scholarly journals Ion transport and regulation in a synaptic vesicle glutamate transporter

Science ◽  
2020 ◽  
Vol 368 (6493) ◽  
pp. 893-897 ◽  
Author(s):  
Fei Li ◽  
Jacob Eriksen ◽  
Janet Finer-Moore ◽  
Roger Chang ◽  
Phuong Nguyen ◽  
...  
Keyword(s):  
Synaptic Transmission ◽  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Structural Information ◽  
Glutamate Transporter ◽  
Excitatory Neurotransmitter ◽  
Glutamate Binding ◽  
Cryo Electron Microscopy ◽  
Vesicle Cycle ◽  
Do So

Synaptic vesicles accumulate neurotransmitters, enabling the quantal release by exocytosis that underlies synaptic transmission. Specific neurotransmitter transporters are responsible for this activity and therefore are essential for brain function. The vesicular glutamate transporters (VGLUTs) concentrate the principal excitatory neurotransmitter glutamate into synaptic vesicles, driven by membrane potential. However, the mechanism by which they do so remains poorly understood owing to a lack of structural information. We report the cryo–electron microscopy structure of rat VGLUT2 at 3.8-angstrom resolution and propose structure-based mechanisms for substrate recognition and allosteric activation by low pH and chloride. A potential permeation pathway for chloride intersects with the glutamate binding site. These results demonstrate how the activity of VGLUTs can be coordinated with large shifts in proton and chloride concentrations during the synaptic vesicle cycle to ensure normal synaptic transmission.

scholarly journals Counting the Number of Glutamate Molecules in Single Synaptic Vesicles

2019 ◽  
Author(s):  
Yuanmo Wang ◽  
Hoda fathali ◽  
devesh mishra ◽  
Thomas Olsson ◽  
Jacqueline Keighron ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
High Speed ◽  
Glutamate Release ◽  
Dependent Manner ◽  
Excitatory Neurotransmitter ◽  
Quantitative Measurements ◽  
Sensor Technique ◽  
Drug Treatments ◽  
Analytical Tools

<div><p>Analytical tools for direct quantitative measurements of glutamate, the principal excitatory neurotransmitter in brain, are lacking. Here, we introduce a new enzyme-based amperometric sensor technique for direct counting of the number of glutamate molecules stored inside single synaptic vesicles. An ultra-fast enzyme-based glutamate sensor is placed into a solution of isolated synaptic vesicles, which stochastically rupture at the sensor surface in a potential dependent manner by applying a constant negative potential. High-speed (10 kHz) amperometry is used to record sub-millisecond current spikes, which represent glutamate release from single vesicles that burst open. Glutamate quantification is achieved by a calibration curve that is based on measurements of glutamate release from vesicles pre-filled with various concentrations of glutamate. Our measurements show that a single synaptic vesicle encapsulates about 8000 glutamate molecules, which is comparable to the measured exocytotic quantal glutamate release in the nucleus accumbens of mouse brain tissue. Hence, this new methodology introduces the means to quantify ultra-small amounts of glutamate and to study synaptic vesicle physiology, pathogenesis and drug treatments for neuronal disorders where glutamate is involved.</p></div>

scholarly journals Dual pools of actin at presynaptic terminals

2012 ◽  
Vol 107 (12) ◽  
pp. 3479-3492 ◽  
Author(s):  
Adam Bleckert ◽  
Huzefa Photowala ◽  
Simon Alford
Keyword(s):  
Synaptic Transmission ◽  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Repetitive Stimulation ◽  
Filamentous Actin ◽  
Cortical Actin ◽  
Vesicle Recycling ◽  
Latrunculin A ◽  
Kinetics Of

