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.     

Regulation of Synaptic Transmission by Presynaptic CaMKII and BK Channels

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and the BK channel are enriched at the presynaptic nerve terminal, where CaMKII associates with synaptic vesicles whereas the BK channel colocalizes with voltage-sensitive Ca²⁺ channels in the plasma membrane. Mounting evidence suggests that these two proteins play important roles in controlling neurotransmitter release. Presynaptic BK channels primarily serve as a negative regulator of neurotransmitter release. In contrast, presynaptic CaMKII either enhances or inhibits neurotransmitter release and synaptic plasticity depending on experimental or physiological conditions and properties of specific synapses. The different functions of presynaptic CaMKII appear to be mediated by distinct downstream proteins, including the BK channel.     

Lipid rafts/caveolae as microdomains of calcium signaling

Ca²⁺ is a major signaling molecule in both excitable and non-excitable cells, where it serves critical functions ranging from cell growth to differentiation to cell death. The physiological functions of these cells are tightly regulated in response to changes in cytosolic Ca²⁺ that is achieved by the activation of several plasma membrane (PM) Ca²⁺ channels as well as release of Ca²⁺ from the internal stores. One such channel is referred to as store-operated Ca²⁺ channel that is activated by the release of endoplasmic reticulum (ER) Ca²⁺ which initiates store-operated Ca²⁺ entry (SOCE). Recent advances in the field suggest that some members of TRPCs and Orai channels function as SOCE channels. However, the molecular mechanisms that regulate channel activity and the exact nature of where these channels are assembled and regulated remain elusive. Research from several laboratories has demonstrated that key proteins involved in Ca²⁺ signaling are localized in discrete PM lipid rafts/caveolar microdomains. Lipid rafts are cholesterol and sphingolipid-enriched microdomains that function as unique signal transduction platforms. In addition lipid rafts are dynamic in nature which tends to scaffold certain signaling molecules while excluding others. By such spatial segregation, lipid rafts not only provide a favorable environment for intra-molecular cross-talk but also aid to expedite the signal relay. Importantly, Ca²⁺ signaling is shown to initiate from these lipid raft microdomains. Clustering of Ca²⁺ channels and their regulators in such microdomains can provide an exquisite spatiotemporal regulation of Ca²⁺-mediated cellular function. Thus in this review we discuss PM lipid rafts and caveolae as Ca²⁺-signaling microdomains and highlight their importance in organizing and regulating SOCE channels.     

Calcium microdomains in regulated exocytosis

Katz and co-workers showed that Ca²⁺ triggers exocytosis. The existence of sub-micrometer domains of greater than 100 μM [Ca²⁺]_i was postulated on theoretical grounds. Using a modified, low-affinity aequorin, Llinas et al. were the first to demonstrate the existence of Ca²⁺ ‘microdomains’ in squid presynaptic terminals. Over the past several years, it has become clear that individual Ca²⁺ nano- and microdomains forming around the mouth of voltage-gated Ca²⁺ channels ascertain the tight coupling of fast synaptic vesicle release to membrane depolarization by action potentials. Recent work has established different geometric arrangements of vesicles and Ca²⁺ channels at different central synapses and pointed out the role of Ca²⁺ syntillas – localized, store operated Ca²⁺ signals – in facilitation and spontaneous release. The coupling between Ca²⁺ increase and evoked exocytosis is more sluggish in peripheral terminals and neuroendocrine cells, where channels are less clustered and Ca²⁺ comes from different sources, including Ca²⁺ influx via the plasma membrane and the mobilization of Ca²⁺ from intracellular stores. Finally, also non- (electrically) excitable cells display highly localized Ca²⁺ signaling domains. We discuss in particular the organization of structural microdomains of Bergmann glia, specialized astrocytes of the cerebellum that have only recently been considered as secretory cells. Glial microdomains are the spatial substrate for functionally segregated Ca²⁺ signals upon metabotropic activation. Our review emphasizes the large diversity of different geometric arrangements of vesicles and Ca²⁺ sources, leading to a wide spectrum of Ca²⁺ signals triggering release.     

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.     

