Calcium dependence of neurotransmitter release and rate of spontaneous vesicle fusions are altered in Drosophila synaptotagmin mutants (original) (raw)
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The Journal of Comparative Neurology, 2006
Synaptotagmin I is a synaptic vesicle protein postulated to mediate vesicle docking, vesicle recycling, and the Ca 2ϩ sensing required to trigger vesicle fusion. Analysis of synaptotagmin I knockouts (sytI NULL mutants) in both Drosophila and mice led to these hypotheses. Although much research on the mechanisms of synaptic transmission in Drosophila is performed at the third instar neuromuscular junction, the ultrastructure of this synapse has never been analyzed in sytI NULL mutants. Here we report severe synaptic vesicle depletion, an accumulation of large vesicles, and decreased vesicle docking at sytI NULL third instar neuromuscular junctions. Mutations in synaptotagmin I's C 2 B Ca 2ϩ -binding motif nearly abolish synaptic transmission and decrease the apparent Ca 2ϩ affinity of neurotransmitter release. Although this result is consistent with disruption of the Ca 2ϩ sensor, synaptic vesicle depletion and/or redistribution away from the site of Ca 2ϩ influx could produce a similar phenotype. To address this question, we examined vesicle distributions at neuromuscular junctions from third instar C 2 B Ca 2ϩ -binding motif mutants and transgenic wild-type controls. The number of docked vesicles and the overall number of synaptic vesicles in the vicinity of active zones was unchanged in the mutants. We conclude that the near elimination of synaptic transmission and the decrease in the Ca 2ϩ affinity of release observed in C 2 B Ca 2ϩ -binding motif mutants is not due to altered synaptic vesicle distribution but rather is a direct result of disrupting synaptotagmin I's ability to bind Ca 2ϩ . Thus, Ca 2ϩ binding by the C 2 B domain mediates a postdocking step in fusion.
Journal of Neuroscience, 2011
The vesicle protein synaptotagmin I is the Ca 2ϩ sensor that triggers fast, synchronous release of neurotransmitter. Specifically, Ca 2ϩ binding by the C 2 B domain of synaptotagmin is required at intact synapses, yet the mechanism whereby Ca 2ϩ binding results in vesicle fusion remains controversial. Ca 2ϩ -dependent interactions between synaptotagmin and SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment receptor) complexes and/or anionic membranes are possible effector interactions. However, no effectorinteraction mutations to date impact synaptic transmission as severely as mutation of the C 2 B Ca 2ϩ -binding motif, suggesting that these interactions are facilitatory rather than essential. Here we use Drosophila to show the functional role of a highly conserved, hydrophobic residue located at the tip of each of the two Ca 2ϩ -binding pockets of synaptotagmin. Mutation of this residue in the C 2 A domain (F286) resulted in a ϳ50% decrease in evoked transmitter release at an intact synapse, again indicative of a facilitatory role. Mutation of this hydrophobic residue in the C 2 B domain (I420), on the other hand, blocked all locomotion, was embryonic lethal even in syt I heterozygotes, and resulted in less evoked transmitter release than that in syt null mutants, which is more severe than the phenotype of C 2 B Ca 2ϩ -binding mutants. Thus, mutation of a single, C 2 B hydrophobic residue required for Ca 2ϩ -dependent penetration of anionic membranes results in the most severe disruption of synaptotagmin function in vivo to date. Our results provide direct support for the hypothesis that plasma membrane penetration, specifically by the C 2 B domain of synaptotagmin, is the critical effector interaction for coupling Ca 2ϩ binding with vesicle fusion.
