The probability of quantal secretion near a single calcium channel of an active zone

A Monte Carlo analysis has been made of calcium dynamics and quantal secretion at microdomains in which the calcium reaches very high concentrations over distances of <50 nm from a channel and for which calcium dynamics are dominated by diffusion. The kinetics of calcium ions in microdomains due to either the spontaneous or evoked opening of a calcium channel, both of which are stochastic events, are described in the presence of endogenous fixed and mobile buffers. Fluctuations in the number of calcium ions within 50 nm of a channel are considerable, with the standard deviation about half the mean. Within 10 nm of a channel these numbers of ions can give rise to calcium concentrations of the order of 100 microM. The temporal changes in free calcium and calcium bound to different affinity indicators in the volume of an entire varicosity or bouton following the opening of a single channel are also determined. A Monte Carlo analysis is also presented of how the dynamics of calcium ions at active zones, after the arrival of an action potential and the stochastic opening of a calcium channel, determine the probability of exocytosis from docked vesicles near the channel. The synaptic vesicles in active zones are found docked in a complex with their calcium-sensor associated proteins and a voltage-sensitive calcium channel, forming a secretory unit. The probability of quantal secretion from an isolated secretory unit has been determined for different distances of an open calcium channel from the calcium sensor within an individual unit: a threefold decrease in the probability of secretion of a quantum occurs with a doubling of the distance from 25 to 50 nm. The Monte Carlo analysis also shows that the probability of secretion of a quantum is most sensitive to the size of the single-channel current compared with its sensitivity to either the binding rates of the sites on the calcium-sensor protein or to the number of these sites that must bind a calcium ion to trigger exocytosis of a vesicle.     

Exocytotic dynamics and calcium cooperativity effects in the calyx of Held synapse: a modelling study

A stochastic computational approach to the study of secretory processes at the calyx of Held synapse is presented in this paper. The calyx of Held is a giant synapse located in the brainstem which is widely used for experimental recording of neurotransmitter release. We focus on the study of the exocytotic dynamics for a pool of readily releasable vesicles using a Monte Carlo simulation scheme that includes models for the P-type calcium channels, the kinetic reactions of endogenous and exogenous (mobile) buffers, the kinetic reactions for the secretory vesicles, as well as the microscopic diffusion of mobile buffers and calcium ions. The simulations are performed in a 3-D orthogonal grid which approximates a cylindrical domain representing an active zone of the presynaptic terminal of the calyx. For this domain, we quantify the release rates related to calcium currents in response to depolarizing voltage pulses. The influence on simulated pulse/action potential depolarization protocols of the kinetic scheme for the calcium sensor of vesicles and the geometry of calcium channels for the kinetic cooperativity for release, is analyzed at a microdomain level. Among other aspects, our results suggest that the spatial organization of Ca ^2 + channels could have measurable effects in the kinetic cooperativity which could reflect developing changes in the calyx of Held synapse.     

Mechanisms of calcium sequestration during facilitation at active zones of an amphibian neuromuscular junction

The calcium transients (Delta[Ca(2+)](i)) at active zones of amphibian (Bufo marinus) motor-nerve terminals that accompany impulses, visualized using a low-affinity calcium indicator injected into the terminal, are described and the pathways of subsequent sequestration of the residual calcium determined, allowing development of a quantitative model of the sequestering processes. Blocking the endoplasmic reticulum calcium pump with thapsigargin did not affect Delta[Ca(2+)](i) for a single impulse but increased its amplitude during short trains. Blocking the uptake of calcium by mitochondria with CCCP had little effect on Delta[Ca(2+)](i) of a single impulse but greatly increased its amplitude during short trains. This present compartmental model is compatible with our previous Monte Carlo diffusion model of Ca(2+) sequestration during facilitation [Bennett, M.R., Farnell, L., Gibson, W.G., 2004. The facilitated probability of quantal secretion within an array of calcium channels of an active zone at the amphibian neuromuscular junction. Biophys. J. 86(5), 2674-2690], with the single plasmalemma pump in that model now replaced by separate pumps for the plasmalemma and endoplasmic reticulum, as well as the introduction of a mitochondrial uniporter.     