A quaternary SNARE-synaptotagmin-Ca2+-phospholipid complex in neurotransmitter release

The function of synaptotagmin as a Ca²⁺ sensor in neurotransmitter release involves Ca²⁺-dependent phospholipid binding to its two C₂ domains, but this activity alone does not explain why Ca²⁺ binding to the C₂B domain is more critical for release than Ca²⁺ binding to the C₂A domain. Synaptotagmin also binds to SNARE complexes, which are central components of the membrane fusion machinery, and displaces complexins from the SNAREs. However, it is unclear how phospholipid binding to synaptotagmin is coupled to SNARE binding and complexin displacement. Using supported lipid bilayers deposited within microfluidic channels, we now show that Ca²⁺ induces simultaneous binding of synaptotagmin to phospholipid membranes and SNARE complexes, resulting in an intimate quaternary complex that we name SSCAP complex. Mutagenesis experiments show that Ca²⁺ binding to the C₂B domain is critical for SSCAP complex formation and displacement of complexin, providing a clear rationale for the preponderant role of the C₂B domain in release. This and other correlations between the effects of mutations on SSCAP complex formation and their functional effects in vivo suggest a key role for this complex in release. We propose a model whereby the highly positive electrostatic potential at the tip of the SSCAP complex helps to induce membrane fusion during release.     

Imaging single-channel calcium microdomains

The Ca²⁺ microdomains generated around the mouth of open ion channels represent the basic building blocks from which cytosolic Ca²⁺ signals are constructed. Recent improvements in optical imaging techniques now allow these microdomains to be visualized as single channel calcium fluorescence transients (SCCaFTs), providing information about channel properties that was previously accessible only by electrophysiological patch-clamp recordings. We review recent advances in single channel Ca²⁺ imaging methodologies, with emphasis on total internal reflection fluorescence microscopy (TIRFM) as the technique of choice for recording SCCaFTs from voltage- and ligand-gated plasmalemmal ion channels. This technique of ‘optical patch-clamp recording’ is massively parallel, permitting simultaneous imaging of hundreds of channels; provides millisecond resolution of gating kinetics together with sub-micron spatial resolution of channel locations; and is applicable to diverse families of membrane channels that display partial permeability to Ca²⁺ ions.     

Ca microdomains in smooth muscle

In smooth muscle, Ca²⁺ controls diverse activities including cell division, contraction and cell death. Of particular significance in enabling Ca²⁺ to perform these multiple functions is the cell's ability to localize Ca²⁺ signals to certain regions by creating high local concentrations of Ca²⁺ (microdomains), which differ from the cytoplasmic average. Microdomains arise from Ca²⁺ influx across the plasma membrane or release from the sarcoplasmic reticulum (SR) Ca²⁺ store. A single Ca²⁺ channel can create a microdomain of several micromolar near (200 nm) the channel. This concentration declines quickly with peak rates of several thousand micromolar per second when influx ends. The high [Ca²⁺] and the rapid rates of decline target Ca²⁺ signals to effectors in the microdomain with rapid kinetics and enable the selective activation of cellular processes. Several elements within the cell combine to enable microdomains to develop. These include the brief open time of ion channels, localization of Ca²⁺ by buffering, the clustering of ion channels to certain regions of the cell and the presence of membrane barriers, which restrict the free diffusion of Ca²⁺. In this review, the generation of microdomains arising from Ca²⁺ influx across the plasma membrane and the release of the ion from the SR Ca²⁺ store will be discussed and the contribution of mitochondria and the Golgi apparatus as well as endogenous modulators (e.g. cADPR and channel binding proteins) will be considered.     

Astrocyte calcium microdomains are inhibited by Bafilomycin A1 and cannot be replicated by low-level Schaffer collateral stimulation in situ

Astrocyte Gq GPCR and IP₃ receptor-dependent Ca²⁺ elevations occur spontaneously in situ and in vivo. These events vary considerably in size, often remaining confined to small territories of astrocyte processes called “microdomains” and sometimes propagating over longer distances that can include the soma. It has remained unclear whether these events are driven by constitutive (basal) GPCR signaling activity, neuronal action potential-dependent or quantal vesicular release, or some combination of these mechanisms. Here, we applied manipulations to increase or inhibit neuronal vesicular neurotransmitter release together with low-level stimulation of Schaffer collaterals in acute mouse hippocampal slices in an effort to determine the mechanisms underlying spontaneous astrocyte Ca²⁺ events. We found no significant change in spontaneous microdomain astrocyte Ca²⁺ elevations when neuronal action potentials were significantly enhanced or blocked. The astrocyte Ca²⁺ activity was also not affected by inhibitors of group I mGluRs. However, blockade of miniature neurotransmitter release using Bafilomycin A1 significantly reduced the frequency of microdomain astrocyte Ca²⁺ elevations. We then tested whether astrocyte Ca²⁺ microdomains can be evoked by low intensity SC stimulation. Importantly, microdomains could not be reproduced even using single, low intensity pulses to the SCs at a minimum distance from the astrocyte. Evoked astrocyte Ca²⁺ responses most often included the cell soma, were reduced by group I mGluR antagonists, and were larger in size compared to spontaneous Ca²⁺ microdomains. Overall, our findings suggest that spontaneous microdomain astrocyte Ca²⁺ elevations are not driven by neuronal action potentials but require quantal release of neurotransmitter which cannot be replicated by stimulation of Schaffer collaterals.     

