechanisms ◽  
Crayfish Neuromuscular Junction

Peptides that inhibit the SNAP-stimulated ATPase activity of N-ethylmaleimide-sensitive fusion protein (NSF-2, NSF-3) were injected intra-axonally to study the role of this protein in the release of glutamate at the crayfish neuromuscular junction. Macropatch recording was used to establish the quantal content and to construct synaptic delay histograms. NSF-2 or NSF-3 injection reduced the quantal content, evoked by either direct depolarization of a single release bouton or by axonal action potentials, on average by 66 ± 12% (mean ± SD; n = 32), but had no effect on the time course of release. NSF-2 had no effect on the amplitude or shape of the presynaptic action potential nor on the excitatory nerve terminal current. Neither NSF-2 nor NSF-3 affected the shape or amplitude of single quantal currents. Injection of a peptide with the same composition as NSF-2, but with a scrambled amino acid sequence, failed to alter the quantal content. We conclude that, at the crayfish neuromuscular junction, NSF-dependent reactions regulate quantal content without contributing to the presynaptic mechanisms that control the time course of release.

scholarly journals Role of Rab3 GDP/GTP Exchange Protein in Synaptic Vesicle Trafficking at the Mouse Neuromuscular Junction

2001 ◽  
Vol 12 (5) ◽  
pp. 1421-1430 ◽  
Author(s):  
Miki Tanaka ◽  
Jun Miyoshi ◽  
Hiroyoshi Ishizaki ◽  
Atsushi Togawa ◽  
Katsunori Ohnishi ◽  
...  
Keyword(s):  
Neuromuscular Junction ◽  
Neurotransmitter Release ◽  
Synaptic Vesicles ◽  
Action Potentials ◽  
Axonal Conduction ◽  
Small G Protein ◽  
Presynaptic Plasma Membrane ◽  
Gastrocnemius Muscles ◽  
Exchange Protein

The Rab3 small G protein family consists of four members, Rab3A, -3B, -3C, and -3D. Of these members, Rab3A regulates Ca2+-dependent neurotransmitter release. These small G proteins are activated by Rab3 GDP/GTP exchange protein (Rab3 GEP). To determine the function of Rab3 GEP during neurotransmitter release, we have knocked out Rab3 GEP in mice. Rab3 GEP−/− mice developed normally but died immediately after birth. Embryos at E18.5 showed no evoked action potentials of the diaphragm and gastrocnemius muscles in response to electrical stimulation of the phrenic and sciatic nerves, respectively. In contrast, axonal conduction of the spinal cord and the phrenic nerve was not impaired. Total numbers of synaptic vesicles, especially those docked at the presynaptic plasma membrane, were reduced at the neuromuscular junction ∼10-fold compared with controls, whereas postsynaptic structures and functions appeared normal. Thus, Rab3 GEP is essential for neurotransmitter release and probably for formation and trafficking of the synaptic vesicles.

Role of SNAP-23 in trafficking of H+-ATPase in cultured inner medullary collecting duct cells

2001 ◽  
Vol 280 (4) ◽  
pp. C775-C781 ◽  
Author(s):  
Abhijit Banerjee ◽  
Guangmu Li ◽  
Edward A. Alexander ◽  
John H. Schwartz
Keyword(s):  
Botulinum Toxin ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Snare Complex ◽  
Attachment Protein ◽  
Regulated Exocytosis ◽  
Inner Medullary Collecting Duct ◽  
Sensitive Factor ◽  
Medullary Collecting Duct

The trafficking of H+-ATPase vesicles to the apical membrane of inner medullary collecting duct (IMCD) cells utilizes a mechanism similar to that described in neurosecretory cells involving soluble N-ethylmaleimide-sensitive factor attachment protein target receptor (SNARE) proteins. Regulated exocytosis of these vesicles is associated with the formation of SNARE complexes. Clostridial neurotoxins that specifically cleave the target (t-) SNARE, syntaxin-1, or the vesicle SNARE, vesicle-associated membrane protein-2, reduce SNARE complex formation, H+-ATPase translocation to the apical membrane, and inhibit H+ secretion. The purpose of these experiments was to characterize the physiological role of a second t-SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP)-23, a homologue of the neuronal SNAP-25, in regulated exocytosis of H+-ATPase vesicles. Our experiments document that 25–50 nM botulinum toxin (Bot) A or E cleaves rat SNAP-23 and thereby reduces immunodetectable and35S-labeled SNAP-23 by >60% within 60 min. Addition of 25 nM BotE to IMCD homogenates reduces the amount of the 20 S-like SNARE complex that can be immunoprecipitated from the homogenate. Treatment of intact IMCD monolayers with BotE reduces the amount of H+-ATPase translocated to the apical membrane by 52 ± 2% of control and reduces the rate of H+ secretion by 77 ± 3% after acute cell acidification. We conclude that SNAP-23 is a substrate for botulinum toxin proteolysis and has a critical role in the regulation of H+-ATPase exocytosis and H+ secretion in these renal epithelial cells.

