Additive effects on the energy barrier for synaptic vesicle fusion cause supralinear effects on the vesicle fusion rate.
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ABSTRACT:
SUBMITTER: Schotten S
PROVIDER: S-EPMC4426983 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Previously, we showed that modulation of the energy barrier for synaptic vesicle fusion boosts release rates supralinearly (Schotten, 2015). Here we show that mouse hippocampal synapses employ this principle to trigger Ca2+-dependent vesicle release and post-tetanic potentiation (PTP). We assess energy barrier changes by fitting release kinetics in response to hypertonic sucrose. Mimicking activation of the C2A domain of the Ca2+-sensor Synaptotagmin-1 (Syt1), by adding a positive charge (Syt1D232N) or increasing its hydrophobicity (Syt14W), lowers the energy barrier. Removing Syt1 or impairing its release inhibitory function (Syt19Pro) increases spontaneous release without affecting the fusion barrier. Both phorbol esters and tetanic stimulation potentiate synaptic strength, and lower the energy barrier equally well in the presence and absence of Syt1. We propose a model where tetanic stimulation activates Syt1-independent mechanisms that lower the energy barrier and act additively with Syt1-dependent mechanisms to produce PTP by exerting multiplicative effects on release rates.
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Project description:Synaptic transmission can occur in a binary or graded fashion, depending on whether transmitter release is triggered by action potentials or by gradual changes in membrane potential. Molecular differences of these two types of fusion events and their differential regulation in a physiological context have yet to be addressed. Complexin is a conserved SNARE-binding protein that has been proposed to regulate both spontaneous and stimulus-evoked synaptic vesicle (SV) fusion.Here we examine complexin function at a graded synapse in C. elegans. Null complexin (cpx-1) mutants are viable, although nervous system function is significantly impaired. Loss of CPX-1 results in a 3-fold increase in the rate of tonic synaptic transmission at the neuromuscular junction, whereas stimulus-evoked SV fusion is decreased 10-fold. A truncated CPX-1 missing its C-terminal domain can rescue stimulus-evoked synaptic vesicle exocytosis but fails to suppress tonic activity, demonstrating that these two modes of exocytosis can be distinguished at the molecular level. A CPX-1 variant with impaired SNARE binding also rescues evoked, but not tonic, neurotransmitter release. Finally, tonic, but not evoked, release can be rescued in a syntaxin point mutant by removing CPX-1. Rescue of either form of exocytosis partially restores locomotory behavior, indicating that both types of synaptic transmission are relevant.These observations suggest a dual role for CPX-1: suppressing SV exocytosis, driven by low levels of endogenous neural activity, while promoting synchronous fusion of SVs driven by a depolarizing stimulus. Thus, patterns of synaptic activity regulate complexin's inhibitory and permissive roles at a graded synapse.
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Project description:Synaptic vesicle (SV) fusion is a fine-tuned process requiring a concert of fusion machineries. Using cortical neurons from snapin-deficient mice, we reveal a role for Snapin in facilitating synchronous release. In addition to reduced frequency of miniature excitatory postsynaptic currents (mini-EPSCs) and smaller release-ready vesicle pool (RRP) size, snapin deficiency results in EPSCs with multiple peaks and increased rise and decay times, reflecting "desynchronized" SV fusion. These defects impair both synaptic precision and efficacy during sustained neurotransmission. Transient expression of Snapin not only rescues the slowed kinetics of EPSCs, but also further accelerates the rate found in wild-type neurons. Furthermore, expression of Snapin-C66A, a dimerization-defective mutant with impaired interactions with SNAP-25 and Synaptotagmin, reduces the RRP size but exhibits less effect on synchronized fusion. Our studies provide mechanistic insights into a dual role of Snapin in enhancing the efficacy of SV priming and in fine-tuning synchronous SV fusion.
