Single calcium channel domain gating of synaptic vesicle fusion at fast synapses; analysis by graphic modeling

At fast-transmitting presynaptic terminals Ca²⁺ enter through voltage gated calcium channels (CaVs) and bind to a synaptic vesicle (SV) -associated calcium sensor (SV-sensor) to gate fusion and discharge. An open CaV generates a high-concentration plume, or nanodomain of Ca²⁺ that dissipates precipitously with distance from the pore. At most fast synapses, such as the frog neuromuscular junction (NMJ), the SV sensors are located sufficiently close to individual CaVs to be gated by single nanodomains. However, at others, such as the mature rodent calyx of Held (calyx of Held), the physiology is more complex with evidence that CaVs that are both close and distant from the SV sensor and it is argued that release is gated primarily by the overlapping Ca²⁺ nanodomains from many CaVs. We devised a 'graphic modeling' method to sum Ca²⁺ from individual CaVs located at varying distances from the SV-sensor to determine the SV release probability and also the fraction of that probability that can be attributed to single domain gating. This method was applied first to simplified, low and high CaV density model release sites and then to published data on the contrasting frog NMJ and the rodent calyx of Held native synapses. We report 3 main predictions: the SV-sensor is positioned very close to the point at which the SV fuses with the membrane; single domain-release gating predominates even at synapses where the SV abuts a large cluster of CaVs, and even relatively remote CaVs can contribute significantly to single domain-based gating.     

An Exclusion Zone for Ca2+ Channels around Docked Vesicles Explains Release Control by Multiple Channels at a CNS Synapse

The spatial arrangement of Ca²⁺ channels and vesicles remains unknown for most CNS synapses, despite of the crucial importance of this geometrical parameter for the Ca²⁺ control of transmitter release. At a large model synapse, the calyx of Held, transmitter release is controlled by several Ca²⁺ channels in a "domain overlap" mode, at least in young animals. To study the geometrical constraints of Ca²⁺ channel placement in domain overlap control of release, we used stochastic MCell modelling, at active zones for which the position of docked vesicles was derived from electron microscopy (EM). We found that random placement of Ca²⁺ channels was unable to produce high slope values between release and presynaptic Ca²⁺ entry, a hallmark of domain overlap, and yielded excessively large release probabilities. The simple assumption that Ca²⁺ channels can be located anywhere at active zones, except below a critical distance of ~ 30 nm away from docked vesicles ("exclusion zone"), rescued high slope values and low release probabilities. Alternatively, high slope values can also be obtained by placing all Ca²⁺ channels into a single supercluster, which however results in significantly higher heterogeneity of release probabilities. We also show experimentally that high slope values, and the sensitivity to the slow Ca²⁺ chelator EGTA-AM, are maintained with developmental maturation of the calyx synapse. Taken together, domain overlap control of release represents a highly organized active zone architecture in which Ca²⁺ channels must obey a certain distance to docked vesicles. Furthermore, domain overlap can be employed by near-mature, fast-releasing synapses.     

RIM‐binding proteins recruit BK‐channels to presynaptic release sites adjacent to voltage‐gated Ca2+‐channels

The active zone of presynaptic nerve terminals organizes the neurotransmitter release machinery, thereby enabling fast Ca²⁺‐triggered synaptic vesicle exocytosis. BK‐channels are Ca²⁺‐activated large‐conductance K⁺‐channels that require close proximity to Ca²⁺‐channels for activation and control Ca²⁺‐triggered neurotransmitter release by accelerating membrane repolarization during action potential firing. How BK‐channels are recruited to presynaptic Ca²⁺‐channels, however, is unknown. Here, we show that RBPs (for RIM‐binding proteins), which are evolutionarily conserved active zone proteins containing SH3‐ and FN3‐domains, directly bind to BK‐channels. We find that RBPs interact with RIMs and Ca²⁺‐channels via their SH3‐domains, but to BK‐channels via their FN3‐domains. Deletion of RBPs in calyx of Held synapses decreased and decelerated presynaptic BK‐currents and depleted BK‐channels from active zones. Our data suggest that RBPs recruit BK‐channels into a RIM‐based macromolecular active zone complex that includes Ca²⁺‐channels, synaptic vesicles, and the membrane fusion machinery, thereby enabling tight spatio‐temporal coupling of Ca²⁺‐influx to Ca²⁺‐triggered neurotransmitter release in a presynaptic terminal.     