The facilitated probability of quantal secretion within an array of calcium channels of an active zone at the amphibian neuromuscular junction

A Monte Carlo analysis has been made of the phenomenon of facilitation, whereby a conditioning impulse leaves nerve terminals in a state of heightened release of quanta by a subsequent test impulse, this state persisting for periods of hundreds of milliseconds. It is shown that a quantitative account of facilitation at the amphibian neuromuscular junction can be given if the exocytosis is triggered by the combined action of a low-affinity calcium-binding molecule at the site of exocytosis and a high-affinity calcium-binding molecule some distance away. The kinetic properties and spatial distribution of these molecules at the amphibian neuromuscular junction are arrived at by considering the appropriate values that the relevant parameters must take to successfully account for the experimentally observed amplitude and time course of decline of F1 and F2 facilitation after a conditioning impulse, as well as the growth of facilitation during short trains of impulses. This model of facilitation correctly predicts the effects on facilitation of exogenous buffers such as BAPTA during short trains of impulses. In addition, it accounts for the relative invariance of the kinetics of quantal release due to test-conditioning sequences of impulses as well as due to change in the extent of calcium influx during an impulse.     

The molecular mechanisms of quantum mediator secretion in the synapse

The modern condition of knowledge about the molecular mechanisms underlying the quantal transmitter release in the central and the peripheric synapses is analysed. The data about the synaptic vesicles types, their forming, transporting to the sites of release at the nerve endings, exo- and endocytosis processes are presented. Ultrastructural and molecular organization of active zone of nerve ending and transmitter release morphofunctional unit--secretosome, which includes synaptic vesicle, exocytosis protein complex and calcium channels, are described. The basic proteins involved in the exo- and endocytosis and their interactions during transmitter release are examined. The role of the intracellular buffer systems, calcium micro- and macrodomains in the quantal transmitter secretion are considered. The reasons of the active zones functional non-uniformity and plasticity and factors reduced transmitter release in the active zone to the single quantum are analysed.     

Probabilistic secretion of quanta and the synaptosecretosome hypothesis: evoked release at active zones of varicosities, boutons, and endplates.

A quantum of transmitter may be released upon the arrival of a nerve impulse if the influx of calcium ions through a nearby voltage-dependent calcium channel is sufficient to activate the vesicle-associated calcium sensor protein that triggers exocytosis. A synaptic vesicle, together with its calcium sensor protein, is often found complexed with the calcium channel in active zones to form what will be called a "synaptosecretosome." In the present work, a stochastic analysis is given of the conditions under which a quantum is released from the synaptosecretosome by a nerve impulse. The theoretical treatment considers the rise of calcium at the synaptosecretosome after the stochastic opening of a calcium channel at some time during the impulse, followed by the stochastic binding of calcium to the vesicle-associated protein and the probability of this leading to exocytosis. This allows determination of the probabilities that an impulse will release 0, 1, 2,... quanta from an active zone, whether this is in a varicosity, a bouton, or a motor endplate. A number of experimental observations of the release of transmitter at the active zones of sympathetic varicosities and boutons as well as somatic motor endplates are described by this analysis. These include the likelihood of the secretion of only one quantum at an active zone of endplates and of more than one quantum at an active zone of a sympathetic varicosity. The fourth-power relationship between the probability of transmitter release at the active zones of sympathetic varicosities and motor endplates and the external calcium concentration is also explained by this approach. So, too, is the fact that the time course of the increased rate of quantal secretion from a somatic active zone after an impulse is invariant with changes in the amount of calcium that enters through its calcium channel, whether due to changes consequent on the actions of autoreceptor agents such as adenosine or to facilitation. The increased probability of quantal release that occurs during F1 facilitation at the active zones of motor endplates and sympathetic boutons is predicted by the residual binding of calcium to a high-affinity site on the vesicle-associated protein. The concept of the stochastic operation of a synaptosecretosome can accommodate most phenomena involving the release of transmitter quanta at these synapses.     