We investigated actin's function in vesicle recycling and exocytosis at lamprey synapses and show that FM1-43 puncta and phalloidin-labeled filamentous actin (F-actin) structures are colocalized, yet recycling vesicles are not contained within F-actin clusters. Additionally, phalloidin also labels a plasma membrane-associated cortical actin. Injection of fluorescent G-actin revealed activity-independent dynamic actin incorporation into presynaptic synaptic vesicle clusters but not into cortical actin. Latrunculin-A, which sequesters G-actin, dispersed vesicle-associated actin structures and prevented subsequent labeled G-actin and phalloidin accumulation at presynaptic puncta, yet cortical phalloidin labeling persisted. Dispersal of presynaptic F-actin structures by latrunculin-A did not disrupt vesicle clustering or recycling or alter the amplitude or kinetics of excitatory postsynaptic currents (EPSCs). However, it slightly enhanced release during repetitive stimulation. While dispersal of presynaptic actin puncta with latrunculin-A failed to disperse synaptic vesicles or inhibit synaptic transmission, presynaptic phalloidin injection blocked exocytosis and reduced endocytosis measured by action potential-evoked FM1-43 staining. Furthermore, phalloidin stabilization of only cortical actin following pretreatment with latrunculin-A was sufficient to inhibit synaptic transmission. Conversely, treatment of axons with jasplakinolide, which induces F-actin accumulation but disrupts F-actin structures in vivo, resulted in increased synaptic transmission accompanied by a loss of phalloidin labeling of cortical actin but no loss of actin labeling within vesicle clusters. Marked synaptic deficits seen with phalloidin stabilization of cortical F-actin, in contrast to the minimal effects of disruption of a synaptic vesicle-associated F-actin, led us to conclude that two structurally and functionally distinct pools of actin exist at presynaptic sites.

Recent Breakthroughs in Neurotransmitter Release: Paradigm for Regulated Exocytosis?

Physiology ◽  
1995 ◽  
Vol 10 (1) ◽  
pp. 42-46
Author(s):  
G Thiel
Keyword(s):  
Synaptic Vesicle ◽  
Brain Function ◽  
Neurotransmitter Release ◽  
Synaptic Vesicles ◽  
Vesicle Trafficking ◽  
Regulated Exocytosis ◽  
Intracellular Vesicle ◽  
Vesicle Cycle ◽  
Synaptic Vesicle Cycle

Synaptic vesicles play a fundamental role in brain function by mediating the release of neurotransmitters. Neurons do not use an entirely unique secretion apparatus but rather a modification of the general secretion machinery. Moreover, the synaptic vesicle cycle has many similarities with intracellular vesicle trafficking pathways.

scholarly journals The synaptic vesicle cycle: a single vesicle budding step involving clathrin and dynamin.

1996 ◽  
Vol 133 (6) ◽  
pp. 1237-1250 ◽  
Author(s):  
K Takei ◽  
O Mundigl ◽  
L Daniell ◽  
P De Camilli
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Hippocampal Neurons ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
Nerve Terminals ◽  
Vesicle Budding ◽  
Vesicle Cycle ◽  
Single Vesicle

Strong evidence implicates clathrin-coated vesicles and endosome-like vacuoles in the reformation of synaptic vesicles after exocytosis, and it is generally assumed that these vacuoles represent a traffic station downstream from clathrin-coated vesicles. To gain insight into the mechanisms of synaptic vesicle budding from endosome-like intermediates, lysed nerve terminals and nerve terminal membrane subfractions were examined by EM after incubations with GTP gamma S. Numerous clathrin-coated budding intermediates that were positive for AP2 and AP180 immunoreactivity and often collared by a dynamin ring were seen. These were present not only on the plasma membrane (Takei, K., P.S. McPherson, S.L.Schmid, and P. De Camilli. 1995. Nature (Lond.). 374:186-190), but also on internal vacuoles. The lumen of these vacuoles retained extracellular tracers and was therefore functionally segregated from the extracellular medium, although narrow connections between their membranes and the plasmalemma were sometimes visible by serial sectioning. Similar observations were made in intact cultured hippocampal neurons exposed to high K+ stimulation. Coated vesicle buds were generally in the same size range of synaptic vesicles and positive for the synaptic vesicle protein synaptotagmin. Based on these results, we suggest that endosome-like intermediates of nerve terminals originate by bulk uptake of the plasma membrane and that clathrin- and dynamin-mediated budding takes place in parallel from the plasmalemma and from these internal membranes. We propose a synaptic vesicle recycling model that involves a single vesicle budding step mediated by clathrin and dynamin.

scholarly journals How do you recognize and reconstitute a synaptic vesicle after fusion?

