Ca sources for the exocytotic release of glutamate from astrocytes

Astrocytes can exocytotically release the gliotransmitter glutamate from vesicular compartments. Increased cytosolic Ca²⁺ concentration is necessary and sufficient for this process. The predominant source of Ca²⁺ for exocytosis in astrocytes resides within the endoplasmic reticulum (ER). Inositol 1,4,5-trisphosphate and ryanodine receptors of the ER provide a conduit for the release of Ca²⁺ to the cytosol. The ER store is (re)filled by the store-specific Ca²⁺-ATPase. Ultimately, the depleted ER is replenished by Ca²⁺ which enters from the extracellular space to the cytosol via store-operated Ca²⁺ entry; the TRPC1 protein has been implicated in this part of the astrocytic exocytotic process. Voltage-gated Ca²⁺ channels and plasma membrane Na⁺/Ca²⁺ exchangers are additional means for cytosolic Ca²⁺ entry. Cytosolic Ca²⁺ levels can be modulated by mitochondria, which can take up cytosolic Ca²⁺ via the Ca²⁺ uniporter and release Ca²⁺ into cytosol via the mitochondrial Na⁺/Ca²⁺ exchanger, as well as by the formation of the mitochondrial permeability transition pore. The interplay between various Ca²⁺ sources generates cytosolic Ca²⁺ dynamics that can drive Ca²⁺-dependent exocytotic release of glutamate from astrocytes. An understanding of this process in vivo will reveal some of the astrocytic functions in health and disease of the brain. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Cytosolic Ca2+ concentration during Ca2+ depletion of isolated rat hearts

Reperfusion of isolated mammalian hearts with a Ca²⁺-containing solution after a short Ca²⁺-free period at 37°:C results in massive influx of Ca²⁺ into the cells and irreversible cell damage: the Ca²⁺paradox. Information about the free intracellular, cytosolic [Ca²⁺] ([Ca²⁺]_i) during Ca²⁺ depletion is essential to assess the possibility of Ca²⁺ influx through reversed Na⁺/Ca²⁺ exchange upon Ca²⁺ repletion. Furthermore, the increase in end-diastolic pressure often seen during Ca²⁺-free perfusion of intact hearts may be similar to that seen during ischemia and caused by liberation of Ca²⁺ from intracellular stores. Therefore, in this study, we measured [Ca²⁺]i during Ca²⁺- free perfusion of isolated rat hearts. To this end, the fluorescent indicator Indo-1 was loaded into isolated Langendorff-perfused hearts and Ca²⁺-transients were recorded. Ca²⁺-transients disappeared within 1 min of Ca²⁺ depletion. Systolic [Ca²⁺]i during control perfusion was 268±54 nM. Diastolic [Ca²⁺]i during control perfusion was 114±34 nM and decreased to 53±19 nM after 10 min of Ca²⁺ depletion. Left ventricular end-diastolic pressure (LVEDP) significantly increased from 13±4 mmHg during control perfusion after Indo-1 AM loading to 31±5 mmHg after 10 min Ca²⁺ depletion. Left ventricular developed pressure did not recover during Ca²⁺ repletion, indicating a full Ca²⁺ paradox. These results show that LVEDP increased during Ca²⁺ depletion despite a decrease in [Ca²⁺]i, and is therefore not comparable to the contracture seen during ischemia. Furthermore, calculation of the driving force for the Na⁺/Ca²⁺ exchanger showed that reversed Na⁺/Ca²⁺ exchange during Ca²⁺ repletion is not able to increase [Ca²⁺]i to cytotoxic levels.     