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Project description:During synaptic vesicle fusion, the soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) protein syntaxin-1 exhibits two conformations that both bind to Munc18-1: a "closed" conformation outside the SNARE complex and an "open" conformation in the SNARE complex. Although SNARE complexes containing open syntaxin-1 and Munc18-1 are essential for exocytosis, the function of closed syntaxin-1 is unknown. We generated knockin/knockout mice that expressed only open syntaxin-1B. Syntaxin-1B(Open) mice were viable but succumbed to generalized seizures at 2 to 3 months of age. Binding of Munc18-1 to syntaxin-1 was impaired in syntaxin-1B(Open) synapses, and the size of the readily releasable vesicle pool was decreased; however, the rate of synaptic vesicle fusion was dramatically enhanced. Thus, the closed conformation of syntaxin-1 gates the initiation of the synaptic vesicle fusion reaction, which is then mediated by SNARE-complex/Munc18-1 assemblies.
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Project description:Both constitutive secretion and Ca(2+)-regulated exocytosis require the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes. At present, little is known about how the SNARE complexes mediating these two distinct pathways differ in structure. Using the Drosophila neuromuscular synapse as a model, we show that a mutation modifying a hydrophobic layer in syntaxin 1A regulates the rate of vesicle fusion. Syntaxin 1A molecules share a highly conserved threonine in the C-terminal +7 layer near the transmembrane domain. Mutation of this threonine to isoleucine results in a structural change that more closely resembles those found in syntaxins ascribed to the constitutive secretory pathway. Flies carrying the I254 mutant protein have increased levels of SNARE complexes and dramatically enhanced rate of both constitutive and evoked vesicle fusion. In contrast, overexpression of the T254 wild-type protein in neurons reduces vesicle fusion only in the I254 mutant background. These results are consistent with molecular dynamics simulations of the SNARE core complex, suggesting that T254 serves as an internal brake to dampen SNARE zippering and impede vesicle fusion, whereas I254 favors fusion by enhancing intermolecular interaction within the SNARE core complex.
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Project description:Recent structural and functional studies of the synaptic vesicle fusion machinery suggest an inhibited tripartite complex consisting of neuronal soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs), synaptotagmin, and complexin prior to Ca2+-triggered synaptic vesicle fusion. We speculate that Ca2+-triggered fusion commences with the release of inhibition by Ca2+ binding to synaptotagmin C2 domains. Subsequently, fusion is assisted by SNARE complex zippering and by active membrane remodeling properties of synaptotagmin. This additional, inhibitory role of synaptotagmin may be a general principle since other recent studies suggest that Ca2+ binding to extended synaptotagmin C2 domains enables lipid transport by releasing an inhibited state of the system, and that Munc13 may nominally be in an inhibited state, which is released upon Ca2+ binding to one of its C2 domains.
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Project description:Exocytosis at synapses involves fusion between vesicles and the plasma membrane. Although compound fusion between vesicles was proposed to occur at ribbon-type synapses, whether it exists, how it is mediated, and what role it plays at conventional synapses remain unclear. Here we report the existence of compound fusion, its underlying mechanism, and its role at a nerve terminal containing conventional active zones in rats and mice. We found that high potassium application and high frequency firing induced giant capacitance up-steps, reflecting exocytosis of vesicles larger than regular ones, followed by giant down-steps, reflecting bulk endocytosis. These intense stimuli also induced giant vesicle-like structures, as observed with electron microscopy, and giant miniature excitatory postsynaptic currents (mEPSCs), reflecting more transmitter release. Calcium and its sensor for vesicle fusion, synaptotagmin, were required for these giant events. After high frequency firing, calcium/synaptotagmin-dependent mEPSC size increase was paralleled by calcium/synaptotagmin-dependent post-tetanic potentiation. These results suggest a new route of exocytosis and endocytosis composed of three steps. First, calcium/synaptotagmin mediates compound fusion between vesicles. Second, exocytosis of compound vesicles increases quantal size, which increases synaptic strength and contributes to the generation of post-tetanic potentiation. Third, exocytosed compound vesicles are retrieved via bulk endocytosis. We suggest that this vesicle cycling route be included in models of synapses in which only vesicle fusion with the plasma membrane is considered.