An Atypical Role for Collapsin Response Mediator Protein 2 (CRMP-2) in Neurotransmitter Release via Interaction with Presynaptic Voltage-gated Calcium Channels

Collapsin response mediator proteins (CRMPs) specify axon/dendrite fate and axonal growth of neurons through protein-protein interactions. Their functions in presynaptic biology remain unknown. Here, we identify the presynaptic N-type Ca²⁺ channel (CaV2.2) as a CRMP-2-interacting protein. CRMP-2 binds directly to CaV2.2 in two regions: the channel domain I-II intracellular loop and the distal C terminus. Both proteins co-localize within presynaptic sites in hippocampal neurons. Overexpression in hippocampal neurons of a CRMP-2 protein fused to enhanced green fluorescent protein caused a significant increase in Ca²⁺ channel current density, whereas lentivirus-mediated CRMP-2 knockdown abolished this effect. Interestingly, the increase in Ca²⁺ current density was not due to a change in channel gating. Rather, cell surface biotinylation studies showed an increased number of CaV2.2 at the cell surface in CRMP-2-overexpressing neurons. These neurons also exhibited a significant increase in vesicular release in response to a depolarizing stimulus. Depolarization of CRMP-2-enhanced green fluorescent protein-overexpressing neurons elicited a significant increase in release of glutamate compared with control neurons. Toxin block of Ca²⁺ entry via CaV2.2 abolished this stimulated release. Thus, the CRMP-2-Ca²⁺ channel interaction represents a novel mechanism for modulation of Ca²⁺ influx into nerve terminals and, hence, of synaptic strength.     

Gating Kinetics of Single Large-Conductance Ca2+-Activated K+ Channels in High Ca2+ Suggest a Two-Tiered Allosteric Gating Mechanism?

The Ca²⁺-dependent gating mechanism of large-conductance calcium-activated K⁺ (BK) channels from cultured rat skeletal muscle was examined from low (4 μM) to high (1,024 μM) intracellular concentrations of calcium (Ca²⁺ _i) using single-channel recording. Open probability (P _o) increased with increasing Ca²⁺ _i (K _0.5 11.2 ± 0.3 μM at +30 mV, Hill coefficient of 3.5 ± 0.3), reaching a maximum of ∼0.97 for Ca²⁺ _i ∼ 100 μM. Increasing Ca²⁺ _i further to 1,024 μM had little additional effect on either P _o or the single-channel kinetics. The channels gated among at least three to four open and four to five closed states at high levels of Ca²⁺ _i (>100 μM), compared with three to four open and five to seven closed states at lower Ca²⁺ _i. The ability of kinetic schemes to account for the single-channel kinetics was examined with simultaneous maximum likelihood fitting of two-dimensional (2-D) dwell-time distributions obtained from low to high Ca²⁺ _i. Kinetic schemes drawn from the 10-state Monod-Wyman-Changeux model could not describe the dwell-time distributions from low to high Ca²⁺ _i. Kinetic schemes drawn from Eigen''s general model for a ligand-activated tetrameric protein could approximate the dwell-time distributions but not the dependency (correlations) between adjacent intervals at high Ca²⁺ _i. However, models drawn from a general 50 state two-tiered scheme, in which there were 25 closed states on the upper tier and 25 open states on the lower tier, could approximate both the dwell-time distributions and the dependency from low to high Ca²⁺ _i. In the two-tiered model, the BK channel can open directly from each closed state, and a minimum of five open and five closed states are available for gating at any given Ca²⁺ _i. A model that assumed that the apparent Ca²⁺-binding steps can reach a maximum rate at high Ca²⁺ _i could also approximate the gating from low to high Ca²⁺ _i. The considered models can serve as working hypotheses for the gating of BK channels.     

Two ryanodine receptor isoforms in nonmammalian vertebrate skeletal muscle: possible roles in excitation-contraction coupling and other processes

The ryanodine receptor (RyR) is a Ca²⁺ release channel in the sarcoplasmic reticulum in vertebrate skeletal muscle and plays an important role in excitation–contraction (E–C) coupling. Whereas mammalian skeletal muscle predominantly expresses a single RyR isoform, RyR1, skeletal muscle of many nonmammalian vertebrates expresses equal amounts of two distinct isoforms, α-RyR and β-RyR, which are homologues of mammalian RyR1 and RyR3, respectively. In this review we describe our current understanding of the functions of these two RyR isoforms in nonmammalian vertebrate skeletal muscle. The Ca²⁺ release via the RyR channel can be gated by two distinct modes: depolarization-induced Ca²⁺ release (DICR) and Ca²⁺-induced Ca²⁺ release (CICR). In frog muscle, α-RyR acts as the DICR channel, whereas β-RyR as the CICR channel. However, several lines of evidence suggest that CICR by β-RyR may make only a minor contribution to Ca²⁺ release during E–C coupling. Comparison of frog and mammalian RyR isoforms highlights the marked differences in the patterns of Ca²⁺ release mediated by RyR1 and RyR3 homologues. Interestingly, common features in the Ca²⁺ release patterns are noticed between β-RyR and RyR1. We will discuss possible roles and significance of the two RyR isoforms in E–C coupling and other processes in nonmammalian vertebrate skeletal muscle.     