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 1734 ◽  
Author(s):  
Natali L. Chanaday ◽  
Ege T. Kavalali
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Membrane Composition ◽  
Synaptic Vesicle Recycling ◽  
Vesicle Recycling ◽  
Molecular Identity ◽  
Daunting Task ◽  
Vesicle Biogenesis ◽  
Vesicle Cycle

Synaptic vesicle recycling is essential for sustained and reliable neurotransmission. A key component of synaptic vesicle recycling is the synaptic vesicle biogenesis process that is observed in synapses and that maintains the molecular identity of synaptic vesicles. However, the mechanisms by which synaptic vesicles are retrieved and reconstituted after fusion remain unclear. The complex molecular composition of synaptic vesicles renders their rapid biogenesis a daunting task. Therefore, in this context, kiss-and-run type transient fusion of synaptic vesicles with the plasma membrane without loss of their membrane composition and molecular identity remains a viable hypothesis that can account for the fidelity of the synaptic vesicle cycle. In this article, we discuss the biological implications of this problem as well as its possible molecular solutions.

scholarly journals Counting the Number of Glutamate Molecules in Single Synaptic Vesicles

2019 ◽  
Author(s):  
Yuanmo Wang ◽  
Hoda fathali ◽  
devesh mishra ◽  
Thomas Olsson ◽  
Jacqueline Keighron ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
High Speed ◽  
Glutamate Release ◽  
Dependent Manner ◽  
Excitatory Neurotransmitter ◽  
Quantitative Measurements ◽  
Sensor Technique ◽  
Drug Treatments ◽  
Analytical Tools

<div><p>Analytical tools for direct quantitative measurements of glutamate, the principal excitatory neurotransmitter in brain, are lacking. Here, we introduce a new enzyme-based amperometric sensor technique for direct counting of the number of glutamate molecules stored inside single synaptic vesicles. An ultra-fast enzyme-based glutamate sensor is placed into a solution of isolated synaptic vesicles, which stochastically rupture at the sensor surface in a potential dependent manner by applying a constant negative potential. High-speed (10 kHz) amperometry is used to record sub-millisecond current spikes, which represent glutamate release from single vesicles that burst open. Glutamate quantification is achieved by a calibration curve that is based on measurements of glutamate release from vesicles pre-filled with various concentrations of glutamate. Our measurements show that a single synaptic vesicle encapsulates about 8000 glutamate molecules, which is comparable to the measured exocytotic quantal glutamate release in the nucleus accumbens of mouse brain tissue. Hence, this new methodology introduces the means to quantify ultra-small amounts of glutamate and to study synaptic vesicle physiology, pathogenesis and drug treatments for neuronal disorders where glutamate is involved.</p></div>

scholarly journals Wnt-7aModulates the Synaptic Vesicle Cycle and Synaptic Transmission in Hippocampal Neurons

2007 ◽  
Vol 283 (9) ◽  
pp. 5918-5927 ◽  
Author(s):  
Waldo Cerpa ◽  
Juan A. Godoy ◽  
Iván Alfaro ◽  
Ginny G. Farías ◽  
María J. Metcalfe ◽  
...  
Keyword(s):  
Synaptic Transmission ◽  
Synaptic Vesicle ◽  
Hippocampal Neurons ◽  
Vesicle Cycle ◽  
Synaptic Vesicle Cycle

scholarly journals Synaptotagmin 7 is targeted to the axonal plasma membrane through γ-secretase processing to promote synaptic vesicle docking in mouse hippocampal neurons

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Jason D Vevea ◽  
Grant F Kusick ◽  
Kevin C Courtney ◽  
Erin Chen ◽  
Shigeki Watanabe ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Hippocampal Neurons ◽  
Glutamate Release ◽  
Vesicle Docking ◽  
Presynaptic Function ◽  
Asynchronous Release ◽  
Vesicle Cycle ◽  
Activity Dependent

Synaptotagmin 7 (SYT7) has emerged as a key regulator of presynaptic function, but its localization and precise role in the synaptic vesicle cycle remain the subject of debate. Here, we used iGluSnFR to optically interrogate glutamate release, at the single-bouton level, in SYT7KO-dissociated mouse hippocampal neurons. We analyzed asynchronous release, paired-pulse facilitation, and synaptic vesicle replenishment and found that SYT7 contributes to each of these processes to different degrees. ‘Zap-and-freeze’ electron microscopy revealed that a loss of SYT7 diminishes docking of synaptic vesicles after a stimulus and inhibits the recovery of depleted synaptic vesicles after a stimulus train. SYT7 supports these functions from the axonal plasma membrane, where its localization and stability require both γ-secretase-mediated cleavage and palmitoylation. In summary, SYT7 is a peripheral membrane protein that controls multiple modes of synaptic vesicle (SV) exocytosis and plasticity, in part, through enhancing activity-dependent docking of SVs.

