Ca2+–Calmodulin regulates SNARE assembly and spontaneous neurotransmitter release via v-ATPase subunit V0a1. (original) (raw)
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Ca2+-Triggered Synaptic Vesicle Fusion Initiated by Release of Inhibition
Trends in Cell Biology, 2018
Recent structural and functional studies of the synaptic vesicle fusion machinery suggest an inhibited tripartite complex consisting of neuronal soluble Nethylmaleimide sensitive factor attachment protein receptors (SNAREs), synaptotagmin, and complexin prior to Ca 2+-triggered synaptic vesicle fusion. We speculate that Ca 2+-triggered fusion commences with the release of inhibition by Ca 2+ 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 Ca 2+ 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 Ca 2+ binding to one of its C2 domains. Synaptic Transmission and Calcium Triggering Synaptic transmission between presynaptic and postsynaptic neurons occurs when the presynaptic neuron terminal is temporarily depolarized upon an action potential, opening Ca 2+ channels near the active zones of synapses. Since the extracellular Ca 2+ concentration is much higher than the cytoplasmic concentration, Ca 2+ will flow into the cytoplasm. In turn, Ca 2+ will trigger fusion of neurotransmitter-filled synaptic vesicles with the presynaptic membrane in less than a millisecond [1,2]. Upon fusion, neurotransmitter molecules are released into the synaptic cleft, and then bind to receptors that are located in the postsynaptic membrane. Many, if not most, of the key factors of the core synaptic fusion machinery have been identified, including fusogenic SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptor), the Ca 2+-sensor synaptotagmin, the activator/regulator complexin, the assembly factors Munc18 (mammalian uncoordinated-18) and Munc13 (mammalian uncoordinated-13), and the disassembly factors NSF (N-ethylmaleimide-sensitive factor) and SNAP (soluble NSF adaptor protein). Yet, the molecular mechanisms of Ca 2+ triggering, regulation, and membrane fusion are still unclear. Central to these questions is the role of synaptotagmin, which in the past has been primarily viewed as an activating factor upon Ca 2+ binding, for example, by bending membranes [3-6] or bridging membranes [7-9]. However, such an activating role does not explain the effect of certain dominant negative mutants of synaptotagmin-1 that abolish evoked release in the background of endogenous wild-type synaptotagmin-1 [10-12]. We note that genetic deletion of synaptotagmin increased the frequency of spontaneous release in flies [13,14], and a similar phenotype was observed upon deletion of synaptotagmin-1 in mouse neurons [15]. However, expression of a dominant negative synaptotagmin-1 mutant also increased spontaneous release in mouse neurons in a Ca 2+-dependent fashion [16], suggesting that a Ca 2+ sensor other than synaptotagmin-1 is important for spontaneous release. Since the molecular mechanisms of spontaneous release are less certain at this time, we primarily Highlights The recent structure of the prefusion complex of neuronal SNAREs, complexin-1, and synaptotagmin-1, along with functional studies, suggests that Ca 2+-triggered fusion is initiated by release of inhibition.
Synaptic Vesicles Are Constitutively Active Fusion Machines that Function Independently of Ca2+
Current Biology, 2008
Background-In neurones, release of neurotransmitter occurs through the fusion of synaptic vesicles with the plasma membrane. Many proteins required for this process have been identified, with the SNAREs syntaxin 1, SNAP-25 and synaptobrevin thought to constitute the core fusion machinery. However, there is still a large gap between our understanding of individual proteinprotein interactions and the functions of these proteins revealed by perturbations in intact synaptic preparations. To bridge this gap, we have used purified synaptic vesicles, together with artificial membranes containing co-reconstituted SNAREs as reaction partners, in fusion assays.
Journal of Cell Biology, 1998
Cortical vesicles (CV) possess components critical to the mechanism of exocytosis. The homotypic fusion of CV centrifuged or settled into contact has a sigmoidal Ca 2 ϩ activity curve comparable to exocytosis (CV-PM fusion). Here we show that Sr 2 ϩ and Ba 2 ϩ also trigger CV-CV fusion, and agents affecting different steps of exocytotic fusion block Ca 2 ϩ , Sr 2 ϩ , and Ba 2 ϩtriggered CV-CV fusion. The maximal number of active fusion complexes per vesicle, Max , was quantified by NEM inhibition of fusion, showing that CV-CV fusion satisfies many criteria of a mathematical analysis developed for exocytosis. Both Max and the Ca 2 ϩ sensitivity of fusion complex activation were comparable to that determined for CV-PM fusion. Using Ca 2 ϩinduced SNARE complex disruption, we have analyzed the relationship between membrane fusion (CV-CV and CV-PM) and the SNARE complex. Fusion and complex disruption have different sensitivities to Ca 2 ϩ , Sr 2 ϩ , and Ba 2 ϩ , the complex remains Ca 2 ϩ -sensitive on fusion-incompetent CV, and disruption does not correlate with the quantified activation of fusion complexes. Under conditions which disrupt the SNARE complex, CV on the PM remain docked and fusion competent, and isolated CV still dock and fuse, but with a markedly reduced Ca 2 ϩ sensitivity. Thus, in this system, neither the formation, presence, nor disruption of the SNARE complex is essential to the Ca 2 ϩ -triggered fusion of exocytotic membranes. Therefore the SNARE complex alone cannot be the universal minimal fusion machine for intracellular fusion. We suggest that this complex modulates the Ca 2 ϩ sensitivity of fusion.