大电导钾离子通道(BK)结构与功能的研究进展

大电导钙离子激活钾通道(BK)是细胞膜上唯一接受细胞内Ca2+和膜电位双重调控的离子通道.最新发表的关于BK通道电镜结构及其胞质功能域的晶体结构的文章,第一次展示了BK通道各亚基的组装,并证实通道各功能域在通道门控机制中存在紧密的相互作用.近年来,针对BK通道的功能调节及其门控动力学模拟的研究取得较多进展,有助于更好地理解BK通道发挥生理功能的门控机制,并揭示BK通道相关疾病的病理生理学基础.     

A sensory cell diversifies its output by varying Ca2+ influx‐release coupling among active zones

The cochlea encodes sound pressures varying over six orders of magnitude by collective operation of functionally diverse spiral ganglion neurons (SGNs). The mechanisms enabling this functional diversity remain elusive. Here, we asked whether the sound intensity information, contained in the receptor potential of the presynaptic inner hair cell (IHC), is fractionated via heterogeneous synapses. We studied the transfer function of individual IHC synapses by combining patch‐clamp recordings with dual‐color Rhod‐FF and iGluSnFR imaging of presynaptic Ca²⁺ signals and glutamate release. Synapses differed in the voltage dependence of release: Those residing at the IHC'' pillar side activated at more hyperpolarized potentials and typically showed tight control of release by few Ca²⁺ channels. We conclude that heterogeneity of voltage dependence and release site coupling of Ca²⁺ channels among the synapses varies synaptic transfer within individual IHCs and, thereby, likely contributes to the functional diversity of SGNs. The mechanism reported here might serve sensory cells and neurons more generally to diversify signaling even in close‐by synapses.     

Divergence of Ca2+ selectivity and equilibrium Ca2+ blockade in a Ca2+ release-activated Ca2+ channel

Prevailing models postulate that high Ca²⁺ selectivity of Ca²⁺ release-activated Ca²⁺ (CRAC) channels arises from tight Ca²⁺ binding to a high affinity site within the pore, thereby blocking monovalent ion flux. Here, we examined the contribution of high affinity Ca²⁺ binding for Ca²⁺ selectivity in recombinant Orai3 channels, which function as highly Ca²⁺-selective channels when gated by the endoplasmic reticulum Ca²⁺ sensor STIM1 or as poorly Ca²⁺-selective channels when activated by the small molecule 2-aminoethoxydiphenyl borate (2-APB). Extracellular Ca²⁺ blocked Na⁺ currents in both gating modes with a similar inhibition constant (K_i; ∼25 µM). Thus, equilibrium binding as set by the K_i of Ca²⁺ blockade cannot explain the differing Ca²⁺ selectivity of the two gating modes. Unlike STIM1-gated channels, Ca²⁺ blockade in 2-APB–gated channels depended on the extracellular Na⁺ concentration and exhibited an anomalously steep voltage dependence, consistent with enhanced Na⁺ pore occupancy. Moreover, the second-order rate constants of Ca²⁺ blockade were eightfold faster in 2-APB–gated channels than in STIM1-gated channels. A four-barrier, three–binding site Eyring model indicated that lowering the entry and exit energy barriers for Ca²⁺ and Na⁺ to simulate the faster rate constants of 2-APB–gated channels qualitatively reproduces their low Ca²⁺ selectivity, suggesting that ion entry and exit rates strongly affect Ca²⁺ selectivity. Noise analysis indicated that the unitary Na⁺ conductance of 2-APB–gated channels is fourfold larger than that of STIM1-gated channels, but both modes of gating show a high open probability (P_o; ∼0.7). The increase in current noise during channel activation was consistent with stepwise recruitment of closed channels to a high P_o state in both cases, suggesting that the underlying gating mechanisms are operationally similar in the two gating modes. These results suggest that both high affinity Ca²⁺ binding and kinetic factors contribute to high Ca²⁺ selectivity in CRAC channels.     

