Synaptotagmin I functions as a calcium sensor to synchronize neurotransmitter release

To characterize Ca(2+)-mediated synaptic vesicle fusion, we analyzed Drosophila synaptotagmin I mutants deficient in specific interactions mediated by its two Ca(2+) binding C2 domains. In the absence of synaptotagmin I, synchronous release is abolished and a kinetically distinct delayed asynchronous release pathway is uncovered. Synapses containing only the C2A domain of synaptotagmin partially recover synchronous fusion, but have an abolished Ca(2+) cooperativity. Mutants that disrupt Ca(2+) sensing by the C2B domain have synchronous release with normal Ca(2+) cooperativity, but with reduced release probability. Our data suggest the Ca(2+) cooperativity of neurotransmitter release is likely mediated through synaptotagmin-SNARE interactions, while phospholipid binding and oligomerization trigger rapid fusion with increased release probability. These results indicate that synaptotagmin is the major Ca(2+) sensor for evoked release and functions to trigger synchronous fusion in response to Ca(2+), while suppressing asynchronous release.     

Doc2 is a Ca2+ sensor required for asynchronous neurotransmitter release

Synaptic transmission involves a fast synchronous phase and a slower asynchronous phase of neurotransmitter release that are regulated by distinct Ca(2+) sensors. Though the Ca(2+) sensor for rapid exocytosis, synaptotagmin I, has been studied in depth, the sensor for asynchronous release remains unknown. In a screen for neuronal Ca(2+) sensors that respond to changes in [Ca(2+)] with markedly slower kinetics than synaptotagmin I, we observed that Doc2--another Ca(2+), SNARE, and lipid-binding protein--operates on timescales consistent with asynchronous release. Moreover, up- and downregulation of Doc2 expression levels in hippocampal neurons increased or decreased, respectively, the slow phase of synaptic transmission. Synchronous release, when triggered by single action potentials, was unaffected by manipulation of Doc2 but was enhanced during repetitive stimulation in Doc2 knockdown neurons, potentially due to greater vesicle availability. In summary, we propose that Doc2 is a Ca(2+) sensor that is kinetically tuned to regulate asynchronous neurotransmitter release.     

Synaptotagmin-1, -2, and -9: Ca(2+) sensors for fast release that specify distinct presynaptic properties in subsets of neurons

Synaptotagmin-1 and -2 are known Ca(2+) sensors for fast synchronous neurotransmitter release, but the potential Ca(2+)-sensor functions of other synaptotagmins in release remain uncharacterized. We now show that besides synaptotagmin-1 and -2, only synaptotagmin-9 (also called synaptotagmin-5) mediates fast Ca(2+) triggering of release. Release induced by the three different synaptotagmin Ca(2+) sensors exhibits distinct kinetics and apparent Ca(2+) sensitivities, suggesting that the synaptotagmin isoform expressed by a neuron determines the release properties of its synapses. Conditional knockout mice producing GFP-tagged synaptotagmin-9 revealed that synaptotagmin-9 is primarily expressed in the limbic system and striatum. Acute deletion of synaptotagmin-9 in striatal neurons severely impaired fast synchronous release without changing the size of the readily-releasable vesicle pool. These data show that in mammalian brain, only synaptotagmin-1, -2, and -9 function as Ca(2+) sensors for fast release, and that these synaptotagmins are differentially expressed to confer distinct release properties onto synapses formed by defined subsets of neurons.     

Complexin clamps asynchronous release by blocking a secondary Ca(2+) sensor via its accessory α helix

Complexin activates and clamps neurotransmitter release; impairing complexin function decreases synchronous, but increases spontaneous and asynchronous synaptic vesicle exocytosis. Here, we show that complexin-different from the Ca(2+) sensor synaptotagmin-1-activates synchronous exocytosis by promoting synaptic vesicle priming, but clamps spontaneous and asynchronous exocytosis-similar to synaptotagmin-1-by blocking a secondary Ca(2+) sensor. Activation and clamping functions of complexin depend on distinct, autonomously acting sequences, namely its N-terminal region and accessory α helix, respectively. Mutations designed to test whether the accessory α helix of complexin clamps exocytosis by inserting into SNARE-complexes support this hypothesis, suggesting that the accessory α helix blocks completion of trans-SNARE-complex assembly until Ca(2+) binding to synaptotagmin relieves this block. Moreover, a juxtamembranous mutation in the SNARE-protein synaptobrevin-2, which presumably impairs force transfer from nascent trans-SNARE complexes onto fusing membranes, also unclamps spontaneous fusion by disinhibiting a secondary Ca(2+) sensor. Thus, complexin performs mechanistically distinct activation and clamping functions that operate in conjunction with synaptotagmin-1 by controlling trans-SNARE-complex assembly.     