Distinct Initial SNARE Configurations Underlying the Diversity of Exocytosis

2012 ◽  
Vol 92 (4) ◽  
pp. 1915-1964 ◽  
Author(s):  
Haruo Kasai ◽  
Noriko Takahashi ◽  
Hiroshi Tokumaru
Keyword(s):  
Membrane Trafficking ◽  
Synaptic Vesicles ◽  
Cell Types ◽  
Snare Proteins ◽  
Attachment Protein ◽  
Protein Receptor ◽  
Large Dense Core Vesicles ◽  
Sensitive Factor ◽  
The Common ◽  
Kinetics Of

The dynamics of exocytosis are diverse and have been optimized for the functions of synapses and a wide variety of cell types. For example, the kinetics of exocytosis varies by more than five orders of magnitude between ultrafast exocytosis in synaptic vesicles and slow exocytosis in large dense-core vesicles. However, in all cases, exocytosis is mediated by the same fundamental mechanism, i.e., the assembly of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. It is often assumed that vesicles need to be docked at the plasma membrane and SNARE proteins must be preassembled before exocytosis is triggered. However, this model cannot account for the dynamics of exocytosis recently reported in synapses and other cells. For example, vesicles undergo exocytosis without prestimulus docking during tonic exocytosis of synaptic vesicles in the active zone. In addition, epithelial and hematopoietic cells utilize cAMP and kinases to trigger slow exocytosis of nondocked vesicles. In this review, we summarize the manner in which the diversity of exocytosis reflects the initial configurations of SNARE assembly, including trans-SNARE, binary-SNARE, unitary-SNARE, and cis-SNARE configurations. The initial SNARE configurations depend on the particular SNARE subtype (syntaxin, SNAP25, or VAMP), priming proteins (Munc18, Munc13, CAPS, complexin, or snapin), triggering proteins (synaptotagmins, Doc2, and various protein kinases), and the submembraneous cytomatrix, and they are the key to determining the kinetics of subsequent exocytosis. These distinct initial configurations will help us clarify the common SNARE assembly processes underlying exocytosis and membrane trafficking in eukaryotic cells.

scholarly journals Fife organizes synaptic vesicles and calcium channels for high-probability neurotransmitter release

2016 ◽  
Vol 216 (1) ◽  
pp. 231-246 ◽  
Author(s):  
Joseph J. Bruckner ◽  
Hong Zhan ◽  
Scott J. Gratz ◽  
Monica Rao ◽  
Fiona Ukken ◽  
...  
Keyword(s):  
Neural Plasticity ◽  
Active Zone ◽  
Neurotransmitter Release ◽  
Synaptic Vesicles ◽  
Motor Behavior ◽  
Molecular Organization ◽  
Electrophysiological Studies ◽  
Circuit Function ◽  
Synaptic Vesicle Release ◽  
Ca2 Channels

The strength of synaptic connections varies significantly and is a key determinant of communication within neural circuits. Mechanistic insight into presynaptic factors that establish and modulate neurotransmitter release properties is crucial to understanding synapse strength, circuit function, and neural plasticity. We previously identified Drosophila Piccolo-RIM-related Fife, which regulates neurotransmission and motor behavior through an unknown mechanism. Here, we demonstrate that Fife localizes and interacts with RIM at the active zone cytomatrix to promote neurotransmitter release. Loss of Fife results in the severe disruption of active zone cytomatrix architecture and molecular organization. Through electron tomographic and electrophysiological studies, we find a decrease in the accumulation of release-ready synaptic vesicles and their release probability caused by impaired coupling to Ca2+ channels. Finally, we find that Fife is essential for the homeostatic modulation of neurotransmission. We propose that Fife organizes active zones to create synaptic vesicle release sites within nanometer distance of Ca2+ channel clusters for reliable and modifiable neurotransmitter release.

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