Effect of phospholipid surface charge on the conductance and gating of a Ca2+-activated K+ channel in planar lipid bilayers

Summary A Ca-activated, K-selective channel from plasma membrane of rat skeletal muscle was studied in artificial lipid bilayers formed from either phosphatidylethanolamine (PE) or phosphatidylserine (PS). In PE, the single-channel conductance exhibited a complex dependence on symmetrical K⁺ concentration that could not be described by simple Michaelis-Menten saturation. At low K⁺ concentrations the channel conductance was higher in PS membranes, but approached the same conductance observed in PE above 0.4m KCl. At the same Ca²⁺ concentration and voltage, the probability of channel opening was significantly greater in PS than PE. The differences in the conduction and gating, observed in the two lipids, can be explained by the negative surface charge of PS compared to the neutral PE membrane. Model calculations of the expected concentrations of K⁺ and Ca²⁺ at various distances from a PS membrane surface, using Gouy-Chapman-Stern theory, suggest that the K⁺-conduction and Ca²⁺-activation sites sense a similar fraction of the surface potential, equivalent to the local electrostatic potential at a distance of 9 Å from the surface.     

Store-operated Ca2+ influx and subplasmalemmal mitochondria

Calcium depletion of the endoplasmic reticulum (ER) induces oligomerisation, puncta formation and translocation of the ER Ca²⁺ sensor proteins, STIM1 and -2 into plasma membrane (PM)-adjacent regions of the ER, where they activate the Orai1, -2 or -3 proteins present in the opposing PM. These proteins form ion channels through which store-operated Ca²⁺ influx (SOC) occurs. Calcium ions exert negative feed-back on SOC. Here we examined whether subplasmalemmal mitochondria, which reduce this feed-back by Ca²⁺ uptake, are located within or out of the high-Ca²⁺ microdomains (HCMDs) formed between the ER and plasmalemmal Orai1 channels. For this purpose, COS-7 cells were cotransfected with Orai1, STIM1 labelled with YFP or mRFP and the mitochondrially targeted Ca²⁺ sensitive fluorescent protein inverse-Pericam. Depletion of ER Ca²⁺ with ATP + thapsigargin (in Ca²⁺-free medium) induced the appearance of STIM1 puncta in the ≤100 nm wide subplasmalemmal space, as examined with TIRF. Mitochondria were located either in the gaps between STIM1-tagged puncta or in remote, STIM1-free regions. After addition of Ca²⁺ mitochondrial Ca²⁺ concentration increased irrespective of the mitochondrion–STIM1 distance. These observations indicate that mitochondria are exposed to Ca²⁺ diffused laterally from the HCMDs formed between the PM and the subplasmalemmal ER.     

Voltage-insensitive Gating after Charge-neutralizing Mutations in the S4 Segment of Shaker Channels

Shaker channel mutants, in which the first (R362), second (R365), and fourth (R371) basic residues in the S4 segment have been neutralized, are found to pass potassium currents with voltage-insensitive kinetics when expressed in Xenopus oocytes. Single channel recordings clarify that these channels continue to open and close from −160 to +80 mV with a constant opening probability (P _o). Although P _o is low (∼0.15) in these mutants, mean open time is voltage independent and similar to that of control Shaker channels. Additionally, these mutant channels retain characteristic Shaker channel selectivity, sensitivity to block by 4-aminopyridine, and are partially blocked by external Ca²⁺ ions at very negative potentials. Furthermore, mean open time is approximately doubled, in both mutant channels and control Shaker channels, when Rb⁺ is substituted for K⁺ as the permeant ion species. Such strong similarities between mutant channels and control Shaker channels suggests that the pore region has not been substantially altered by the S4 charge neutralizations. We conclude that single channel kinetics in these mutants may indicate how Shaker channels would behave in the absence of voltage sensor input. Thus, mean open times appear primarily determined by voltage-insensitive transitions close to the open state rather than by voltage sensor movement, even in control, voltage-sensitive Shaker channels. By contrast, the low and voltage-insensitive P _o seen in these mutant channels suggests that important determinants of normal channel opening derive from electrostatic coupling between S4 charges and the pore domain.     