The Journal of Neuroscience, 2001
Synaptotagmin has been proposed to function as a Ca 2ϩ sensor that regulates synaptic vesicle exocytosis, whereas the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is thought to form the core of a conserved membrane fusion machine. Little is known concerning the functional relationships between synaptotagmin and SNAREs. Here we report that synaptotagmin can facilitate SNARE complex formation in vitro and that synaptotagmin mutations disrupt SNARE complex formation in vivo. Synaptotagmin oligomers efficiently bind SNARE complexes, whereas Ca 2ϩ acting via synaptotagmin triggers cross-linking of SNARE complexes into dimers. Mutations in Drosophila that delete the C2B domain of synaptotagmin disrupt clathrin AP-2 binding and endocytosis. In contrast, a mutation that blocks Ca 2ϩtriggered conformational changes in C2B and diminishes Ca 2ϩ-triggered synaptotagmin oligomerization results in a postdocking defect in neurotransmitter release and a decrease in SNARE assembly in vivo. These data suggest that Ca 2ϩdriven oligomerization via the C2B domain of synaptotagmin may trigger synaptic vesicle fusion via the assembly and clustering of SNARE complexes.
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.
Role of synaptotagmin in Ca2+-triggered exocytosis
Biochemical Journal, 2002
The Ca2+-binding synaptic-vesicle protein synaptotagmin I has attracted considerable interest as a potential Ca2+ sensor that regulates exocytosis from neurons and neuroendocrine cells. Recent studies have shed new light on the structure, biochemical/biophysical properties and function of synaptotagmin, and the emerging view is that it plays an important role in both exocytosis and endocytosis. At least a dozen additional isoforms exist, some of which are expressed outside of the nervous system, suggesting that synaptotagmins might regulate membrane traffic in a variety of cell types. Here we provide an overview of the members of this gene family, with particular emphasis on the question of whether and how synaptotagmin I functions during the final stages of membrane fusion: does it regulate the Ca2+-triggered opening and dilation of fusion pores?
A molecular mechanism for calcium-mediated synaptotagmin-triggered exocytosis
Nature Structural & Molecular Biology, 2018
The regulated exocytotic release of neurotransmitter and hormones is accomplished by a complex protein machinery consisting in its core of SNARE proteins and the calcium sensor synaptotagmin-1. We propose a mechanism where the lipid membrane is intimately involved in coupling calcium sensing to release. We demonstrate that fusion of dense core vesicles, derived from rat PC12 cells is strongly linked to the angle between the cytoplasmic domain of the SNARE complex and the plane of the target membrane. We propose that, as this tilt angle increases, force is exerted on the SNARE transmembrane domains to drive the merger of the two bilayers. The tilt angle dramatically increases upon calcium-mediated binding of synaptotagmin to membranes, strongly depends on the surface electrostatics of the membrane, and is strictly coupled to lipid order of the target membrane.
Journal of Neuroscience, 2008
Synaptotagmin I is the Ca 2ϩ sensor for fast, synchronous release of neurotransmitter; however, the molecular interactions that couple Ca 2ϩ binding to membrane fusion remain unclear. The structure of synaptotagmin is dominated by two C 2 domains that interact with negatively charged membranes after binding Ca 2ϩ . In vitro work has implicated a conserved basic residue at the tip of loop 3 of the Ca 2ϩ -binding pocket in both C 2 domains in coordinating this electrostatic interaction with anionic membranes. Although results from cultured cells suggest that the basic residue of the C 2 A domain is functionally significant, such studies provide contradictory results regarding the importance of the C 2 B basic residue during vesicle fusion. To directly test the functional significance of each of these residues at an intact synapse in vivo, we neutralized either the C 2 A or the C 2 B basic residue and assessed synaptic transmission at the Drosophila neuromuscular junction. The conserved basic residues at the tip of the Ca 2ϩ -binding pocket of both the C 2 A and C 2 B domains mediate Ca 2ϩ -dependent interactions with anionic membranes and are required for efficient evoked transmitter release. Our results directly support the hypothesis that the interactions between synaptotagmin and the presynaptic membrane, which are mediated by the basic residues at the tip of both the C 2 A and C 2 B Ca 2ϩ -binding pockets, are critical for coupling Ca 2ϩ influx with vesicle fusion during synaptic transmission in vivo. Our model for synaptotagmin's direct role in coupling Ca 2ϩ binding to vesicle fusion incorporates this finding with results from multiple in vitro and in vivo studies.