Ca2+ influx-dependent refilling of intracellular Ca2+ stores determines the frequency of Ca2+ oscillations in fertilized mouse eggs

On mammalian fertilization, long-lasting Ca²⁺ oscillations are induced in the egg by the fusing spermatozoon. While each transient Ca²⁺ increase in Ca²⁺ concentration ([Ca²⁺]) in the cytosol is due to Ca²⁺ release from the endoplasmic reticulum (ER), Ca²⁺ influx from outside is required for Ca²⁺ oscillations to persist. In this study, we investigated how Ca²⁺ influx is interrelated to the cycle of Ca²⁺ release and uptake by the intracellular Ca²⁺ stores during Ca²⁺ oscillations in fertilized mouse eggs. In addition to monitoring cytosolic [Ca²⁺] with fura-2, the influx rate was evaluated using Mn²⁺ quenching technique, and the change in [Ca²⁺] in the ER lumen was visualized with a targeted fluorescent probe. We found that the influx was stimulated after each transient Ca²⁺ release and then diminished gradually to the basal level, and demonstrated that the ER Ca²⁺ stores once depleted by Ca²⁺ release were gradually refilled until the next Ca²⁺ transient to be initiated. Experiments altering extracellular [Ca²⁺] in the middle of Ca²⁺ oscillations revealed the dependence of both the refilling rate and the oscillation frequency on the rate of Ca²⁺ influx, indicating the crucial role of Ca²⁺ influx in determining the intervals of Ca²⁺ transients. As for the influx pathway supporting Ca²⁺ oscillations to persist, STIM1/Orai1-mediated store-operated Ca²⁺ entry (SOCE) may not significantly contribute, since neither known SOCE blockers nor the expression of protein fragments that interfere the interaction between STIM1 and Orai1 inhibited the oscillation frequency or the influx rate.     

Calcium uptake mechanisms of mitochondria

The ability of mitochondria to capture Ca²⁺ ions has important functional implications for cells, because mitochondria shape cellular Ca²⁺ signals by acting as a Ca²⁺ buffer and respond to Ca²⁺ elevations either by increasing the cell energy supply or by triggering the cell death program of apoptosis. A mitochondrial Ca²⁺ channel known as the uniporter drives the rapid and massive entry of Ca²⁺ ions into mitochondria. The uniporter operates at high, micromolar cytosolic Ca²⁺ concentrations that are only reached transiently in cells, near Ca²⁺ release channels. Mitochondria can also take up Ca²⁺ at low, nanomolar concentrations, but this high affinity mode of Ca²⁺ uptake is not well characterized. Recently, leucine-zipper-EF hand-containing transmembrane region (Letm1) was proposed to be an electrogenic 1:1 mitochondrial Ca²⁺/H⁺ antiporter that drives the uptake of Ca²⁺ into mitochondria at nanomolar cytosolic Ca²⁺ concentrations. In this article, we will review the properties of the Ca²⁺ import systems of mitochondria and discuss how Ca²⁺ uptake via an electrogenic 1:1 Ca²⁺/H⁺ antiport challenges our current thinking of the mitochondrial Ca²⁺ uptake mechanism.     

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.     

Decoding of calcium oscillations by phosphorylation cycles: analytic results

Experimental studies have demonstrated that Ca²⁺-regulated proteins are sensitive to the frequency of Ca²⁺ oscillations, and several mathematical models for specific proteins have provided insight into the mechanisms involved. Because of the large number of Ca²⁺-regulated proteins in signal transduction, metabolism and gene expression, it is desirable to establish in general terms which molecular properties shape the response to oscillatory Ca²⁺ signals. Here we address this question by analyzing in detail a model of a prototypical Ca²⁺-decoding module, consisting of a target protein whose activity is controlled by a Ca²⁺-activated kinase and the counteracting phosphatase. We show that this module can decode the frequency of Ca²⁺ oscillations, at constant average Ca²⁺ signal, provided that the Ca²⁺ spikes are narrow and the oscillation frequency is sufficiently low—of the order of the phosphatase rate constant or below. Moreover, Ca²⁺ oscillations activate the target more efficiently than a constant signal when Ca²⁺ is bound cooperatively and with low affinity. Thus, the rate constants and the Ca²⁺ affinities of the target-modifying enzymes can be tuned in such a way that the module responds optimally to Ca²⁺ spikes of a certain amplitude and frequency. Frequency sensitivity is further enhanced when the limited duration of the external stimulus driving Ca²⁺ signaling is accounted for. Thus, our study identifies molecular parameters that may be involved in establishing the specificity of cellular responses downstream of Ca²⁺ oscillations.     

