Nanodomain coupling between Ca2+ channels and Ca2+ sensors promotes fast and efficient transmitter release at a cortical GABAergic synapse

It is generally thought that transmitter release at mammalian central synapses is triggered by Ca2+ microdomains, implying loose coupling between presynaptic Ca2+ channels and Ca2+ sensors of exocytosis. Here we show that Ca2+ channel subunit immunoreactivity is highly concentrated in the active zone of GABAergic presynaptic terminals of putative parvalbumin-containing basket cells in the hippocampus. Paired recording combined with presynaptic patch pipette perfusion revealed that GABA release at basket cell-granule cell synapses is sensitive to millimolar concentrations of the fast Ca2+ chelator BAPTA but insensitive to the slow Ca2+ chelator EGTA. These results show that Ca2+ source and Ca2+ sensor are tightly coupled at this synapse, with distances in the range of 10-20 nm. Models of Ca2+ inflow-exocytosis coupling further reveal that the tightness of coupling increases efficacy, speed, and temporal precision of transmitter release. Thus, tight coupling contributes to fast feedforward and feedback inhibition in the hippocampal network.     

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.     

A novel dual Ca2+ sensor system regulates Ca2+-dependent neurotransmitter release

Ca²⁺-dependent neurotransmitter release requires synaptotagmins as Ca²⁺ sensors to trigger synaptic vesicle (SV) exocytosis via binding of their tandem C2 domains—C2A and C2B—to Ca²⁺. We have previously demonstrated that SNT-1, a mouse synaptotagmin-1 (Syt1) homologue, functions as the fast Ca²⁺ sensor in Caenorhabditis elegans. Here, we report a new Ca²⁺ sensor, SNT-3, which triggers delayed Ca²⁺-dependent neurotransmitter release. snt-1;snt-3 double mutants abolish evoked synaptic transmission, demonstrating that C. elegans NMJs use a dual Ca²⁺ sensor system. SNT-3 possesses canonical aspartate residues in both C2 domains, but lacks an N-terminal transmembrane (TM) domain. Biochemical evidence demonstrates that SNT-3 binds both Ca²⁺ and the plasma membrane. Functional analysis shows that SNT-3 is activated when SNT-1 function is impaired, triggering SV release that is loosely coupled to Ca²⁺ entry. Compared with SNT-1, which is tethered to SVs, SNT-3 is not associated with SV. Eliminating the SV tethering of SNT-1 by removing the TM domain or the whole N terminus rescues fast release kinetics, demonstrating that cytoplasmic SNT-1 is still functional and triggers fast neurotransmitter release, but also exhibits decreased evoked amplitude and release probability. These results suggest that the fast and slow properties of SV release are determined by the intrinsically different C2 domains in SNT-1 and SNT-3, rather than their N-termini–mediated membrane tethering. Our findings therefore reveal a novel dual Ca²⁺ sensor system in C. elegans and provide significant insights into Ca²⁺-regulated exocytosis.     

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 is a Ca2+ sensor for somatodendritic dopamine release

1. Download : Download high-res image (144KB)
2. Download : Download full-size image

     

Type-3 ryanodine receptor involved in Ca2+-induced Ca2+ release and transmitter exocytosis at frog motor nerve terminals

Ca(2+)-induced Ca2+ release (CICR) occurs in frog motor nerve terminals after ryanodine receptors (RyRs) are primed for activation by conditioning large Ca2+ entry. We studied which type of RyR exists, whether CICR occurs without conditioning Ca2+ entry and how RyRs are primed. Immunohistochemistry revealed the existence of RyR3 in motor nerve terminals and axons and both RyR1 and RyR3 in muscle fibers. A blocker of RyR, 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) slightly decreased rises in intracellular Ca2+ ([Ca2+]i) induced by a short tetanus (50 Hz, 1-2s), but not after treatment with ryanodine. Repetitive tetani (50 Hz for 15s every 20s) produced repetitive rises in [Ca2+]i, whose amplitude overall waxed and waned. TMB-8 blocked the waxing and waning components. Ryanodine suppressed a slow increase in end-plate potentials (EPPs) induced by stimuli (33.3 Hz, 15s) in a low Ca2+, high Mg2+ solution. KN-62, a blocker of Ca(2+)/calmoduline-activated protein kinase II (CaMKII), slightly reduced short tetanus-induced rises in [Ca2+]i, but markedly the slow waxing and waning rises produced by repetitive tetani in both normal and low Ca2+, high Mg2+ solutions. Likewise, KN-62, but not KN-04, an inactive analog, suppressed slow increases in EPP amplitude and miniature EPP frequency during long tetanus. Thus, CICR normally occurs weakly via RyR3 activation by single impulse-induced Ca2+ entry in frog motor nerve terminals and greatly after the priming of RyR via CaMKII activation by conditioning Ca2+ entry, thus, facilitating transmitter exocytosis and its plasticity.     

STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered Ca2+ influx

Ca(2+) signaling in nonexcitable cells is typically initiated by receptor-triggered production of inositol-1,4,5-trisphosphate and the release of Ca(2+) from intracellular stores. An elusive signaling process senses the Ca(2+) store depletion and triggers the opening of plasma membrane Ca(2+) channels. The resulting sustained Ca(2+) signals are required for many physiological responses, such as T cell activation and differentiation. Here, we monitored receptor-triggered Ca(2+) signals in cells transfected with siRNAs against 2,304 human signaling proteins, and we identified two proteins required for Ca(2+)-store-depletion-mediated Ca(2+) influx, STIM1 and STIM2. These proteins have a single transmembrane region with a putative Ca(2+) binding domain in the lumen of the endoplasmic reticulum. Ca(2+) store depletion led to a rapid translocation of STIM1 into puncta that accumulated near the plasma membrane. Introducing a point mutation in the STIM1 Ca(2+) binding domain resulted in prelocalization of the protein in puncta, and this mutant failed to respond to store depletion. Our study suggests that STIM proteins function as Ca(2+) store sensors in the signaling pathway connecting Ca(2+) store depletion to Ca(2+) influx.     

Synaptotagmin C2A loop 2 mediates Ca2+-dependent SNARE interactions essential for Ca2+-triggered vesicle exocytosis

Synaptotagmins contain tandem C2 domains and function as Ca(2+) sensors for vesicle exocytosis but the mechanism for coupling Ca(2+) rises to membrane fusion remains undefined. Synaptotagmins bind SNAREs, essential components of the membrane fusion machinery, but the role of these interactions in Ca(2+)-triggered vesicle exocytosis has not been directly assessed. We identified sites on synaptotagmin-1 that mediate Ca(2+)-dependent SNAP25 binding by zero-length cross-linking. Mutation of these sites in C2A and C2B eliminated Ca(2+)-dependent synaptotagmin-1 binding to SNAREs without affecting Ca(2+)-dependent membrane binding. The mutants failed to confer Ca(2+) regulation on SNARE-dependent liposome fusion and failed to restore Ca(2+)-triggered vesicle exocytosis in synaptotagmin-deficient PC12 cells. The results provide direct evidence that Ca(2+)-dependent SNARE binding by synaptotagmin is essential for Ca(2+)-triggered vesicle exocytosis and that Ca(2+)-dependent membrane binding by itself is insufficient to trigger fusion. A structure-based model of the SNARE-binding surface of C2A provided a new view of how Ca(2+)-dependent SNARE and membrane binding occur simultaneously.     

Ca2+ release via ryanodine receptors and Ca2+ entry: major mechanisms in NAADP-mediated Ca2+ signaling in T-lymphocytes

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+ mobilizing nucleotide essentially involved in T cell activation. Using combined microinjection and single cell calcium imaging, we demonstrate that co-injection of NAADP and the D-myo-inositol 1,4,5-trisphosphate antagonist heparin did not inhibit Ca2+ mobilization. In contrast, co-injection of the ryanodine receptor antagonist ruthenium red efficiently blocked NAADP induced Ca2+ signalling. This pharmacological approach was confirmed using T cell clones stably transfected with plasmids expressing antisense mRNA targeted specifically against ryanodine receptors. NAADP induced Ca2+ signaling was strongly reduced in these clones. In addition, inhibition of Ca2+ entry by SK&F 96365 resulted in a dramatically decreased Ca2+ signal upon NAADP injection. Gd3+, a known blocker of Ca2+ release activated Ca2+ entry, only partially inhibited NAADP mediated Ca2+ signaling. These data indicate that in T cells (i) ryanodine receptor are the major intracellular Ca2+ release channels involved in NAADP induced Ca2+ signals, and that (ii) such Ca2+ release events are largely amplified by Ca2+ entry.     