Presynaptic active zones in invertebrates and vertebrates

The regulated release of neurotransmitter occurs via the fusion of synaptic vesicles (SVs) at specialized regions of the presynaptic membrane called active zones (AZs). These regions are defined by a cytoskeletal matrix assembled at AZs (CAZ), which functions to direct SVs toward docking and fusion sites and supports their maturation into the readily releasable pool. In addition, CAZ proteins localize voltage‐gated Ca²⁺ channels at SV release sites, bringing the fusion machinery in close proximity to the calcium source. Proteins of the CAZ therefore ensure that vesicle fusion is temporally and spatially organized, allowing for the precise and reliable release of neurotransmitter. Importantly, AZs are highly dynamic structures, supporting presynaptic remodeling, changes in neurotransmitter release efficacy, and thus presynaptic forms of plasticity. In this review, we discuss recent advances in the study of active zones, highlighting how the CAZ molecularly defines sites of neurotransmitter release, endocytic zones, and the integrity of synapses.     

The calyx of Held

The calyx of Held is a large glutamatergic synapse in the mammalian auditory brainstem. By using brain slice preparations, direct patch-clamp recordings can be made from the nerve terminal and its postsynaptic target (principal neurons of the medial nucleus of the trapezoid body). Over the last decade, this preparation has been increasingly employed to investigate basic presynaptic mechanisms of transmission in the central nervous system. We review here the background to this preparation and summarise key findings concerning voltage-gated ion channels of the nerve terminal and the ionic mechanisms involved in exocytosis and modulation of transmitter release. The accessibility of this giant terminal has also permitted Ca²⁺-imaging and -uncaging studies combined with electrophysiological recording and capacitance measurements of exocytosis. Together, these studies convey the panopoly of presynaptic regulatory processes underlying the regulation of transmitter release, its modulatory control and short-term plasticity within one identified synaptic terminal.     

The Gating Kinetics of the Slow Vacuolar Channel. A Novel Mechanism for SV Channel Functioning?

Although there is consensus that the slow vacuolar or SV channel is a Ca²⁺ release channel, the underlying mechanism of operation is still controversial. The main reason is that the voltage sensitivity of SV gating seems to exclude activation at hyperpolarized (physiological) membrane potentials. Inspired by a study of Gambale et al. (1993) and supported by simulation studies presented here, we interpreted SV activation and deactivation kinetics in terms of a cyclic state diagram originally applied to animal cation-selective channels. A cyclic state diagram allows two pathways of activation operating in opposite directions. One pathway represents the frequently observed slow activation at moderate depolarization (<130 mV). With the open state (O) next to the closed state initially occupied (C ₁), direct transitions from C ₁ to O can account for the fast activation observed at higher depolarized potentials (>130 mV). We hypothesize that similar state transitions directly to O may also occur during hyperpolarization. The implication of this proposed mechanism is that SV accomplishes its physiological role during hyperpolarization-evoked deactivation. Despite their rare occurrence and possibly short duration, these opening events may last long enough to substantially raise the local cytosolic free Ca²⁺ level at the channel mouth by as much as 600 nM/ms. Because under in vivo conditions the Ca²⁺ flux is inwardly directed, the mechanism presented here revives the notion that the SV channel can be subject to calcium-induced calcium release. Present address for H. M.: BioMade Technology Foundation, Nijenborgh 4, 9747 AG Groningen, The Netherlands     

Ion Channel Selectivity through Stepwise Changes in Binding Affinity

Voltage-gated Ca²⁺ channels select Ca²⁺ over competing, more abundant ions by means of a high affinity binding site in the pore. The maximum off rate from this site is ∼1,000× slower than observed Ca²⁺ current. Various theories that explain how high Ca²⁺ current can pass through such a sticky pore all assume that flux occurs from a condition in which the pore''s affinity for Ca²⁺ transiently decreases because of ion interactions. Here, we use rate theory calculations to demonstrate a different mechanism that requires no transient changes in affinity to quantitatively reproduce observed Ca²⁺ channel behavior. The model pore has a single high affinity Ca²⁺ binding site flanked by a low affinity site on either side; ions permeate in single file without repulsive interactions. The low affinity sites provide steps of potential energy that speed the exit of a Ca²⁺ ion off the selectivity site, just as potential energy steps accelerate other chemical reactions. The steps could be provided by weak binding in the nonselective vestibules that appear to be a general feature of ion channels, by specific protein structures in a long pore, or by stepwise rehydration of a permeating ion. The previous ion-interaction models and this stepwise permeation model demonstrate two general mechanisms, which might well work together, to simultaneously generate high flux and high selectivity in single file pores.     