Role ofDrosophila TRP in inositide-mediated Ca2+ entry

Inositol lipid signaling relies on an InsP₃-induced Ca²⁺ release from intracellular stores and on extracellular Ca²⁺ entry, which takes place when the Ca²⁺ stores become depleted of Ca²⁺. This interplay between Ca²⁺ release and Ca²⁺ entry has been termed capacitative Ca²⁺ entry and the inward current calcium release activated current (CRAC) to indicate gating of Ca²⁺ entry by Ca²⁺-store depletion. The signaling pathway and the gating mechanism of capacitative Ca²⁺ entry, however, are largely unknown and the molecular participants in this process have not been identified. In this article we review genetic, molecular, and functional studies of wild-type and mutantDrosophila photoreceptors, suggesting that thetransient receptor potential mutant (trp) is the first putative capacitative Ca²⁺ entry mutant. Furthermore, several lines of evidence suggest that thetrp gene product TRP is a candidate subunit of the plasma membrane channel that is activated by Ca²⁺ store depletion.     

Ca signaling and STIM1

An increase in the intracellular calcium ion concentration ([Ca²⁺]) impacts a diverse range of cell functions, including adhesion, motility, gene expression and proliferation. Elevation of intracellular calcium ion (Ca²⁺) regulates various cellular events after the stimulation of cells. Initial increase in Ca²⁺ comes from the endoplasmic reticulum (ER), intracellular storage space. However, the continuous influx of extracellular Ca²⁺ is required to maintain the increased level of Ca²⁺ inside cells. Store-operated Ca²⁺ entry (SOCE) manages this process, and STIM1, a newly discovered molecule, has a unique and essential role in SOCE. STIM1 can sense the exhaustion of Ca²⁺ in the ER, and activate the SOC channel in the plasma membrane, leading to the continuous influx of extracellular Ca²⁺. STIM1 senses the status of the intracellular Ca²⁺ stores via a luminal N-terminal Ca²⁺-binding EF-hand domain. Dissociation of Ca²⁺ from this domain induces the clustering of STIM1 to regions of the ER that lie close to the plasma membrane, where it regulates the activity of the store-operated Ca²⁺ channels/entry (calcium-release-activated calcium channels/entry). In this review, we summarize the mechanism by which STIM1 regulates SOCE, and also its role in the control of mast cell functions and allergic responses.     

Mg2+-sensitive alterations in Ca2+ regulation associated with cell transformation

The intracellular Ca²⁺ content of nontransformed Balb/c3T3 cells is two to three times higher than that of a spontaneously transformed derivative. Depriving either cell type of extracellular Mg²⁺ causes a 2- to 3-fold increase in their Ca²⁺ content over a 24-hr period. Restoring Mg²⁺ to the medium decreases the Ca²⁺ content of the cells to their original values in about the same time. The increase in Ca²⁺ content is not blocked by cycloheximide suggesting that normal rates of protein synthesis are not required to produce this effect. Mg²⁺ deprivation also decreases the initial rate of Ca²⁺ efflux from the transformed cells and increases the size of the slowly exchanging fraction of Ca²⁺ to the levels found in the nontransformed cells. Since Mg²⁺ deprivation normalizes the appearance and growth behavior of the transformed cells, the possible intermediary role of Ca²⁺ in this normalization was studied. Large changes in extracellular Ca²⁺ produced large changes in the Ca²⁺ content of the transformed cells with little change in appearance or thymidine incorporation rate. Ca²⁺ deprivation did inhibit thymidine incorporation in early passage nontransformed cells; however with repeated passage, this effect decreased, as did the Ca²⁺ content of these cells. The possible role of Mg²⁺ in regulating cellular Ca²⁺ content and distribution is discussed, as is the relation of Ca²⁺ content and distribution to the development of the transformed state.     