scholarly journals C2B Polylysine Motif of Synaptotagmin Facilitates a Ca2+-independent Stage of Synaptic Vesicle Priming In Vivo

2006 ◽  
Vol 17 (12) ◽  
pp. 5211-5226 ◽  
Author(s):  
Carin A. Loewen ◽  
Soo-Min Lee ◽  
Yeon-Kyun Shin ◽  
Noreen E. Reist
Keyword(s):  
Synaptic Vesicle ◽  
Membrane Trafficking ◽  
Synaptic Vesicles ◽  
Attachment Protein ◽  
Release Probability ◽  
Synaptotagmin I ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Vesicle Cycle

Synaptotagmin I, a synaptic vesicle protein required for efficient synaptic transmission, contains a highly conserved polylysine motif necessary for function. Using Drosophila, we examined in which step of the synaptic vesicle cycle this motif functions. Polylysine motif mutants exhibited an apparent decreased Ca2+ affinity of release, and, at low Ca2+, an increased failure rate, increased facilitation, and increased augmentation, indicative of a decreased release probability. Disruption of Ca2+ binding, however, cannot account for all of the deficits in the mutants; rather, the decreased release probability is probably due to a disruption in the coupling of synaptotagmin to the release machinery. Mutants exhibited a major slowing of recovery from synaptic depression, which suggests that membrane trafficking before fusion is disrupted. The disrupted process is not endocytosis because the rate of FM 1-43 uptake was unchanged in the mutants, and the polylysine motif mutant synaptotagmin was able to rescue the synaptic vesicle depletion normally found in sytnull mutants. Thus, the polylysine motif functions after endocytosis and before fusion. Finally, mutation of the polylysine motif inhibits the Ca2+-independent ability of synaptotagmin to accelerate SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-mediated fusion. Together, our results demonstrate that the polylysine motif is required for efficient Ca2+-independent docking and/or priming of synaptic vesicles in vivo.

scholarly journals Synaptotagmin 7 is enriched at the plasma membrane through γ-secretase processing to promote vesicle docking and control synaptic plasticity in mouse hippocampal neurons

2021 ◽  
Author(s):  
Jason D. Vevea ◽  
Grant F. Kusick ◽  
Erin Chen ◽  
Kevin C. Courtney ◽  
Shigeki Watanabe ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Hippocampal Neurons ◽  
Synaptic Function ◽  
List Type ◽  
Paired Pulse ◽  
Paired Pulse Facilitation ◽  
Asynchronous Release ◽  
Vesicle Cycle

Abstract Synaptotagmin (SYT) 7 has emerged as key regulator of presynaptic function, but its localization and precise function in the synaptic vesicle cycle remain unclear. Here, we used iGluSnFR to optically and directly interrogate glutamate release, at the single bouton level, in SYT7 KO dissociated mouse hippocampal neurons. We analyzed asynchronous release, paired pulse facilitation, and synaptic vesicle replenishment, and found that SYT7 contributes to each of these processes to different degrees. ‘Zap-and-freeze’ electron microscopy revealed that loss of SYT7 impairs the docking of synaptic vesicles after a stimulus and the recovery of depleted synaptic vesicles after a stimulus train. To execute these functions, SYT7 must be targeted to the plasma membrane via γ-secretase-mediated cleavage of the amino terminus, followed by palmitoylation. The complex sorting itinerary of SYT7 endows this Ca2+-sensor with the ability to control crucial forms of synaptic function and plasticity. SYT7 mediated asynchronous release, paired pulse facilitation, and synaptic vesicle replenishment was observed optically at individual hippocampal synapses Localization, trafficking, and stability of SYT7 is dependent on processing by γ-secretase Short term plasticity defects arise in SYT7KOs due to decreased docking of synaptic vesicles after stimulation SYT7 promotes paired-pulse facilitation and asynchronous release via distinct mechanisms

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