Surface potential of phosphatidylserine monolayers. I. Divalent ion binding effect

Surface potentials of phosphatidylserine monolayers have been measured in the presence of different divalent ion concentrations in order to determine the way in which divalent ions bind to the membrane surface. The association constants for divalent ions (Mg²⁺, Ca²⁺ and Mn²⁺) with the phosphatidylserine membrane have been obtained from the experimental data and simple ion binding theory. The order of divalent ion binding to the membrane is Mn²⁺ > Ca²⁺ > Mg²⁺. However, none of the divalent ions used completely neutralized the negative charge of phosphatidylserine even at relatively high concentrations. The amounts of the divalent ions bound depended upon the concentration of the monovalent ions present in the subphase. It is suggested that the amounts of bound ions obtained from the use of radioisotope tracer methods may include a considerable contribution from the excess free ions in the double layer region of the phosphatidylserine membrane.     

Clustered Distribution of Calcium Sensitivities: An Indication of Hetero-tetrameric Gating Components in Ca2+-activated K+ Channels Reconstituted from Avian Nasal Gland Cells

High-conductance, Ca²⁺-activated K⁺ channels from the basolateral membrane of rabbit distal colon epithelial cells were reconstituted into planar phospholipid bilayers to examine the effect of Mg²⁺ on the single-channel properties. Mg²⁺ decreases channel current and conductance in a concentration-dependent manner from both the cytoplasmic and the extracellular side of the channel. In contrast to other K⁺ channels, Mg²⁺ does not cause rectification of current through colonic Ca²⁺-activated K⁺ channels. In addition, cytoplasmic Mg²⁺ decreases the reversal potential of the channel. The Mg²⁺-induced decrease in channel conductance is relieved by high K⁺ concentrations, indicating competitive interaction between K⁺ and Mg²⁺. The monovalent organic cation choline also decreases channel conductance and reversal potential, suggesting that the effect is unspecific. The inhibition of channel current by Mg²⁺ and choline most likely is a result of electrostatic screening of negative charges located superficially in the channel entrance. But in addition to charge, other properties appear to be necessary for channel inhibition, as Na⁺ and Ba²⁺ are no (or only weak) inhibitors. Mg²⁺ and possibly other cations may play a role in the regulation of current through these channels. Received: 25 August 1995/Revised: 16 November 1995     

The influence of surface charges on quaternary ammonium block of shaker K+ channels

Block of K⁺ channels can be influenced by the ability of charged residues on the protein surface to accumulate cationic blocking ions to concentrations greater than those in bulk solution. We examined the ionic strength dependence of extracellular block of Shaker K⁺ channels by tetraethylammonium ions (TEA⁺) and by a trivalent quaternary ammonium ion, gallamine³⁺. Wild-type and mutant channels were expressed in Xenopus oocytes and currents recorded with the cut-open oocyte technique. Channel block by both compounds was substantially increased when the bathing electrolyte ionic strength was lowered, but with a much larger effect for trivalent gallamine. These data were quantitatively well described by a simple electrostatic model, accounting for accumulation of blocking ions near the pore of the channel by surface charges. The surface charge density of the wild-type channel consistent with the results was −0.1 e nm⁻². Shaker channels with T449Y mutations have an increased affinity for both TEA and gallamine but the ionic strength dependence of block was described with the same surface charge density as wild-type channels. Much of the increased sensitivity of Shaker K⁺ channels to gallamine may be due to a larger local accumulation of the trivalent ion. The negative charge at position 431 contributes to the sensitivity of channels to TEA (MacKinnon & Yellen, 1990). A charge reversal mutation at this location had little effect on the ionic strength dependence of quaternary ammonium ion block, suggesting that the charge on this amino acid may directly affect binding affinity but not local ion accumulation. Received: 7 December 2000/Revised: 27 April 2001     

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.     