Monte carlo simulation of 3-D buffered Ca(2+) diffusion in neuroendocrine cells

Buffered Ca(2+) diffusion in the cytosol of neuroendocrine cells is a plausible explanation for the slowness and latency in the secretion of hormones. We have developed a Monte Carlo simulation to treat the problem of 3-D diffusion and kinetic reactions of ions and buffers. The 3-D diffusion is modeled as a random walk process that follows the path of each ion and buffer molecule, combined locally with a stochastic treatment of the first-order kinetic reactions involved. Such modeling is able to predict [Ca(2+)] and buffer concentration time courses regardless of how low the calcium influx is, and it is therefore a convenient method for dealing with physiological calcium currents and concentrations. We study the effects of the diffusional and kinetic parameters of the model on the concentration time courses as well as on the local equilibrium of buffers with calcium. An in-mobile and fast endogenous buffer as described by, Biophys. J. 72:674-690) was able to reach local equilibrium with calcium; however, the exogenous buffers considered are displaced drastically from equilibrium at the start of the calcium pulse, particularly below the pores. The versatility of the method also allows the effect of different arrangements of calcium channels on submembrane gradients to be studied, including random distribution of calcium channels and channel clusters. The simulation shows how the particular distribution of channels or clusters can be of relevance for secretion in the case where the distribution of release granules is correlated with the channels or clusters.     

Synaptic vesicle recruitment for release explored by Monte Carlo stimulation at the crayfish neuromuscular junction

Neurotransmission at chemically transmitting synapses requires calcium-mediated fusion of synaptic vesicles with the presynaptic membrane. Utilizing ultrastructural information available for the crustacean excitatory neuromuscular junction, we developed a model that employs the Monte Carlo simulation technique to follow the entry and movement of Ca2+ ions at a presynaptic active zone, where synaptic vesicles are preferentially docked for release. The model includes interaction of Ca2+ with an intracellular buffer, and variable separation between calcium channels and vesicle-associated Ca(2+)-binding targets that react with Ca2+ to trigger vesicle fusion. The end point for vesicle recruitment for release was binding of four Ca2+ ions to the target controlling release. The results of the modeling experiments showed that intracellular structures that interfere with Ca2+ diffusion (in particular synaptic vesicles) influence recruitment or priming of vesicles for release. Vesicular recruitment is strongly influenced by the separation distance between an opened calcium channel and the target controlling release, and by the concentration and binding properties of the intracellular buffers, as in previous models. When a single opened calcium channel is very close to the target, a single synaptic vesicle can be recruited. However, many of the single-channel openings actuated by a nerve impulse are likely to be ineffective for release, although they contribute to the buildup of total intracellular Ca2+. Thus, the overall effectiveness of single calcium channels in causing vesicles to undergo exocytosis is likely quite low.     

Endogenous calcium buffers regulate fast exocytosis in the synaptic terminal of retinal bipolar cells.

Calcium-triggered exocytosis at the synapse is suppressed by addition of calcium chelators, but the effects of endogenous Ca(2+) buffers have not been tested. We find that 80% of Ca(2+) binding sites in the synaptic terminal of retinal bipolar cells were associated with mobile molecules that suppressed activation of Ca(2+)-sensitive K(+) channels with an efficiency equivalent to approximately 1.2 mM BAPTA. Removing these buffers caused a 30-fold increase in the number of vesicles released by Ca(2+) tail currents lasting approximately 0.5 ms and a 2-fold increase in the rapidly releasable pool of vesicles (RRP). The effects of BAPTA and EGTA indicate that vesicles comprising the RRP were docked at variable distances from Ca(2+) channels. We propose that endogenous Ca(2+) buffers regulate the size of the RRP by suppressing the release of vesicles toward the periphery of the active zone.     

Calcium-dependent regulation of exocytosis

A rapid increase in intracellular calcium directly triggers regulated exocytosis. In addition, changes in intracellular calcium concentration can adjust the extent of exocytosis (quantal content) or the magnitude of individual release events (quantal size) in both the short- and long-term. It is generally agreed that calcium achieves this regulation via an interaction with a number of different molecular targets located at or near to the site of membrane fusion. We review here the synaptic proteins with defined calcium-binding domains and protein kinases activated by calcium, summarize what is known about their function in membrane fusion and the experimental evidence in support of their involvement in synaptic plasticity.     