Calcium regulation of spontaneous and asynchronous neurotransmitter release

The molecular machinery underlying action potential-evoked, synchronous neurotransmitter release, has been intensely studied. It was presumed that two other forms of exocytosis, delayed (asynchronous) and spontaneous transmission, were mediated by the same voltage-activated Ca(2+) channels (VACCs), intracellular Ca(2+) sensors and vesicle pools. However, a recent explosion in the study of spontaneous and asynchronous release has shown these presumptions to be incorrect. Furthermore, the finding that different forms of synaptic transmission may mediate distinct physiological functions emphasizes the importance of identifying the mechanisms by which Ca(2+) regulates spontaneous and asynchronous release. In this article, we will briefly summarize new and published data on the role of Ca(2+) in regulating spontaneous and asynchronous release at a number of different synapses. We will discuss how an increase of extracellular [Ca(2+)] increases spontaneous and asynchronous release, show that VACCs are involved at only some synapses, and identify regulatory roles for other ion channels and G protein-coupled receptors. In particular, we will focus on two novel pathways that play important roles in the regulation of non-synchronous release at two exemplary synapses: one modulated by the Ca(2+)-sensing receptor and the other by transient receptor potential cation channel sub-family V member 1.     

Genetic analysis of synaptotagmin 2 in spontaneous and Ca2+-triggered neurotransmitter release

Synaptotagmin 2 resembles synaptotagmin 1, the Ca2+ sensor for fast neurotransmitter release in forebrain synapses, but little is known about synaptotagmin 2 function. Here, we describe a severely ataxic mouse strain that harbors a single, destabilizing amino-acid substitution (I377N) in synaptotagmin 2. In Calyx of Held synapses, this mutation causes a delay and a decrease in Ca2+-induced but not in hypertonic sucrose-induced release, suggesting that synaptotagmin 2 mediates Ca2+ triggering of evoked release in brainstem synapses. Unexpectedly, we additionally observed in synaptotagmin 2 mutant synapses a dramatic increase in spontaneous release. Synaptotagmin 1-deficient excitatory and inhibitory cortical synapses also displayed a large increase in spontaneous release, demonstrating that this effect was shared among synaptotagmins 1 and 2. Our data suggest that synaptotagmin 1 and 2 perform equivalent functions in the Ca2+ triggering of action potential-induced release and in the restriction of spontaneous release, consistent with a general role of synaptotagmins in controlling 'release slots' for synaptic vesicles at the active zone.     

Calcium control of neurotransmitter release

Upon entering a presynaptic terminal, an action potential opens Ca(2+) channels, and transiently increases the local Ca(2+) concentration at the presynaptic active zone. Ca(2+) then triggers neurotransmitter release within a few hundred microseconds by activating synaptotagmins Ca(2+). Synaptotagmins bind Ca(2+) via two C2-domains, and transduce the Ca(2+) signal into a nanomechanical activation of the membrane fusion machinery; this activation is mediated by the Ca(2+)-dependent interaction of the synaptotagmin C2-domains with phospholipids and SNARE proteins. In triggering exocytosis, synaptotagmins do not act alone, but require an obligatory cofactor called complexin, a small protein that binds to SNARE complexes and simultaneously activates and clamps the SNARE complexes, thereby positioning the SNARE complexes for subsequent synaptotagmin action. The conserved function of synaptotagmins and complexins operates generally in most, if not all, Ca(2+)-regulated forms of exocytosis throughout the body in addition to synaptic vesicle exocytosis, including in the degranulation of mast cells, acrosome exocytosis in sperm cells, hormone secretion from endocrine cells, and neuropeptide release.     