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Project description:Measuring the biophysical properties of macromolecular complexes at work is a major challenge of modern biology. The protein complex composed of vesicle-associated membrane protein 2, synaptosomal-associated protein of 25 kDa, and syntaxin 1 [soluble N-ethyl-maleimide-sensitive factor attachment protein receptor (SNARE) complex] is essential for docking and fusion of neurotransmitter-filled synaptic vesicles with the presynaptic membrane. To better understand the fusion mechanisms, we reconstituted the synaptic SNARE complex in the imaging chamber of an atomic force microscope and measured the interaction forces between its components. Each protein was tested against the two others, taken either individually or as binary complexes. This approach allowed us to determine specific interaction forces and dissociation kinetics of the SNAREs and led us to propose a sequence of interactions. A theoretical model based on our measurements suggests that a minimum of four complexes is probably necessary for fusion to occur. We also showed that the regulatory protein neuronal Sec1 injected into the atomic force microscope chamber prevented the complex formation. Finally, we measured the effect of tetanus toxin protease on the SNARE complex and its activity by on-line registration during tetanus toxin injection. These experiments provide a basis for the functional study of protein microdomains and also suggest opportunities for sensitive screening of drugs that can modulate protein-protein interactions.
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Project description:Rod photoreceptors (PRs) use ribbon synapses to transmit visual information. To signal 'no light detected' they release glutamate continually to activate post-synaptic receptors. When light is detected glutamate release pauses. How a rod's individual ribbon enables this process was studied here by recording evoked changes in whole-cell membrane capacitance from wild-type and ribbonless (Ribeye-ko) mice. Wild-type rods filled with high (10 mM) or low (0.5 mM) concentrations of the Ca2+-buffer EGTA created a readily releasable pool (RRP) of 87 synaptic vesicles (SVs) that emptied as a single kinetic phase with a τ<0.4 ms. The lower concentration of EGTA accelerated Cav channel opening and facilitated release kinetics. In contrast, ribbonless rods created a much smaller RRP of 22 SVs, and they lacked Cav channel facilitation; however, Ca2+ channel-release coupling remained tight. These release deficits caused a sharp attenuation of rod-driven scotopic light responses. We conclude that the synaptic ribbon facilitates Ca2+-influx and establishes a large RRP of SVs.
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Project description:The interaction of the presynaptic membrane proteins SNAP-25 and syntaxin with the synaptic vesicle protein synaptobrevin (VAMP) plays a key role in the regulated exocytosis of neurotransmitters. Clostridial neurotoxins, which proteolyze these polypeptides, are potent inhibitors of neurotransmission. The cytoplasmic domains of the three membrane proteins join into a tight SDS-resistant complex (Hayashi et al., 1994). Here, we show that this reconstituted complex, as well as heterodimers composed of syntaxin and SNAP-25, can be disassembled by the concerted action of the N-ethylmaleimide-sensitive factor, NSF, and the soluble NSF attachment protein, alpha-SNAP. alpha-SNAP binds to predicted alpha-helical coiled-coil regions of syntaxin and SNAP-25, shown previously to be engaged in their direct interaction. Synaptobrevin, although incapable of binding alpha-SNAP individually, induced a third alpha-SNAP binding site when associated with syntaxin and SNAP-25 into heterotrimers. NSF released prebound alpha-SNAP from full-length syntaxin but not from a syntaxin derivative truncated at the N-terminus. Disassembly of complexes containing this syntaxin mutant was impaired, indicating a critical role for the N-terminal domain in the alpha-SNAP/NSF-mediated dissociation process. Complexes containing C-terminally deleted SNAP-25 derivatives, as generated by botulinal toxins type A and E, were dissociated more efficiently. In contrast, the N-terminal fragment generated from synaptobrevin by botulinal toxin type F produced an SDS-sensitive complex that was poorly dissociated.
| S-EPMC398339 | biostudies-other