Probabilistic secretion of quanta: spontaneous release at active zones of varicosities, boutons, and endplates.

The amplitude-frequency histogram of spontaneous miniature endplate potentials follows a Gaussian distribution at mature endplates. This distribution gives the mean and variance of the quantum of transmitter. According to the vesicle hypothesis, this quantum is due to exocytosis of the contents of a single synaptic vesicle. Multimodal amplitude-frequency histograms are observed in varying degrees at developing endplates and at peripheral and central synapses, each of which has a specific active zone structure. These multimodal histograms may be due to the near synchronous exocytosis of more than one vesicle. In the present work, a theoretical treatment is given of the rise of intraterminal calcium after the stochastic opening of a calcium channel within a particular active zone geometry. The stochastic interaction of this calcium with the vesicle-associated proteins involved in exocytosis is then used to calculate the probability of quantal secretions from one or several vesicles at each active zone type. It is shown that this procedure can account for multiquantal spontaneous release that may occur at varicosities and boutons, compared with that at the active zones of motor nerve terminals.     

The synaptic bouton acts like a salt shaker

The physiological quantal responses at the neuromuscular junction and the bouton-neuron show two classes based on amplitude such that the larger class is about 10 times that of the smaller class; and, the larger class is composed of the smaller class. The ratio of the two classes changes with synaptogenesis, degeneration, nerve stimulation, and is readily altered with various challenges (ionic, tonicity, pharmacological agents). Statistical analyses demonstrate that each bouton or release site at the neruomuscular junction (NMJ) secretes a standard amount of transmitter (one quantum) with each action potential. The amount of transmitter secreted (quantal size) is frequency dependent. The quantal-vesicular-exocytotic (QVE) hypothesis posits that the packet of secreted transmitter is released from one vesicle by exocytosis. The QVE hypothesis neither explains two quantal classes and subunits nor exocytosis of only one vesicle at each site. The latter observation requires a mechanism to select one vesicle from each array. Our porocytosis hypothesis states that the quantal packet is pulsed from an array of secretory pores. A salt shaker delivers a standard pinch of salt with each shake because salt flows through all openings in the cap. The variation in the pinch of salt or transmitter decreases with an increase in array size. The docked vesicles, paravesicular matrix, and porosomes (pores) of a release site form the secretory unit. In analogy with the sacromere as the functional unit of skeletal muscle, we term the array of docked vesicles and paravesicular grid along with the array of postsynaptic receptors a synaptomere. Pulsed secretion from an array explains the substructure of the postsynaptic response (quantum). The array guarantees a constant amount of secretion with each action potential and permits a given synapse to function in different responses because different frequencies would secrete signature amounts of transmitter. Our porocytosis hypothesis readily explains a change in quantal size during learning and memory with an increase in the number of elements (docked vesicles) composing the array.     

The synaptic bouton acts like a slat shaker

The physiological quantal responses at the neuromuscular junction and the bouton-neuron show two classes based on amplitude such that the larger class is about 10 times that of the smaller class; and, the larger class is composed of the smaller class. The ratio of the two classes changes with synaptogenesis, degeneration, nerve stimulation, and is readily altered with various challenges (ionic, tonicity, pharmacological agents). Statistical analyses demonstrate that each bouton or release site at the neruomuscular junction (NMJ) secretes a standard amount of transmitter (one quantum) with each action potential. The amount of transmitter secreted (quantal size) is frequency dependent. The quantal-vesicular-exocytotic (QVE) hypothesis posits that the packet of secreted transmitter is released from one vesicle by exocytosis. The QVE hypothesis neither explains two quantal classes and subunits nor exocytosis of only one vesicle at each site. The latter observation requires a mechanism to select one vesicle from each array. Our porocytosis hypothesis states that the quantal packet is pulsed from an array of secretory pores. A salt shaker delivers a standard pinch of salt with each shake because salt flows through all openings in the cap. The variation in the pinch of salt or transmitter decreases with an increase in array size. The docked vesicles, paravesicular matrix, and porosomes (pores) of a release site form the secretory unit. In analogy with the sacromere as the functional unit of skeletal muscle, we term the array of docked vesicles and paravesicular grid along with the array of postsynaptic receptors a synaptomere. Pulsed secretion from an array explains the substructure of the postsynaptic response (quantum). The array guarantees a constant amount of secretion with each action potential and permits a given synapse to function in different responses because different frequencies would secrete signature amounts of transmitter. Our porocytosis hypothesis readily explains a change in quantal size during learning and memory with an increase in the number of elements (docked vesicles) composing the array.     