Sodium-dependent action potentials induced by brevetoxin-3 trigger both IP3 increase and intracellular Ca2+ release in rat skeletal myotubes

Brevetoxin-3 (PbTx-3), described to increase the open probability of voltage-dependent sodium channels, caused trains of action potentials and fast oscillatory changes in fluorescence intensity of fluo-3-loaded rat skeletal muscle cells in primary culture, indicating that the toxin increased intracellular Ca(2+) levels. PbTx-3 did not elicit calcium transients in dysgenic myotubes (GLT cell line), lacking the alpha1 subunit of the dihydropyridine receptor (DHPR), but after transfection of the alpha1DHPR cDNA to GLT cells, PbTx-3 induced slow calcium transients that were similar to those of normal cells. Ca(2+) signals evoked by PbTx-3 were inhibited by blocking either IP(3) receptors, with 2-aminoethoxydiphenyl borate, or phospholipase C with U73122. PbTx-3 caused a tetrodotoxin-sensitive increase in intracellular IP(3) mass levels, dependent on extra-cellular Na(+). A similar increase in IP(3) mass was induced by high K(+) depolarization but no action potential trains (nor calcium signals) were elicited by prolonged depolarization under current clamp conditions. The increase in IP(3) mass induced by either PbTx-3 or K(+) was also detected in Ca(2+)-free medium. These results establish that the effect of the toxin on both intracellular Ca(2+) and IP(3) levels occurs via a membrane potential sensor instead of directly by Na(+) flux and supports the notion of a train of action potentials being more efficient as a stimulus than sustained depolarization, suggesting that tetanus is the physiological stimulus for the IP(3)-dependent calcium signal involved in regulation of gene expression.     

Please release me

In this issue of Neuron, Südhof and colleagues determine which of the eight Ca(2+)-binding synaptotagmin isoforms expressed in brain can support synchronous neurotransmitter release at mammalian CNS synapses. Unexpectedly, only three-synaptotagmin-1, -2, and -9-can serve as Ca(2+) sensors for fast transmission. Further characterization reveals the unique ability of each isoform to shape neurotransmission.     

Tag team action at the synapse

Communication between neurons relies on chemical synapses and the release of neurotransmitters into the synaptic cleft. Neurotransmitter release is an exquisitely regulated membrane fusion event that requires the linking of an electrical nerve stimulus to Ca(2+) influx, which leads to the fusion of neurotransmitter-filled vesicles with the cell membrane. The timing of neurotransmitter release is controlled through the regulation of the soluble N-ethylmaleimide sensitive factor attachment receptor (SNARE) proteins-the core of the membrane fusion machinery. Assembly of the fusion-competent SNARE complex is regulated by several neuronal proteins, including complexin and the Ca(2+)-sensor synaptotagmin. Both complexin and synaptotagmin bind directly to SNAREs, but their mechanism of action has so far remained unclear. Recent studies revealed that synaptotagmin-Ca(2+) and complexin collaborate to regulate membrane fusion. These compelling new results provide a molecular mechanistic insight into the functions of both proteins: complexin 'clamps' the SNARE complex in a pre-fusion intermediate, which is then released by the action of Ca(2+)-bound synaptotagmin to trigger rapid fusion.     

Synaptotagmin I: A major Ca2+ sensor for transmitter release at a central synapse

Mice carrying a mutation in the synaptotagmin I gene were generated by homologous recombination. Mutant mice are phenotypically normal as heterozygotes, but die within 48 hr after birth as homozygotes. Studies of hippocampal neurons cultured from homozygous mutant mice reveal that synaptic transmission is severely impaired. The synchronous, fast component of Ca²⁺-dependent neurotransmitter release is decreased, whereas asynchronous release processes, including spontaneous synaptic activity (miniature excitatory postsynaptic current frequency) and release triggered by hypertonic solution or α-latrotoxin, are unaffected. Our findings demonstrate that synaptotagmin I function is required for Ca²⁺-triggering of synchronous neurotransmitter release, but is not essential for asynchronous or Ca²⁺-independent release. We propose that synaptotagmin I is the major low affinity Ca²⁺ sensor mediating Ca²⁺ regulation of synchronous neurotransmitter release in hippocampal neurons.     

