Synaptotagmin 1 oligomers clamp and regulate different modes of neurotransmitter release

AbstractSynaptotagmin1 (Syt1) synchronises neurotransmitter release to action potentials acting as the fast Ca2+ release sensor and as the inhibitor (clamp) of spontaneous and delayed asynchronous release. Whilst the Syt1 Ca2+ activation mechanism has been well characterised, how Syt1 clamps transmitter release remains enigmatic. Here we show that C2B domain-dependent oligomerisation provides the molecular basis for the Syt1 clamping function. This follows from the investigation of a designed mutation (F349A), which selectively destabilises Syt1 oligomerisation. Using combination of fluorescence imaging and electrophysiology in neocortical synapses we show that Syt1F349A is more efficient than wild type Syt1 (Syt1WT) in triggering synchronous transmitter release but fails to clamp spontaneous and Synaptotagmin7 (Syt7)-mediated asynchronous release components both in rescue (Syt1−/− knock-out background) and dominant-interference (Syt1+/+ background) conditions. Thus we conclude that Ca2+-sensitive Syt1 oligomers, acting as an exocytosis clamp, are critical for maintaining the balance among the different modes of neurotransmitter release.

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Synaptotagmin 1 oligomers clamp and regulate different modes of neurotransmitter release

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1920403117 ◽

2020 ◽

Vol 117 (7) ◽

pp. 3819-3827 ◽

Cited By ~ 4

Author(s):

Erica Tagliatti ◽

Oscar D. Bello ◽

Philipe R. F. Mendonça ◽

Dimitrios Kotzadimitriou ◽

Elizabeth Nicholson ◽

...

Keyword(s):

Fluorescence Imaging ◽

Transmitter Release ◽

Neurotransmitter Release ◽

Action Potentials ◽

Molecular Basis ◽

Activation Mechanism ◽

Wild Type ◽

Synaptotagmin 1 ◽

Asynchronous Release

Synaptotagmin 1 (Syt1) synchronizes neurotransmitter release to action potentials (APs) acting as the fast Ca2+ release sensor and as the inhibitor (clamp) of spontaneous and delayed asynchronous release. While the Syt1 Ca2+ activation mechanism has been well-characterized, how Syt1 clamps transmitter release remains enigmatic. Here we show that C2B domain-dependent oligomerization provides the molecular basis for the Syt1 clamping function. This follows from the investigation of a designed mutation (F349A), which selectively destabilizes Syt1 oligomerization. Using a combination of fluorescence imaging and electrophysiology in neocortical synapses, we show that Syt1F349A is more efficient than wild-type Syt1 (Syt1WT) in triggering synchronous transmitter release but fails to clamp spontaneous and synaptotagmin 7 (Syt7)-mediated asynchronous release components both in rescue (Syt1−/− knockout background) and dominant-interference (Syt1+/+ background) conditions. Thus, we conclude that Ca2+-sensitive Syt1 oligomers, acting as an exocytosis clamp, are critical for maintaining the balance among the different modes of neurotransmitter release.

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Structural elements that underlie Doc2β function during asynchronous synaptic transmission

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1502288112 ◽

2015 ◽

Vol 112 (31) ◽

pp. E4316-E4325 ◽

Cited By ~ 11

Author(s):

Renhao Xue ◽

Jon D. Gaffaney ◽

Edwin R. Chapman

Keyword(s):