Pulses of Cell Ca<Superscript>2+</Superscript> and the Dynamics of Tight Junction Opening and Closing

A mathematical modeling of tight junction (TJ) dynamics was elaborated in a previous study (Kassab, F., Marques, R.P., Lacaz-Vieira, F. 2002. Modeling tight junction dynamics and oscillations. J. Gen. Physiol. 120:237–247) to better understand the dynamics of TJ opening and closing, as well as oscillations of TJ permeability that are observed in response to changes of extracellular Ca²⁺ levels. In this model, TJs were assumed to be specifically controlled by the Ca²⁺ concentration levels at the extracellular Ca²⁺ binding sites of zonula adhaerens. Despite the fact that the model predicts all aspects of TJ dynamics, we cannot rule out the likelihood that changes of intracellular Ca²⁺ concentration (Ca²⁺ _cell), which might result from changes \ of extracellular Ca²⁺ concentration (Ca²⁺ _extl), contribute to the observed results. In order to address this aspect of TJ regulation, fast Ca²⁺-switch experiments were performed in which changes of Ca²⁺ _cell were induced using the Ca²⁺ ionophore A23187 or thapsigargin, a specific inhibitor of the sarco-endoplasmic reticulum Ca²⁺-ATPase. The results indicate that the ionophore or thapsigargin per se do not affect basal tissue electrical conductance (G), showing that the sealing of TJs is not affected by a rise in Ca²⁺ _cell. When TJs were kept in a dynamic state, as partially open structures or in oscillation, conditions in which the junctions are very sensitive to disturbances that affect their regulation, a rise of Ca²⁺ _cell never led to a decline of G, indicating that a rise of Ca²⁺ _cell does not trigger per se TJ closure. On the contrary, always the first response to a rise of Ca²⁺ _cell is an increase of G that, in most cases, is a transient response. Despite these observations we cannot assure that a rise of Ca²⁺ _cell is without effect on the TJs, since an increase of Ca²⁺ _cell not only causes a transient increase of G but, in addition, during oscillations a rise of Ca²⁺ _cell induced by the Ca²⁺ ionophore transiently halted the oscillatory pattern of TJs. The main conclusion of this study is that TJ closure that is observed when basolateral Ca²⁺ concentration (Ca²⁺ _bl) is increased after TJs were opened by Ca²⁺ _bl removal cannot be ascribed to a rise of Ca²⁺ _cell and might be a consequence of Ca²⁺ binding to extracellular Ca²⁺ sites.     

A Model of CatSper Channel Mediated Calcium Dynamics in Mammalian Spermatozoa

CatSpers are calcium (Ca²⁺) channels that are located along the principal piece of mammalian sperm flagella and are directly linked to sperm motility and hyperactivation. It has been observed that Ca²⁺ entry through CatSper channels triggers a tail to head Ca²⁺ propagation in mouse sperm, as well as a sustained increase of Ca²⁺ in the head. Here, we develop a mathematical model to investigate this propagation and sustained increase in the head. A 1-d reaction-diffusion model tracking intracellular Ca²⁺ with flux terms for the CatSper channels, a leak flux, and plasma membrane Ca²⁺ clearance mechanism is studied. Results of this simple model exhibit tail to head Ca²⁺ propagation, but no sustained increase in the head. Therefore, in this model, a simple plasma membrane pump-leak system with diffusion in the cytosol cannot account for these experimentally observed results. It has been proposed that Ca²⁺ influx from the CatSper channels induce additional Ca²⁺ release from an internal store. We test this hypothesis by examining the possible role of Ca²⁺ release from the redundant nuclear envelope (RNE), an inositol 1,4,5-trisphosphate (IP₃) gated Ca²⁺ store in the neck. The simple model is extended to include an equation for IP₃ synthesis, degradation, and diffusion, as well as flux terms for Ca²⁺ in the RNE. When IP₃ and the RNE are accounted for, the results of the model exhibit a tail to head Ca²⁺ propagation as well as a sustained increase of Ca²⁺ in the head.     