An apoplastic Ca2+ sensor regulates internal Ca2+ release in aequorin-transformed tobacco cells

Removal of Ca(2+) from tobacco suspension cell medium has two immediate effects on cytosolic Ca(2+) fluxes: (i) externally derived Ca(2+) influx (occurring in response to cold shock or hypo-osmotic shock) is inhibited, and (ii) organellar Ca(2+) release (induced by a fungally derived defense elicitor, caffeine, or hypo-osmotic shock) is elevated. We show here that the enhanced release of internal Ca(2+) is likely due to increased discharge from a caffeine-sensitive store in response to a signal transduced from an extracellular Ca(2+) sensor. Thus, chelation of extracellular Ca(2+) in the absence of any other stimulus directly activates release of intracellular Ca(2+) into the cytosol. Evidence that this chelator-activated Ca(2+) flux is dependent on a signaling pathway includes its abrogation by prior treatment with caffeine, and its inhibition by protein kinase inhibitors (K252a and staurosporine) and anion channel blockers (niflumate and anthracene-9-carboxylate). An unexpected characteristic of tobacco cell adaptation to low external Ca(2+) was the emergence of a new Ca(2+) compartment that was inaccessible to external EGTA, yet responsive to the usual stimulants of extracellular Ca(2+) entry. Thus, cells that are exposed to EGTA for 20 min lose sensitivity to caffeine and defense elicitors, indicating that their intracellular Ca(2+) pools have been depleted. Surprisingly, these same cells simultaneously regain their ability to respond to stimuli that usually activate extracellular Ca(2+) influx even though all external Ca(2+) is chelated. Because this gradual restoration of Ca(2+) influx can be inhibited by the same kinase inhibitors that block EGTA-activated Ca(2+) release, we propose that chelator-activated Ca(2+) release from internal stores leads to deposition of this Ca(2+) into a novel EGTA- and caffeine-insensitive compartment that can subsequently be activated by stimulants of extracellular Ca(2+) entry.     

The voltage-gated Ca2+ channel is the Ca2+ sensor of fast neurotransmitter release

Previously it demonstrated that in the absence of Ca²⁺ entry, evoked secretion occurs neither by membrane depolarization, induction of [Ca²⁺] _i rise, nor by both combined (Ashery, U., Weiss, C., Sela, D., Spira, M. E., and Atlas, D. (1993). Receptors Channels 1:217–220.). These studies designate Ca²⁺ entry as opposed to [Ca²⁺] _i rise, essential for exocytosis. It led us to propose that the channel acts as the Ca²⁺ sensor and modulates secretion through a physical and functional contact with the synaptic proteins. This view was supported by protein–protein interactions reconstituted in the Xenopus oocytes expression system and release experiments in pancreatic cells (Barg, S., Ma, X., Elliasson, L., Galvanovskis, J., Gopel, S. O., Obermuller, S., Platzer, J., Renstrom, E., Trus, M., Atlas, D., Streissnig, G., and Rorsman, P. (2001). Biophys. J.; Wiser, O., Bennett, M. K., and Atlas, D. (1996). EMBO J. 15:4100–4110; Wiser, O., Trus, M., Hernandez, A., Renström, E., Barg, S., Rorsman, P., and Atlas, D. (1999). Proc. Natl. Acad. Sci. U.S.A. 96:248–253). The kinetics of Ca_v1.2 (Lc-type) and Ca_v2.2 (N-type) Ca²⁺ channels were modified in oocytes injected with cRNA encoding syntaxin 1A and SNAP-25. Conserved cysteines (Cys271, Cys272) within the syntaxin 1A transmembrane domain are essential. Synaptotagmin I, a vesicle-associated protein, accelerated the activation kinetics indicating Ca_v2.2 coupling to the vesicle. The unique modifications of Ca_v1.2 and Ca_v2.2 kinetics by syntaxin 1A, SNAP-25, and synaptotagmin combined implied excitosome formation, a primed fusion complex of the channel with synaptic proteins. The Ca_v1.2 cytosolic domain Lc_753–893, acted as a dominant negative modulator, competitively inhibiting insulin release of channel-associated vesicles (CAV), the readily releasable pool of vesicles (RRP) in islet cells. A molecular mechanism is offered to explain fast secretion of vesicles tethered to SNAREs-associated Ca²⁺ channel. The tight arrangement facilitates the propagation of conformational changes induced during depolarization and Ca²⁺-binding at the channel, to the SNAREs to trigger secretion. The results imply a rapid Ca²⁺-dependent CAV (RRP) release, initiated by the binding of Ca²⁺ to the channel, upstream to intracellular Ca²⁺ sensor thus establishing the Ca²⁺ channel as the Ca²⁺ sensor of neurotransmitter release.     