Plasma Membrane ◽

Synaptic Transmission ◽

Neurotransmitter Release ◽

Lipid Binding ◽

Slow Phase ◽

Structural Elements ◽

Excitatory Postsynaptic Current ◽

Synaptotagmin 1 ◽

Asynchronous Release

Double C2-like domain-containing proteins alpha and beta (Doc2α and Doc2β) are tandem C2-domain proteins proposed to function as Ca2+ sensors for asynchronous neurotransmitter release. Here, we systematically analyze each of the negatively charged residues that mediate binding of Ca2+ to the β isoform. The Ca2+ ligands in the C2A domain were dispensable for Ca2+-dependent translocation to the plasma membrane, with one exception: neutralization of D220 resulted in constitutive translocation. In contrast, three of the five Ca2+ ligands in the C2B domain are required for translocation. Importantly, translocation was correlated with the ability of the mutants to enhance asynchronous release when overexpressed in neurons. Finally, replacement of specific Ca2+/lipid-binding loops of synaptotagmin 1, a Ca2+ sensor for synchronous release, with corresponding loops from Doc2β, resulted in chimeras that yielded slower kinetics in vitro and slower excitatory postsynaptic current decays in neurons. Together, these data reveal the key determinants of Doc2β that underlie its function during the slow phase of synaptic transmission.

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Postsynaptic Action Potentials Are Required for Nitric-Oxide-Dependent Long-Term Potentiation in CA1 Neurons of Adult GluR1 Knock-Out and Wild-Type Mice

Journal of Neuroscience ◽

10.1523/jneurosci.3984-08.2008 ◽

2008 ◽

Vol 28 (52) ◽

pp. 14031-14041 ◽

Cited By ~ 26

Author(s):

K. G. Phillips ◽

N. R. Hardingham ◽

K. Fox

Keyword(s):

Nitric Oxide ◽

Action Potentials ◽

Long Term Potentiation ◽

Wild Type ◽

Knock Out ◽

Ca1 Neurons ◽

Term Potentiation ◽

Postsynaptic Action

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Drosophila Synaptotagmin 7 negatively regulates synaptic vesicle fusion and replenishment in a dosage-dependent manner

10.1101/2020.01.30.926246 ◽

2020 ◽

Author(s):

Zhuo Guan ◽

Mónica C. Quiñones-Frías ◽

Yulia Akbergenova ◽

J. Troy Littleton

Keyword(s):

Plasma Membrane ◽

Synaptic Vesicle ◽

Active Zone ◽

Neurotransmitter Release ◽

Dependent Manner ◽

Short Term ◽

Sensor Model ◽

Synaptotagmin 1 ◽

Asynchronous Release ◽

Synaptic Vesicle Fusion

AbstractSynchronous neurotransmitter release is triggered by Ca2+ binding to the synaptic vesicle protein Synaptotagmin 1, while asynchronous fusion and short-term facilitation is hypothesized to be mediated by plasma membrane-localized Synaptotagmin 7 (SYT7). We generated mutations in Drosophila Syt7 to determine if it plays a conserved role as the Ca2+ sensor for these processes. Electrophysiology and quantal imaging revealed evoked release was elevated 2-fold. Syt7 mutants also had a larger pool of readily-releasable vesicles, faster recovery following stimulation, and robust facilitation. Syt1/Syt7 double mutants displayed more release than Syt1 mutants alone, indicating SYT7 does not mediate the residual asynchronous release remaining in the absence of SYT1. SYT7 localizes to an internal membrane tubular network within the peri-active zone, but does not enrich at release sites. These findings indicate the two Ca2+ sensor model of SYT1 and SYT7 mediating all phases of neurotransmitter release and facilitation is not applicable at Drosophila synapses.

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

Journal of Neurophysiology ◽

10.1152/jn.1998.80.6.3233 ◽

1998 ◽

Vol 80 (6) ◽

pp. 3233-3246 ◽

Cited By ~ 42

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.

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Presynaptic calcium-activated potassium channels and calcium channels at a crayfish neuromuscular junction

Journal of Neurophysiology ◽

10.1152/jn.1995.73.1.178 ◽

1995 ◽

Vol 73 (1) ◽

pp. 178-189 ◽

Cited By ~ 20

Author(s):

J. A. Blundon ◽

S. N. Wright ◽

M. S. Brodwick ◽

G. D. Bittner

Keyword(s):