Cellular calcium deficiency plays a role in neuronal death caused by proteasome inhibitors

Cytosolic Ca²⁺ concentration ([Ca²⁺]_i) is reduced in cultured neurons undergoing neuronal death caused by inhibitors of the ubiquitin proteasome system. Activation of calcium entry via voltage‐gated Ca²⁺ channels restores cytosolic Ca²⁺ levels and reduces this neuronal death ( Snider et al. 2002 ). We now show that this reduction in [Ca²⁺]_i is transient and occurs early in the cell death process, before activation of caspase 3. Agents that increase Ca²⁺ influx such as activation of voltage‐gated Ca²⁺ channels or stimulation of Ca²⁺ entry via the plasma membrane Na–Ca exchanger attenuate neuronal death only if applied early in the cell death process. Cultures treated with proteasome inhibitors had reduced current density for voltage‐gated Ca²⁺ channels and a less robust increase in [Ca²⁺]_i after depolarization. Levels of endoplasmic reticulum Ca²⁺ were reduced and capacitative Ca²⁺ entry was impaired early in the cell death process. Mitochondrial Ca²⁺ was slightly increased. Preventing the transfer of Ca²⁺ from mitochondria to cytosol increased neuronal vulnerability to this death while blockade of mitochondrial Ca²⁺ uptake via the uniporter had no effect. Programmed cell death induced by proteasome inhibition may be caused in part by an early reduction in cytosolic and endoplasmic reticulum Ca^2+, possibly mediated by dysfunction of voltage‐gated Ca²⁺ channels. These findings may have implications for the treatment of disorders associated with protein misfolding in which proteasome impairment and programmed cell death may occur.     

Differentiation between Ca2+ transport and ATP-induced Ca2+ binding by sarcoplasmic reticulum

The Ca²⁺ actively accumulated by sarcoplasmic reticulum isolated from skeletal muscle is composed of two fractions; one represented by intravesicular free Ca²⁺ and another represented by Ca²⁺ selectively bound to the membranes. Both of these Ca²⁺ fractions depend on ATP, although it is not clear whether ATP hydrolysis is essential for accumulation of the second Ca²⁺ fraction. The existence of the membrane-bound Ca²⁺ induced by ATP is clearly shown in experiments in which the Ca²⁺ retention by sarcoplasmic reticulum is measured in the presence and in the absence of X-537A, a Ca²⁺ ionophore, which makes the membrane permeable to Ca²⁺. Thus, in the presence of X-537A all Ca²⁺ accumulated due to ATP is bound to the membranes. This membrane-bound Ca²⁺ represents about 30 nmol/mg protein in the range of external $\text{p}\text{Ca}$ values of 7 to 3.5. The magnitude of this Ca²⁺ fraction is slightly higher whether or not the experiments are performed in the presence of oxalate, which greatly increased the intravesicular Ca²⁺ accumulation. Furthermore, taking advantage of the impermeability of sarcoplasmic reticulum to EGTA, it is possible to show the existence of the membrane-bound Ca²⁺ as a distinct fraction from that which exists intravesicularly.     

Mitochondrial Calcium Uniporter MCU Supports Cytoplasmic Ca2+ Oscillations,Store-Operated Ca2+ Entry and Ca2+-Dependent Gene Expression in Response to Receptor Stimulation

Ca²⁺ flux into mitochondria is an important regulator of cytoplasmic Ca²⁺ signals, energy production and cell death pathways. Ca²⁺ uptake can occur through the recently discovered mitochondrial uniporter channel (MCU) but whether the MCU is involved in shaping Ca²⁺ signals and downstream responses to physiological levels of receptor stimulation is unknown. Here, we show that modest stimulation of leukotriene receptors with the pro-inflammatory signal LTC₄ evokes a series of cytoplasmic Ca²⁺ oscillations that are rapidly and faithfully propagated into mitochondrial matrix. Knockdown of MCU or mitochondrial depolarisation, to reduce the driving force for Ca²⁺ entry into the matrix, prevents the mitochondrial Ca²⁺ rise and accelerates run down of the oscillations. The loss of cytoplasmic Ca²⁺ oscillations appeared to be a consequence of enhanced Ca²⁺-dependent inactivation of InsP₃ receptors, which arose from the loss of mitochondrial Ca²⁺ buffering. Ca²⁺ dependent gene expression in response to leukotriene receptor activation was suppressed following knockdown of the MCU. In addition to buffering Ca²⁺ release, mitochondria also sequestrated Ca²⁺ entry through store-operated Ca²⁺ channels and this too was prevented following loss of MCU. MCU is therefore an important regulator of physiological pulses of cytoplasmic Ca²⁺.     