Structural basis for the evolutionary inactivation of Ca2+ binding to synaptotagmin 4

The neuronal protein synaptotagmin 1 functions as a Ca(2+) sensor in exocytosis via two Ca(2+)-binding C(2) domains. The very similar synaptotagmin 4, which includes all the predicted Ca(2+)-binding residues in the C(2)B domain but not in the C(2)A domain, is also thought to function as a neuronal Ca(2+) sensor. Here we show that, unexpectedly, both C(2) domains of fly synaptotagmin 4 exhibit Ca(2+)-dependent phospholipid binding, whereas neither C(2) domain of rat synaptotagmin 4 binds Ca(2+) or phospholipids efficiently. Crystallography reveals that changes in the orientations of critical Ca(2+) ligands, and perhaps their flexibility, render the rat synaptotagmin 4 C(2)B domain unable to form full Ca(2+)-binding sites. These results indicate that synaptotagmin 4 is a Ca(2+) sensor in the fly but not in the rat, that the Ca(2+)-binding properties of C(2) domains cannot be reliably predicted from sequence analyses, and that proteins clearly identified as orthologs may nevertheless have markedly different functional properties.     

Close membrane-membrane proximity induced by Ca(2+)-dependent multivalent binding of synaptotagmin-1 to phospholipids

Synaptotagmin acts as a Ca(2+) sensor in neurotransmitter release through its two C(2) domains. Ca(2+)-dependent phospholipid binding is key for synaptotagmin function, but it is unclear how this activity cooperates with the SNARE complex involved in release or why Ca(2+) binding to the C(2)B domain is more crucial for release than Ca(2+) binding to the C(2)A domain. Here we show that Ca(2+) induces high-affinity simultaneous binding of synaptotagmin to two membranes, bringing them into close proximity. The synaptotagmin C(2)B domain is sufficient for this ability, which arises from the abundance of basic residues around its surface. We propose a model wherein synaptotagmin cooperates with the SNAREs in bringing the synaptic vesicle and plasma membranes together and accelerates membrane fusion through the highly positive electrostatic potential of its C(2)B domain.     

Calcium-induced calcium release contributes to somatic secretion of serotonin in leech Retzius neurons

We analyzed the contribution of calcium (Ca2+)-induced Ca2+ release to somatic secretion in serotonergic Retzius neurons of the leech. Somatic secretion was studied by the incorporation of fluorescent dye FM1-43 upon electrical stimulation with trains of 10 impulses and by electron microscopy. Quantification of secretion with FM1-43 was made in cultured neurons to improve optical resolution. Stimulation in the presence of FM1-43 produced a frequency-dependent number of fluorescent spots. While a 1-Hz train produced 19.5+/-5.0 spots/soma, a 10-Hz train produced 146.7+/-20.2 spots/soma. Incubation with caffeine (10 mM) to induce Ca2+ release from intracellular stores without electrical stimulation and external Ca2+, produced 168+/-21.7 spots/soma. This staining was reduced by 49% if neurons were preincubated with the Ca2+- ATPase inhibitor thapsigargin (200 nM). Moreover, in neurons stimulated at 10 Hz in the presence of ryanodine (100 microM) to block Ca2+-induced Ca2+ release, FM1-43 staining was reduced by 42%. In electron micrographs of neurons at rest or stimulated at 1 Hz in the ganglion, endoplasmic reticulum lay between clusters of dense core vesicles and the plasma membrane. In contrast, in neurons stimulated at 20 Hz, the vesicle clusters were apposed to the plasma membrane and flanked by the endoplasmic reticulum. These results suggest that Ca2+-induced Ca2+ release produces vesicle mobilization and fusion in the soma of Retzius neurons, and supports the idea that neuronal somatic secretion shares common mechanisms with secretion by excitable endocrine cells.     

Synaptotagmin VII as a plasma membrane Ca(2+) sensor in exocytosis

Synaptotagmins I and II are Ca(2+) binding proteins of synaptic vesicles essential for fast Ca(2+)-triggered neurotransmitter release. However, central synapses and neuroendocrine cells lacking these synaptotagmins still exhibit Ca(2+)-evoked exocytosis. We now propose that synaptotagmin VII functions as a plasma membrane Ca(2+) sensor in synaptic exocytosis complementary to vesicular synaptotagmins. We show that alternatively spliced forms of synaptotagmin VII are expressed in a developmentally regulated pattern in brain and are concentrated in presynaptic active zones of central synapses. In neuroendocrine PC12 cells, the C(2)A and C(2)B domains of synaptotagmin VII are potent inhibitors of Ca(2+)-dependent exocytosis, but only when they bind Ca(2+). Our data suggest that in synaptic vesicle exocytosis, distinct synaptotagmins function as independent Ca(2+) sensors on the two fusion partners, the plasma membrane (synaptotagmin VII) versus synaptic vesicles (synaptotagmins I and II).     