Synaptotagmin in Ca2+ -dependent exocytosis: dynamic action in a flash

Synaptotagmins have been the popular candidates for the Ca2+ sensor that couples local rise in Ca2+ to neurotransmitter release. Studies in worm, fly, and mouse corroborate the likely role for synaptotagmin I, the best-studied synaptotagmin prototype, as a Ca2+ trigger for synaptic vesicle exocytosis. Recent investigations have focused on structural domains of synaptotagmin that are critical for its function. Here we provide a brief overview of synaptotagmin I and discuss recent studies within the framework of neurotransmitter release mechanisms for fast synaptic transmission.     

Rate of quantal transmitter release at the mammalian rod synapse.

Under scotopic conditions, the mammalian rod encodes either one photon or none within its integration time. Consequently the signal presented to its synaptic terminal is binary. The synapse has a single active zone that releases neurotransmitter quanta tonically in darkness and pauses briefly in response to a rhodopsin isomerization by a photon. We asked: what minimum tonic rate would allow the postsynaptic bipolar cell to distinguish this pause from an extra-long interval between quanta due to the stochastic timing of release? The answer required a model of the circuit that included the rod convergence onto the bipolar cell and the bipolar cell''s signal-to-noise ratio. Calculations from the model suggest that tonic release must be at least 40 quanta/s. This tonic rate is much higher than at conventional synapses where reliability is achieved by employing multiple active zones. The rod''s synaptic mechanism makes efficient use of space, which in the retina is at a premium.     

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).     

Role of intracellular Ca2+ in stimulation-induced increases in transmitter release at the frog neuromuscular junction

Under conditions of reduced quantal content, repetitive stimulation of a presynaptic nerve can result in a progressive increase in the amount of transmitter released by that nerve in response to stimulation. At the frog neuromuscular junction, this increase in release has been attributed to four different processes: first and second components of facilitation, augmentation, and potentiation (e.g., Zengel, J. E., and K. L. Magleby. 1982. Journal of General Physiology. 80:583-611). It has been suggested that an increased entry of Ca2+ or an accumulation of intraterminal Ca2+ may be responsible for one or more of these processes. To test this hypothesis, we have examined the role of intracellular Ca2+ in mediating changes in end-plate potential (EPP) amplitude during and after repetitive stimulation at the frog neuromuscular junction. We found that increasing the extracellular Ca2+ concentration or exposing the preparation to carbonyl cyanide m- chlorophenylhydrazone, ionomycin, or cyclopiazonic acid all led to a greater increase in EPP amplitude during conditioning trains of 10-200 impulses applied at a frequency of 20 impulses/s. These experimental manipulations, all of which have been shown to increase intracellular levels of Ca2+, appeared to act by increasing primarily the augmentation component of increased release. The results of this study are consistent with previous suggestions that the different components of increased release represent different mechanisms, and that Ca2+ may be acting at more than one site in the nerve terminal.     