Transmitter Release ◽

Neurotransmitter Release ◽

Action Potentials ◽

Presynaptic Terminal ◽

Terminal Region ◽

Ca Channel ◽

Current Clamp ◽

Presynaptic Terminals ◽

Duration Of Action ◽

Test Pulse

1. We used a two-microelectrode current clamp to investigate various characteristics of the Ca(2+)-activated K+ conductance [gK(Ca)] and Ca2+ conductance (gCa), and transmitter release in presynaptic terminals of excitatory neuromuscular junctions in the crayfish walking leg. 2. Voltage-activated Na+ conductances (gNa) and K+ conductances [gK(v)] were blocked with tetrodotoxin and 3,4-diaminopyridine, respectively. Under these conditions, presynaptic depolarization produced by a first (conditioning) pulse admitted Ca2+ into the presynaptic terminals and activated gK(Ca), which modulated the amplitude of the depolarization produced by a second (test) pulse. The relative amount of gK(Ca) measured at the test pulse increased with increased magnitude or duration of the conditioning pulse. 3. A brief hyperpolarization immediately after a conditioning pulse substantially reduced gK(Ca). 4. gK(Ca) activation was blocked by funnel web spider toxin (a Ca2+ channel blocker) or by injection of the presynaptic terminal region with a calcium chelator, bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA). Under current-clamp conditions, gK(Ca) was not blocked by charybdotoxin or iberiotoxin [specific gK(Ca) blockers]. 5. When gK(Ca) was blocked or reduced, the amplitude of the depolarizing afterpotential of action potentials was increased. When gK(v) was blocked or reduced, the duration of action potentials was increased. 6. Intracellular injection of BAPTA into the presynaptic terminal region eliminated evoked neurotransmitter release before test pulse modulation was affected, suggesting that the K(Ca) channel had a greater sensitivity (greater affinity or lower stoichiometry) for Ca2+ than did the transmitter release machinery. BAPTA reduced neurotransmitter release by 66-78%, but did not affect facilitation of neurotransmitter release. 7. When gNa, gK(v), and gK(Ca) were blocked, we detected a membrane depolarization produced by an increase in presynaptic gCa that was eliminated by 2 mM Cd2+ or 0 mM Ca2+.

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cAMP Modulates Intracellular Ca2+ Sensitivity of Fast-Releasing Synaptic Vesicles at the Calyx of Held Synapse

Journal of Neurophysiology ◽

10.1152/jn.00685.2010 ◽

2010 ◽

Vol 104 (6) ◽

pp. 3250-3260 ◽

Cited By ~ 14

Author(s):

Lijun Yao ◽

Takeshi Sakaba

Keyword(s):

Transmitter Release ◽

Neurotransmitter Release ◽

Synaptic Vesicles ◽

Action Potentials ◽

Calyx Of Held ◽

Intracellular Ca ◽

Underlying Mechanisms ◽

Brain Stem Slices ◽

Allosteric Model ◽

Stem Slices

cAMP potentiates neurotransmitter release from the presynaptic terminal in many CNS synapses, but the underlying mechanisms remain unclear. Here we addressed this issue quantitatively by performing double patch-clamp recordings from the pre- and postsynaptic compartments of the calyx of Held synapse in rat brain stem slices in combination with Ca2+ uncaging. We found that elevation of cAMP increased intracellular Ca2+ sensitivity for transmitter release especially at lower Ca2+ concentrations. The change in Ca2+ sensitivity was limited to the fast-releasing synaptic vesicles, which could be released rapidly on action potentials. cAMP did not affect the slowly releasing vesicles. Fit of the data using a simplified allosteric model indicated that cAMP increased the fusion “willingness,” thereby facilitating transmitter release. We suggest that synaptic vesicles have to be positionally primed to the release sites close to the Ca2+ channel cluster for cAMP to modulate intracellular Ca2+ sensitivity of transmitter release.

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The role of the C2A domain of synaptotagmin 1 in asynchronous neurotransmitter release

10.1101/2020.03.11.973461 ◽

2020 ◽

Author(s):

Mallory C. Shields ◽

Matthew R. Bowers ◽

Hannah L. Kramer ◽

McKenzie M. Fulcer ◽

Lara C. Perinet ◽

...