Exocytotic insertion of calcium channels constrains compensatory endocytosis to sites of exocytosis

Proteins inserted into the cell surface by exocytosis are thought to be retrieved by compensatory endocytosis, suggesting that retrieval requires granule proteins. In sea urchin eggs, calcium influx through P-type calcium channels is required for retrieval, and the large size of sea urchin secretory granules permits the direct observation of retrieval. Here we demonstrate that retrieval is limited to sites of prior exocytosis. We tested whether channel distribution can account for the localization of retrieval at exocytotic sites. We find that P-channels reside on secretory granules before fertilization, and are translocated to the egg surface by exocytosis. Our study provides strong evidence that the transitory insertion of P-type calcium channels in the surface membrane plays an obligatory role in the mechanism coupling exocytosis and compensatory endocytosis.     

Interactions between proteins implicated in exocytosis and voltage-gated calcium channels

Neurotransmitter release from synaptic vesicles is triggered by voltage-gated calcium influx through P/Q-type or N-type calcium channels. Purification of N-type channels from rat brain synaptosomes initially suggested molecular interactions between calcium channels and two key proteins implicated in exocytosis: synaptotagmin I and syntaxin 1. Co-immunoprecipitation experiments were consistent with the hypothesis that both N- and P/Q-type calcium channels, but not L-type channels, are associated with the 7S complex containing syntaxin 1, SNAP-25, VAMP and synaptotagmin I or II. Immunofluorescence confocal microscopy at the frog neuromuscular junction confirmed that calcium channels, syntaxin 1 and SNAP-25 are co-localized at active zones of the presynaptic plasma membrane where transmitter release occurs. Experiments with recombinant proteins were performed to map synaptic protein interaction sites on the alpha 1A subunit, which forms the pore of the P/Q-type calcium channel. In vitro-translated 35S-synaptotagmin I bound to a site located on the cytoplasmic loop linking homologous domains II and III of the alpha 1A subunit. This direct link would target synaptotagmin, a putative calcium sensor for exocytosis, to a microdomain of calcium influx close to the channel mouth. Cysteine string proteins (CSPs) contain a J-domain characteristic of molecular chaperones that cooperate with Hsp70. They are located on synaptic vesicles and thought to be involved in modulating the activity of presynaptic calcium channels. CSPs were found to bind to the same domain of the calcium channel as synaptotagmin, and also to associate with VAMP. CSPs may act as molecular chaperones in association with Hsp70 to direct assembly or dissociation of multiprotein complexes at the calcium channel.     

Strength of synaptic transmission at neuromuscular junction of crustaceans and insects in relation to calcium entry

Crustacean and insect neuromuscular junctions typically include numerous small synapses, each of which usually contains one or more active zones, which possess voltage-sensitive calcium channels and are specialized for release of synaptic vesicles. Strength of transmission (the number of quantal units released per synapse by a nerve impulse) varies greatly among different endings of individual neurons, and from one neuron to another. Ultrastructural features of synapses account for some of the physiological differences at endings of individual neurons. The nerve terminals that release more neurotransmitter per impulse have a higher incidence of synapses with more than one active zone, and this is correlated with more calcium build-up during stimulation. However, comparison of synaptic structure in neurons with different physiological phenotypes indicates no major differences in structure that could account for their different levels of neurotransmitter release per impulse, and release per synapse differs among neurons despite similar calcium build-up in their terminals during stimulation. The evidence indicates differences in calcium sensitivity of the release process among neurons as an aspect of physiological specialization.     