Separation of Gating Properties from Permeation and Block in mslo Large Conductance Ca-activated K+ Channels

In this and the following paper we have examined the kinetic and steady-state properties of macroscopic mslo Ca-activated K⁺ currents in order to interpret these currents in terms of the gating behavior of the mslo channel. To do so, however, it was necessary to first find conditions by which we could separate the effects that changes in Ca²⁺ concentration or membrane voltage have on channel permeation from the effects these stimuli have on channel gating. In this study we investigate three phenomena which are unrelated to gating but are manifest in macroscopic current records: a saturation of single channel current at high voltage, a rapid voltage-dependent Ca²⁺ block, and a slow voltage-dependent Ba²⁺ block. Where possible methods are described by which these phenomena can be separated from the effects that changes in Ca²⁺ concentration and membrane voltage have on channel gating. Where this is not possible, some assessment of the impact these effects have on gating parameters determined from macroscopic current measurements is provided. We have also found that without considering the effects of Ca²⁺ and voltage on channel permeation and block, macroscopic current measurements suggest that mslo channels do not reach the same maximum open probability at all Ca²⁺ concentrations. Taking into account permeation and blocking effects, however, we find that this is not the case. The maximum open probability of the mslo channel is the same or very similar over a Ca²⁺ concentration range spanning three orders of magnitude indicating that over this range the internal Ca²⁺ concentration does not limit the ability of the channel to be activated by voltage.     

高锶离子亲和力传感蛋白介导CALYX突触囊泡异步和自发释放

突触囊泡在钙离子(Ca2+)触发下释放神经递质普遍存在着同步和异步两种形式.突触囊泡膜蛋白(synaptotagmin 2,Syt-2)已被证实是Calyx of Held突触囊泡同步释放的Ca2+传感蛋白,而相关的异步释放Ca2+传感蛋白还有待于探索.虽然锶离子(Sr2+)因其物理和化学性质都接近Ca2+,且能触发更多的囊泡异步释放成分而成为研究异步释放机制的常用工具,但有关Sr2+触发异步释放的机制存在着争议.本文在胞外以Sr2+替换Ca2+的条件下,通过对野生型(WT)和Syt-2敲除型(Z2B-/-)小鼠Calyx突触囊泡自发和诱发释放的电生理特性分析,发现Syt-2是介导Sr2+诱发的突触囊泡快速释放的传感蛋白,但不是介导Sr2+相关神经递质异步释放和自发释放的传感蛋白;而未知的触发囊泡异步释放的传感蛋白相比Syt-2对Sr2+具有更高的亲和力,同时也介导突触囊泡的自发释放.这一研究为探索并最终发现触发囊泡异步释放的未知传感蛋白提供了新的线索.     

Slowly activating vacuolar channels can not mediate Ca2+-induced Ca2+ release

In order to test the hypothesis that slowly activating vacuolar (SV) channels mediate Ca²⁺-induced Ca²⁺ release the voltage- and Ca²⁺-dependence of these K⁺ and Ca²⁺- permeable channels were studied in a quantitative manner. The patch-clamp technique was applied to barley (Hordeum vulgare L.) mesophyll vacuoles in the whole vacuole and vacuolar-free patch configuration. Under symmetrical ionic conditions the current-voltage relationship of the open SV channel was characterized by a pronounced inward rectification. The single channel current amplitude was not affected by changes in cytosolic Ca²⁺ whereas an increase in vacuolar Ca²⁺ decreased the unitary current in a voltage-dependent manner. The SV channel open-probability increased with positive potentials and elevated cytosolic Ca²⁺, but not with elevated cytosolic Mg²⁺. An increase of cytosolic Ca²⁺ shifted the half-activation potential to more negative voltages, whereas an increase of vacuolar Ca²⁺ shifted the half-activation potential to more positive voltages. At physiological vacuolar Ca²⁺ activities (50 μM to 2 mM) changes in cytosolic Ca²⁺ (5 μM to 2 mM) revealed an exponential dependence of the SV channel open-probability on the electrochemical potential gradient for Ca²⁺ (Δμ_Ca). At the Ca²⁺ equilibrium potential (Δμ_Ca = 0) the open-probability was as low as 0.4%. Higher open-probabilities required net Ca²⁺ motive forces which would drive Ca²⁺ influx into the vacuole. Under conditions favouring Ca²⁺ release from the vacuole, however, the open-probability further decreased. Based on quantitative analysis, it was concluded that the SV channel is not suited for Ca²⁺-induced Ca²⁺ release from the vacuole.     