SECOND MESSENGERS AND THE REGULATION OF CA 2+ FLUXES BY CA 2+ -MOBILIZING AGONISTS IN RAT LIVER

Knowledge of the mechanism of action of Ca²⁺-mobilizing agonists in liver has progressed considerably following the discovery that their interaction with specific receptors on the plasma membrane is accompanied by the hydrolysis of PIP₂ and the generation of the second messengers diacylglycerol and IP₃, for the activation of protein kinase C and the mobilization of intracellular Ca²⁺, respectively. Although the second messenger functions of diacylglycerol and IP₃ in these actions seem well established, it is not yet clear how the agonists are able to regulate Ca²⁺ influx across the plasma membrane, an event which is crucial for those actions of the agonists which are dependent on the maintenance of an elevated level of cytosolic Ca²⁺, Whilst there is evidence for the existence of more than one pathway for Ca²⁺ influx in liver, it appears that in each instance the Ca²⁺ influx process is regulated differently to the Ca²⁺ influx through the volage-sensitive Ca²⁺ channels that is known to occur in excitable tissues. At present it is not clear whether any of the Ca²⁺ influx pathways in liver is regulated by direct coupling to the agonist receptor mechanism on the outer surface of the plasma membrane, or whether the regulation involves the production of some second messenger(s). However, indirect evidence from a number of tissues appears to favour the involvement of both IP₃ and IP₄ in the regulation of Ca²⁺ influx. The mechanism by which IP₃ and IP₄ may regulate Ca²⁺ influx remains to be established, but it has been proposed that Ca²⁺ entry into the cell occurs through a pathway connecting the plasma membrane and the endoplasmic reticulum, following the release of intracellular Ca²⁺ from the lumen of the endoplasmic reticulum. Although it is not yet known whether glucagon (or cyclic AMP) activates the same pathway for Ca²⁺ influx as Ca²⁺-mobilizing agonists, the marked potentiation by cyclic AMP of the Ca²⁺ influx induced by Ca²⁺-mobilizing agonists has provided a powerful system with which to study the regulation of Ca²⁺ influx in liver. Whether this Ca²⁺ influx process occurs through some ion exchange mechanism (such as Ca²⁺/Na⁺ exchange) remains to be determined. Results from this study suggests that the Ca²⁺ influx is inhibited by neomycin, acidic pH, and a depolarization of the plasma membrane. The observation that cyclic AMP synergistically potentiates the influx of Ca²⁺ induced by Ca²⁺-mobilizing agonists, that this influx appears to correlate with the reported ability of these agonists to induce PIP₂ hydrolysis and accumulation of IP₃, and that cyclic AMP synergistically potentiates the production of IP₄ by vasopressin, are all consistent with the notion that IP₃ and IP₄ are involved in regulating Ca²⁺ influx. Whilst little is known about the Ca²⁺ transport process itself, these studies coupled with the recent finding that Ca²⁺ influx into the liver cell can occur through different pathways, seem set to lead to a better understanding of this important process in the near future.     

Assessment of Sarcoplasmic Reticulum Ca Depletion During Spontaneous Ca Waves in Isolated Permeabilized Rabbit Ventricular Cardiomyocytes