Keyword(s):

Neurotransmitter Release ◽

Spatial Competition ◽

Competition Hypothesis ◽

Inhibition Hypothesis ◽

Synaptotagmin 1 ◽

Asynchronous Release ◽

Opposing Effects ◽

Insight Into

AbstractFollowing nerve stimulation, there are two distinct phases of Ca2+-dependent neurotransmitter release: a fast, synchronous release phase, and a prolonged, asynchronous release phase. Each of these phases is tightly regulated and mediated by distinct mechanisms. Synaptotagmin 1 is the major Ca2+ sensor that triggers fast, synchronous neurotransmitter release upon Ca2+ binding by its C2A and C2B domains. It has also been implicated in the inhibition of asynchronous neurotransmitter release, as blocking Ca2+ binding by the C2A domain of synaptotagmin 1 results in increased asynchronous release. However, the mutation used to block Ca2+ binding in the previous experiments (aspartate to asparagine mutations, sytD-N) had the unintended side effect of mimicking Ca2+ binding, raising the possibility that the increase in asynchronous release was an artifact of ostensibly constitutive Ca2+ binding. To directly test this C2A inhibition hypothesis, we utilized an alternate C2A mutation that we designed to block Ca2+ binding without mimicking it (an aspartate to glutamate mutation, sytD-E). Analysis of both the original sytD-N mutation and our alternate sytD-E mutation at the Drosophila neuromuscular junction showed differential effects on asynchronous release, as well as on synchronous release and the frequency of spontaneous release. Importantly, we found that asynchronous release is not increased in the sytD-E mutant. Thus, our work provides new mechanistic insight into synaptotagmin 1 function during Ca2+-evoked synaptic transmission and demonstrates that Ca2+ binding by the C2A domain of synaptotagmin 1 does not inhibit asynchronous neurotransmitter release in vivo.Significance statementThis study provides mechanistic insights into synaptotagmin function during asynchronous neurotransmitter release and supports a dramatically different hypothesis regarding the mechanisms triggering asynchronous vesicle fusion. Using two distinct C2A mutations that block Ca2+ binding, we report opposing effects on synchronous, spontaneous, and asynchronous neurotransmitter release. Importantly, our data demonstrate that Ca2+ binding by the C2A domain of synaptotagmin does not regulate asynchronous release and thus disprove the current inhibition hypothesis. We propose a spatial competition hypothesis to explain these seemingly discordant results of the differing C2A Ca2+ binding mutations.

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Drosophila Synaptotagmin 7 negatively regulates synaptic vesicle release and replenishment in a dosage-dependent manner

eLife ◽

10.7554/elife.55443 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 1

Author(s):

Zhuo Guan ◽

Monica C Quiñones-Frías ◽

Yulia Akbergenova ◽

J Troy Littleton

Keyword(s):

Synaptic Vesicle ◽

Neurotransmitter Release ◽

Dependent Manner ◽

Short Term ◽

Vesicle Release ◽

Sensor Model ◽

Synaptotagmin 1 ◽

Active Zones ◽

Asynchronous Release ◽

Synaptic Vesicle Release

Synchronous neurotransmitter release is triggered by Ca2+ binding to the synaptic vesicle protein Synaptotagmin 1, while asynchronous fusion and short-term facilitation is hypothesized to be mediated by plasma membrane-localized Synaptotagmin 7 (SYT7). We generated mutations in Drosophila Syt7 to determine if it plays a conserved role as the Ca2+ sensor for these processes. Electrophysiology and quantal imaging revealed evoked release was elevated 2-fold. Syt7 mutants also had a larger pool of readily-releasable vesicles, faster recovery following stimulation, and intact facilitation. Syt1/Syt7 double mutants displayed more release than Syt1 mutants alone, indicating SYT7 does not mediate the residual asynchronous release remaining in the absence of SYT1. SYT7 localizes to an internal membrane tubular network within the peri-active zone, but does not enrich at active zones. These findings indicate the two Ca2+ sensor model of SYT1 and SYT7 mediating all phases of neurotransmitter release and facilitation is not applicable at Drosophila synapses.

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