Synaptotagmins I and IV promote transmitter release independently of Ca2+ binding in the C2A domain
Nature, 2002
At nerve terminals, a focal and transient increase in intracellular Ca 21 triggers the fusion of neurotransmitter-filled vesicles with the plasma membrane. The most extensively studied candidate for the Ca 21 -sensing trigger is synaptotagmin I, whose Ca 21dependent interactions with acidic phospholipids and syntaxin 1 have largely been ascribed to its C 2 A domain 2-6 , although the C 2 B domain also binds Ca 21 (refs 7, 8). Genetic tests of synaptotagmin I have been equivocal as to whether it is the Ca 21 -sensing trigger of fusion . Synaptotagmin IV, a related isoform that does not bind Ca 21 in the C 2 A domain, might be an inhibitor of release . We mutated an essential aspartate of the Ca 21 -binding site of the synaptotagmin I C 2 A domain and expressed it in Drosophila lacking synaptotagmin I. Here we show that, despite the disruption of the binding site, the Ca 21 -dependent properties of transmission were not altered. Similarly, we found that synaptotagmin IV could substitute for synaptotagmin I. We conclude that the C 2 A domain of synaptotagmin is not required for Ca 21 -dependent synaptic transmission, and that synaptotagmin IV promotes rather than inhibits transmission.
A Novel Function for the Second C2 Domain of Synaptotagmin
Journal of Biological …, 1996
Synaptotagmin serves as the major Ca 2؉ sensor for regulated exocytosis from neurons. While the mechanism by which synaptotagmin regulates membrane fusion remains unknown, studies using Drosophila indicate that the molecule functions as a multimeric complex and that its second C2 domain is essential for efficient excitation-secretion coupling. Here we describe biochemical data that may account for these phenomena. We report that Ca 2؉ causes synaptotagmin to oligomerize, primarily forming dimers, via its second C2 domain. This effect is specific for divalent cations that can stimulate exocytosis of synaptic vesicles (Ca 2؉ > > Ba 2؉ , Sr 2؉ > >> Mg 2؉) and occurs with an EC 50 value of 3-10 M Ca 2؉. In contrast, a separate Ca 2؉-dependent interaction between synaptotagmin and syntaxin, a component of the fusion apparatus, occurs with an EC 50 value of ϳ100 M Ca 2؉ and involves the synergistic action of both C2 domains of synaptotagmin. We propose that Ca 2؉ triggers two consecutive protein-protein interactions: the formation of synaptotagmin dimers at low Ca 2؉ concentrations followed by the association of synaptotagmin dimers with syntaxin at higher Ca 2؉concentrations. Our findings, in conjunction with physiological studies, indicate that the Ca 2؉-induced dimerization of synaptotagmin is important for the efficient regulation of exocytosis by Ca 2؉ .
Journal of Neuroscience, 2012
A is not required for triggering synchronous fusion. Based on a structural analysis, we generated a novel mutation of a single Ca 2ϩ -binding residue in C 2 A (D229E in Drosophila) that inhibited Ca 2ϩ binding but maintained the negative charge of the pocket. This C 2 A aspartate-to-glutamate mutation resulted in ϳ80% decrease in synchronous transmitter release and a decrease in the apparent Ca 2ϩ affinity of release. Previous aspartate-to-asparagine mutations in C 2 A partially mimicked Ca 2ϩ binding by decreasing the negative charge of the pocket. We now show that the major function of Ca 2ϩ binding to C 2 A is to neutralize the negative charge of the pocket, thereby unleashing the fusion-stimulating activity of synaptotagmin. Our results demonstrate that Ca 2ϩ binding by C 2 A is a critical component of the electrostatic switch that triggers synchronous fusion. Thus, Ca 2ϩ binding by C 2 B is necessary and sufficient to regulate the precise timing required for coupling vesicle fusion to Ca 2ϩ influx, but Ca 2ϩ binding by both C 2 domains is required to flip the electrostatic switch that triggers efficient synchronous synaptic transmission.