Low pH inhibits compensatory endocytosis at a step between depolarization and calcium influx

Cell function can be modulated by the insertion and removal of ion channels from the cell surface. The mechanism used to keep channels quiescent prior to delivery to the cell surface is not known. In eggs, cortical vesicle exocytosis inserts voltage-gated calcium channels into the cell surface. Calcium influx through these channels triggers compensatory endocytosis. Secretory vesicles contain high concentrations of calcium and hydrogen ions. We propose that lumenal hydrogen ions inhibit vesicular calcium channel gating prior to exocytosis, discharge of lumenal protons upon vesicle-plasma membrane fusion enables calcium channel gating. Consistent with this hypothesis we find that cortical vesicle lumens are acidic, and exocytosis releases lumenal hydrogen ions. Acidic extracellular pH reversibly blocks endocytosis, and the windows of opportunity for inhibition with a calcium-channel blocker or hydrogen ions are indistinguishable. Calcium ionophore treatment circumvents the low pH block, suggesting that calcium influx, or an upstream step, is obstructed. Inhibition of calcium influx by preventing membrane depolarization is unlikely, as elevation of the extracellular potassium concentration failed to overcome the pH block, and low extracellular pH was found to depolarize the membrane potential. We conclude that low pH inhibits endocytosis at a step between membrane depolarization and calcium influx .     

Molecular Mechanism of Active Zone Organization at Vertebrate Neuromuscular Junctions

Organization of presynaptic active zones is essential for development, plasticity, and pathology of the nervous system. Recent studies indicate a trans-synaptic molecular mechanism that organizes the active zones by connecting the pre- and the postsynaptic specialization. The presynaptic component of this trans-synaptic mechanism is comprised of cytosolic active zone proteins bound to the cytosolic domains of voltage-dependent calcium channels (P/Q-, N-, and L-type) on the presynaptic membrane. The postsynaptic component of this mechanism is the synapse organizer (laminin β2) that is expressed by the postsynaptic cell and accumulates specifically on top of the postsynaptic specialization. The pre- and the postsynaptic components interact directly between the extracellular domains of calcium channels and laminin β2 to anchor the presynaptic protein complex in front of the postsynaptic specialization. Hence, the presynaptic calcium channel functions as a scaffolding protein for active zone organization and as an ion-conducting channel for synaptic transmission. In contrast to the requirement of calcium influx for synaptic transmission, the formation of the active zone does not require the calcium influx through the calcium channels. Importantly, the active zones of adult synapses are not stable structures and require maintenance for their integrity. Furthermore, aging or diseases of the central and peripheral nervous system impair the active zones. This review will focus on the molecular mechanisms that organize the presynaptic active zones and summarize recent findings at the neuromuscular junctions and other synapses.     

Low-threshold exocytosis induced by cAMP-recruited CaV3.2 (alpha1H) channels in rat chromaffin cells

We have studied the functional role of CaV3 channels in triggering fast exocytosis in rat chromaffin cells (RCCs). CaV3 T-type channels were selectively recruited by chronic exposures to cAMP (3 days) via an exchange protein directly activated by cAMP (Epac)-mediated pathway. Here we show that cAMP-treated cells had increased secretory responses, which could be evoked even at very low depolarizations (-50, -40 mV). Potentiation of exocytosis in cAMP-treated cells did not occur in the presence of 50 microM Ni2+, which selectively blocks T-type currents in RCCs. This suggests that the "low-threshold exocytosis" induced by cAMP is due to increased Ca2+ influx through cAMP-recruited T-type channels, rather than to an enhanced secretion downstream of Ca2+ entry, as previously reported for short-term cAMP treatments (20 min). Newly recruited T-type channels increase the fast secretory response at low voltages without altering the size of the immediately releasable pool. They also preserve the Ca2+ dependence of exocytosis, the initial speed of vesicle depletion, and the mean quantal size of single secretory events. All this indicates that cAMP-recruited CaV3 channels enhance the secretory activity of RCCs at low voltages by coupling to the secretory apparatus with a Ca2+ efficacy similar to that of already existing high-threshold Ca2+ channels. Finally, using RT-PCRs we found that the fast inactivating low-threshold Ca2+ current component recruited by cAMP is selectively associated to the alpha1H (CaV3.2) channel isoform.