Calcium-triggered Membrane Fusion Proceeds Independently of Specific Presynaptic Proteins
Journal of Biological Chemistry, 2003
Complexes of specific presynaptic proteins have been hypothesized to drive or catalyze the membrane fusion steps of exocytosis. Here we use a stage-specific preparation to test the roles of SNAREs, synaptotagmin, and SNARE-binding proteins in the mechanism of Ca 2؉ -triggered membrane fusion. Excess exogenous proteins, sufficient to block SNARE interactions, did not inhibit either the Ca 2؉ sensitivity, extent, or kinetics of fusion. In contrast, despite a limited effect on SNARE and synaptotagmin densities, treatments with high doses of chymotrypsin markedly inhibited fusion. Conversely, low doses of chymotrypsin had no effect on the Ca 2؉ sensitivity or extent of fusion but did alter the kinetic profile, indicating a more direct involvement of other proteins in the triggered fusion pathway. SNAREs, synaptotagmin, and their immediate binding partners are critical to exocytosis at a stage other than membrane fusion, although they may still influence the triggered steps.
Proceedings of the …, 2011
Understanding the molecular principles of synaptic vesicle fusion is a long-sought goal. It requires the development of a synthetic system that allows manipulations and observations not possible in vivo. Here, we report an in vitro system with reconstituted synaptic proteins that meets the long-sought goal to produce fast content release in the millisecond time regime upon Ca 2þ triggering. Our system simultaneously monitors both content and lipid exchange, and it starts from stable interacting pairs of donor and acceptor vesicles, mimicking the readily releasable pool of synaptic vesicles prior to an action potential. It differentiates between single-vesicle interaction, hemifusion, and complete fusion, the latter mimicking quantized neurotransmitter release upon exocytosis of synaptic vesicles. Prior to Ca 2þ injection, the system is in a state in which spontaneous fusion events between donor and acceptor vesicles are rare. Upon Ca 2þ injection, a rapid burst of complete fusion events emerges, followed by a biphasic decay. The present study focuses on neuronal SNAREs, the Ca 2þ sensor synaptotagmin 1, and the modulator complexin. However, other synaptic proteins could be added and their function examined. Ca 2þ triggering is cooperative, requiring the presence of synaptotagmin, whereas SNAREs alone do not produce a fast fusion burst. Manipulations of the system mimic effects observed in vivo. These results also show that neuronal SNAREs alone do not efficiently produce complete fusion, that the combination of SNAREs with synaptotagmin lowers the activation barriers to full fusion, and that complexin enhances this kinetic control.
Reconstitution of Ca 2+ -Regulated Membrane Fusion by Synaptotagmin and SNAREs
Science, 2004
We investigated the effect of synaptotagmin I on membrane fusion mediated by neuronal SNARE proteins, SNAP-25, syntaxin, and synaptobrevin, which were reconstituted into vesicles. In the presence of Ca 2+ , the cytoplasmic domain of synaptotagmin I (syt) strongly stimulated membrane fusion when synaptobrevin densities were similar to those found in native synaptic vesicles. The Ca 2+ dependence of syt-stimulated fusion was modulated by changes in lipid composition of the vesicles and by a truncation that mimics cleavage of SNAP-25 by botulinum neurotoxin A. Stimulation of fusion was abolished by disrupting the Ca 2+ -binding activity, or by severing the tandem C2 domains, of syt. Thus, syt and SNAREs are likely to represent the minimal protein complement for Ca 2+ -triggered exocytosis.
CaV1 and CaV2 calcium channels mediate the release of distinct pools of synaptic vesicles
Activation of voltage-gated calcium channels at synapses leads to local increases in calcium and the fusion of synaptic vesicles. However, presynaptic output will be determined by the density of calcium channels, the dynamic properties of the channel, the distance to docked vesicles, and the release probability at the docking site. We demonstrate that at C. elegans neuromuscular junctions two different classes of voltage-gated calcium channels, CaV2 and CaV1, mediate the release of distinct pools of synaptic vesicles. CaV2 channels are concentrated in densely packed clusters ∼300 nm in diameter with the active zone proteins Neurexin, α-Liprin, SYDE, ELKS/CAST, RIM-BP, α-Catulin, and MAGI1. CaV2 channels mediate the fusion of vesicles docked adjacent to the dense projection and are colocalized with the synaptic vesicle priming protein UNC-13L. By contrast, CaV1 channels are dispersed in the synaptic varicosity and are coupled to internal calcium stores via the ryanodine receptor. CaV...