Calcium and the tonic release of transmitter at a non-impulsive synapse in the crab

Depolarization-transmitter release coupling was studied in the promotor stretch receptor/motoneuron synapse of the crab. Callinectes sapidus, a preparation in which presynaptic action potentials do not occur. Intracellular microelectrode recordings were made from the presynaptic terminal and from the somata of postsynaptic motoneurons while injecting current pulses into the peripheral stretch receptor dendrite with the aid of the sucrose-gap. 1. For short current pulses, the relationship between presynaptic potential and postsynaptic response was found to be similar to that demonstrated in the giant synapse of the squid stellate ganglion, indicating a common reliance on the properties of voltage-dependent calcium channels. 2. The crab synapse was found to be capable of continuous transmission in the range of seconds and minutes without the pronounced depletion of transmitter seen in the squid, and without inactivation of the release process (i.e., the calcium conductance is non-inactivating). 3. A graded, transient response to depolarising current in the presynaptic fibre was found to be calcium-dependent, and probably to reflect the presence of a separate, inactivating calcium conductance. 4. It was concluded that the graded response of the presynaptic membrane could function in helping to compensate for capacitative distortion of receptor potentials decrementally conducted in the sensory dendrite, and was therefore a specialisation for non-impulsive transmission.     

Ca(2+) channels and transmitter release at the active zone

Ca(2+)-dependent transmitter release is the most important signaling mechanism for fast information transfer between neurons. Transmitter release takes places at highly specialized active zones with sub-micrometer dimension, which contain the molecular machinery for vesicle docking and -fusion, as well as a high density of voltage-gated Ca(2+) channels. In the absence of direct evidence for the ultrastructural localization of Ca(2+) channels at CNS synapses, important insights into Ca(2+) channel-vesicle coupling has come from functional experiments relating presynaptic Ca(2+) current and transmitter release, at large and accessible synapses like the calyx of Held. First, high slope values in log-log plots of transmitter release versus presynaptic Ca(2+) current indicate that multiple Ca(2+) channels are involved in release control of a single vesicle. Second, release kinetics in response to step-like depolarizations revealed fast- and slowly releasable sub-pools of vesicles, FRP and SRP, which, according to the "positional" model, are distinguished by a differential proximity to Ca(2+) channels. Considering recent evidence for a rapid conversion of SRP- to FRP vesicles, however, we highlight that multivesicular release events and clearance of vesicle membrane from the active zone must be taken into account when interpreting kinetic release data. We conclude that the careful kinetic analysis of transmitter release at presynaptically accessible and molecularly targeted synapses has the potential to yield important insights into the molecular physiology of transmitter release.     

Controlling the urge for a Ca2+ surge: all-or-none Ca2+ release in neurons

Changes in the intracellular calcium concentration ([Ca²⁺]_i) convey signals that are essential to the life and death of neurons. Ca²⁺-induced Ca²⁺-release (CICR), a process in which a modest elevation in [Ca²⁺]_i is amplified by a secondary release of Ca²⁺ from stores within the cell, plays a prominent role in shaping neuronal [Ca²⁺]_i signals. When CICR becomes regenerative, an explosive increase in [Ca²⁺]_i generates a Ca²⁺ wave that spreads throughout the cell. A discrete threshold controls activation of this all-or-none behavior and cellular context adjusts the threshold. Thus, the store acts as a switch that determines whether a given pattern of electrical activity will produce a local or global Ca²⁺ signal. This gatekeeper function seems to control some forms of Ca²⁺-triggered plasticity in neurons. BioEssays 21:743–750, 1999. © 1999 John Wiley & Sons, Inc.     

Structure of Ca2+ release channel at 14 A resolution

The 14 A resolution structure of the 2.3 MDa Ca2+ release channel (also known as RyR1) was determined by electron cryomicroscopy and single particle reconstruction. This structure was produced using collected data used for our previous published structures at 22-30 A resolution, but now taking advantage of recent algorithmic improvements in the EMAN software suite. This improved map clearly exhibits more structural detail and allows better defined docking of computationally predicted structural domain folds. Using sequence-based fold recognition, the N-terminal region of RyR1, residues 216-572, was predicted to have significant structural similarity with the IP3-binding core region of the type 1 IP3R. This putative structure was computationally localized to the clamp-shaped region of RyR1, which has been implicated to have a regulatory role in the channel activity.