Dueling Ca2+ sensors in neurotransmitter release

Ca(2+)-triggered neurotransmitter release is characterized by two kinetically distinct components: a fast synchronous phase and a slow asynchronous phase. Yao et al. (2011) now report that double C2 domain (Doc2) proteins function as high-affinity Ca(2+) sensors to specifically regulate the asynchronous component of neurotransmitter release.     

Sr2+ binding to the Ca2+ binding site of the synaptotagmin 1 C2B domain triggers fast exocytosis without stimulating SNARE interactions

Sr(2+) triggers neurotransmitter release similar to Ca(2+), but less efficiently. We now show that in synaptotagmin 1 knockout mice, the fast component of both Ca(2+)- and Sr(2+)-induced release is selectively impaired, suggesting that both cations partly act by binding to synaptotagmin 1. Both the C(2)A and the C(2)B domain of synaptotagmin 1 bind Ca(2+) in phospholipid complexes, but only the C(2)B domain forms Sr(2+)/phospholipid complexes; therefore, Sr(2+) binding to the C(2)B domain is sufficient to trigger fast release, although with decreased efficacy. Ca(2+) induces binding of the synaptotagmin C(2) domains to SNARE proteins, whereas Sr(2+) even at high concentrations does not. Thus, triggering of the fast component of release by Sr(2+) as a Ca(2+) agonist involves the formation of synaptotagmin/phospholipid complexes, but does not require stimulated SNARE binding.     

A complexin/synaptotagmin 1 switch controls fast synaptic vesicle exocytosis

Ca(2+) binding to synaptotagmin 1 triggers fast exocytosis of synaptic vesicles that have been primed for release by SNARE-complex assembly. Besides synaptotagmin 1, fast Ca(2+)-triggered exocytosis requires complexins. Synaptotagmin 1 and complexins both bind to assembled SNARE complexes, but it is unclear how their functions are coupled. Here we propose that complexin binding activates SNARE complexes into a metastable state and that Ca(2+) binding to synaptotagmin 1 triggers fast exocytosis by displacing complexin from metastable SNARE complexes. Specifically, we demonstrate that, biochemically, synaptotagmin 1 competes with complexin for SNARE-complex binding, thereby dislodging complexin from SNARE complexes in a Ca(2+)-dependent manner. Physiologically, increasing the local concentration of complexin selectively impairs fast Ca(2+)-triggered exocytosis but retains other forms of SNARE-dependent fusion. The hypothesis that Ca(2+)-induced displacement of complexins from SNARE complexes triggers fast exocytosis accounts for the loss-of-function and gain-of-function phenotypes of complexins and provides a molecular explanation for the high speed and synchronicity of fast Ca(2+)-triggered neurotransmitter release.     

Two Ca(2+)-dependent steps controlling synaptic vesicle fusion and replenishment at the cerebellar basket cell terminal

Cerebellar basket cells inhibit postsynaptic Purkinje cells in a rapid and precise manner. To investigate the mechanisms of transmitter release underlying this rapid inhibition, Ca(2+) uncaging was employed to measure the intracellular Ca(2+) dependence of transmitter release and the kinetics of synaptic vesicle pool transitions in immature basket cell synapses at room temperature. Vesicle release properties distinct from those previously observed at excitatory synapses were seen, including a relatively high intracellular Ca(2+) sensitivity of vesicle fusion, rapid vesicle pool mobilization with few reluctant vesicles, and vesicle replenishment driven by unusually high Ca(2+) levels from both local and residual Ca(2+) sources during action potential trains. These results suggest that inhibitory basket cell synapses are optimized for rapid and precise temporal and spatial Ca(2+) coordination of synaptic vesicle fusion and replenishment, which may contribute to the unique physiology of inhibitory synaptic transmission, including phasic release during action potential trains and tonic release by residual intracellular Ca(2+).