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.     

Differential contribution of EF-hands to the Ca²⁺-dependent activation in the plant two-pore channel TPC1

Two‐pore channels (TPC) have been established as components of calcium signalling networks in plants and animals. In plants, TPC1 in the vacuolar membrane is gated open upon binding of calcium in a voltage‐dependent manner. Here, we analyzed the molecular mechanism of the Ca²⁺‐dependent activity of TPC1 from Arabidopsis thaliana, using site‐directed mutagenesis of its two canonical EF‐hands. Wild‐type TPC1 and TPC1‐D335A with a mutated first Ca²⁺ ligand in EF‐hand 1 produced channels that retained their voltage‐ and Ca²⁺‐dependent gating characteristics, but were less sensitive at Ca²⁺ concentrations <200 μm . Additional mutation of the first Ca²⁺ ligand in EF‐hand 2 resulted in silent TPC1‐D335A/D376A channels. Similarly, the single mutant TPC1‐D376A could not be activated up to 1 mm Ca²⁺, indicating that the second EF‐hand is essential for the Ca²⁺‐dependent channel gating. Molecular modeling suggests that EF‐hand 1 displays a low‐affinity Ca²⁺/Mg²⁺‐binding site, while EF‐hand 2 represents a high‐affinity Ca²⁺‐binding site. Together, our data prove that EF‐hand 2 is responsible for the Ca²⁺‐receptor characteristics of TPC1, while EF‐hand 1 is a structural site required to enable channel responses at physiological changes in Ca²⁺ concentration.     

Close agreement between deterministic versus stochastic modeling of first-passage time to vesicle fusion

Ca²⁺-dependent cell processes, such as neurotransmitter or endocrine vesicle fusion, are inherently stochastic due to large fluctuations in Ca²⁺ channel gating, Ca²⁺ diffusion, and Ca²⁺ binding to buffers and target sensors. However, previous studies revealed closer-than-expected agreement between deterministic and stochastic simulations of Ca²⁺ diffusion, buffering, and sensing if Ca²⁺ channel gating is not Ca²⁺ dependent. To understand this result more fully, we present a comparative study complementing previous work, focusing on Ca²⁺ dynamics downstream of Ca²⁺ channel gating. Specifically, we compare deterministic (mean-field/mass-action) and stochastic simulations of vesicle exocytosis latency, quantified by the probability density of the first-passage time (FPT) to the Ca²⁺-bound state of a vesicle fusion sensor, following a brief Ca²⁺ current pulse. We show that under physiological constraints, the discrepancy between FPT densities obtained using the two approaches remains small even if as few as ～50 Ca²⁺ ions enter per single channel-vesicle release unit. Using a reduced two-compartment model for ease of analysis, we illustrate how this close agreement arises from the smallness of correlations between fluctuations of the reactant molecule numbers, despite the large magnitude of fluctuation amplitudes. This holds if all relevant reactions are heteroreaction between molecules of different species, as is the case for bimolecular Ca²⁺ binding to buffers and downstream sensor targets. In this case, diffusion and buffering effectively decorrelate the state of the Ca²⁺ sensor from local Ca²⁺ fluctuations. Thus, fluctuations in the Ca²⁺ sensor’s state underlying the FPT distribution are only weakly affected by the fluctuations in the local Ca²⁺ concentration around its average, deterministically computable value.     

Spatial and temporal crosstalk between the cAMP and Ca2+ signaling systems

The ubiquitous secondary messengers, Ca²⁺ and cAMP, play a vital role in shaping a diverse array of physiological processes. More significantly, accumulating evidence over the past several decades underpin extensive crosstalk between these two canonical messengers in discrete sub-cellular nanodomains across various cell types. Within such specialized nanodomains, each messenger fine-tunes signaling to maintain homeostasis by manipulating the activities of cellular machinery accountable for the metabolism or activity of the complementary pathway. Interaction between these messengers is ensured by scaffolding proteins which tether components of the signaling machinery in close proximity. Disruption of dynamic communications between Ca²⁺ and cAMP at these loci consequently is linked to several pathological conditions. This review summarizes recent novel mechanisms underlying effective crosstalk between Ca²⁺ and cAMP in such nanodomains.