Calcium-induced modulation of synaptic transmission

Calcium (Ca²⁺) is a second messenger regulating a wide variety of intracellular processes. Using GABA-and glycinergic synapses as examples, this review analyzes two functions of this unique ion: postsynaptic Ca²⁺-dependent modulation of receptor-operated channels and Ca²⁺-induced retrograde regulation of neurotransmitter release from the presynaptic terminals. Phosphorylation, rapid Ca²⁺-induced modulation via intermediate Ca²⁺-binding proteins, and changes in the number of functional receptors represent the main pathways of short-and long-term plasticity of postsynaptic receptor-operated channel machinery. Retrograde signaling is an example of synaptic modulation triggered by stimulation of postsynaptic cells and mediated via regulation of presynaptic neurotransmitter release. This mechanism provides postsynaptic neurons with efficient tools to control the presynaptic afferents in an activity-dependent mode. Elevation of intracellular Ca²⁺ in a postsynaptic neuron triggers the synthesis of endocannabinoids (derivatives of arachidonic acid). Their retrograde diffusion through the synaptic cleft and consequent activation of presynaptic G-protein coupled to CB1 receptors inhibits the release of neurotransmitter. These mechanisms of double modulation, which include control over the function of postsynaptic ion channels and retrograde suppression of the release machinery, play an important role in Ca²⁺-dependent control of the main excitatory and inhibitory synaptic pathways in the mammalian nervous system.     

Effects of the Electrostatic Repulsion Between Nanoparticles on Colorimetric Sensing: An Investigation of Determination of Hg2+ with Silver Nanoparticles

The electrostatic repulsion between metal nanoparticles has an important effect on the colorimetric assays based on the aggregation of nanoparticles. In this work, an Hg²⁺ colorimetric sensor based on silver nanoparticles (AgNPs) was used as a model system to study the effects of electrostatic repulsion. Adenine nucleotides carrying different numbers of phosphate groups, adenosine monophosphate (AMP), adenosine diphosphate, and adenosine triphosphate, were used to functionalize AgNPs, respectively. Three kinds of AgNPs displayed different responses to Hg²⁺ ions due to the difference in electrostatic repulsion force, which is resulted from the varying of the number of phosphate groups. The density of negative charges on the surface of AgNPs exhibited a great affect on the size of the AgNPs, detection sensitivity and response range for Hg²⁺ ions. On these bases, the AMP-modified AgNPs were developed into highly sensitive colorimetric sensor to determine Hg²⁺ ions in aqueous solution, which showed a high sensitivity of 0.5 nM and excellent selectivity for Hg²⁺. The Hg²⁺ levels in water samples were determined using AMP-AgNPs with satisfied recovery. The studies on the role of electrostatic repulsion in the colorimetric assays will facilitate the development of more sensitive colorimetric sensors.     

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.     

Redox-Regulated Heterogeneous Thresholds for Ligand Recruitment among InsP3R Ca-Release Channels

To clarify the molecular mechanisms behind quantal Ca²⁺ release, the graded Ca²⁺ release from intracellular stores through inositol 1,4,5-trisphosphate receptor (InsP₃R) channels responding to incremental ligand stimulation, single-channel patch-clamp electrophysiology was used to continuously monitor the number and open probability of InsP₃R channels in the same excised cytoplasmic-side-out nuclear membrane patches exposed alternately to optimal and suboptimal cytoplasmic ligand conditions. Progressively more channels were activated by more favorable conditions in patches from insect cells with only one InsP₃R gene or from cells solely expressing one recombinant InsP₃R isoform, demonstrating that channels with identical primary sequence have different ligand recruitment thresholds. Such heterogeneity was largely abrogated, in a fully reversible manner, by treatment of the channels with sulfhydryl reducing agents, suggesting that it was mostly regulated by different levels of posttranslational redox modifications of the channels. In contrast, sulfhydryl reduction had limited effects on channel open probability. Thus, sulfhydryl redox modification can regulate various aspects of intracellular Ca²⁺ signaling, including quantal Ca²⁺ release, by tuning ligand sensitivities of InsP₃R channels. No intrinsic termination of channel activity with a timescale comparable to that for quantal Ca²⁺ release was observed under any steady ligand conditions, indicating that this process is unlikely to contribute.