In this study, Ca²⁺ release due to spontaneous Ca²⁺ waves was measured both from inside the sarcoplasmic reticulum (SR) and from the cytosol of rabbit cardiomyocytes. These measurements utilized Fluo5N-AM for intra-SR Ca²⁺ from intact cells and Fluo5F in the cytosol of permeabilized cells. Restricted subcellular volumes were resolved with the use of laser-scanning confocal microscopy. Local Ca²⁺ signals during spontaneous Ca²⁺ release were compared with those induced by rapid caffeine application. The free cytoplasmic [Ca²⁺] increase during a Ca²⁺ wave was 98.1% ± 0.3% of that observed during caffeine application. Conversion to total Ca²⁺ release suggested that Ca²⁺ release from a Ca²⁺ wave was not significantly different from that released during caffeine application (104% ± 6%). In contrast, the maximum decrease in intra-SR Fluo-5N fluorescence during a Ca²⁺ wave was 82.5% ± 2.6% of that observed during caffeine application. Assuming a maximum free [Ca²⁺] of 1.1 mM, this translates to a 96.2% ± 0.8% change in intra-SR free [Ca²⁺] and a 91.7% ± 1.6% depletion of the total Ca²⁺. This equates to a minimum intra-SR free Ca²⁺ of 46 ± 7 μM during a Ca²⁺ wave. Reduction of RyR2 Ca²⁺ sensitivity by tetracaine (50 μM) reduced the spontaneous Ca²⁺ release frequency while increasing the Ca²⁺ wave amplitude. This did not significantly change the total depletion of the SR (94.5% ± 1.1%). The calculated minimum [Ca²⁺] during these Ca²⁺ waves (87 ± 19 μM) was significantly higher than control (p < 0.05). A computational model incorporating this level of Ca²⁺ depletion during a Ca²⁺ wave mimicked the transient and sustained effects of tetracaine on spontaneous Ca²⁺ release. In conclusion, spontaneous Ca²⁺ release results in substantial but not complete local Ca²⁺ depletion of the SR. Furthermore, measurements suggest that Ca²⁺ release terminates when luminal [Ca²⁺] reaches ∼50 μM.     

Modeling the calcium sequestration system in isolated guinea pig cardiac mitochondria

Under high Ca²⁺ load conditions, Ca²⁺ concentrations in the extra-mitochondrial and mitochondrial compartments do not display reciprocal dynamics. This is due to a paradoxical increase in the mitochondrial Ca²⁺ buffering power as the Ca²⁺ load increases. Here we develop and characterize a mechanism of the mitochondrial Ca²⁺ sequestration system using an experimental data set from isolated guinea pig cardiac mitochondria. The proposed mechanism elucidates this phenomenon and others in a mathematical framework and is integrated into a previously corroborated model of oxidative phosphorylation including the Na⁺/Ca²⁺ cycle. The integrated model reproduces the Ca²⁺ dynamics observed in both compartments of the isolated mitochondria respiring on pyruvate after a bolus of CaCl₂ followed by ruthenium red and a bolus of NaCl. The model reveals why changes in mitochondrial Ca²⁺ concentration of Ca²⁺ loaded mitochondria appear significantly mitigated relative to the corresponding extra-mitochondrial Ca²⁺ concentration changes after Ca²⁺ efflux is initiated. The integrated model was corroborated by simulating the set-point phenomenon. The computational results support the conclusion that the Ca²⁺ sequestration system is composed of at least two classes of Ca²⁺ buffers. The first class represents prototypical Ca²⁺ buffering, and the second class encompasses the complex binding events associated with the formation of amorphous calcium phosphate. With the Ca²⁺ sequestration system in mitochondria more precisely defined, computer simulations can aid in the development of innovative therapeutics aimed at addressing the myriad of complications that arise due to mitochondrial Ca²⁺ overload.     

Model for the allosteric regulation of the Na+/Ca2+ exchanger NCX

The Na⁺/Ca²⁺ exchanger provides a major Ca²⁺ extrusion pathway in excitable cells and plays a key role in the control of intracellular Ca²⁺ concentrations. In Canis familiaris, Na⁺/Ca²⁺ exchanger (NCX) activity is regulated by the binding of Ca²⁺ to two cytosolic Ca²⁺‐binding domains, CBD1 and CBD2, such that Ca²⁺‐binding activates the exchanger. Despite its physiological importance, little is known about the exchanger's global structure, and the mechanism of allosteric Ca²⁺‐regulation remains unclear. It was found previously that for NCX in the absence of Ca²⁺ the two domains CBD1 and CBD2 of the cytosolic loop are flexibly linked, while after Ca²⁺‐binding they adopt a rigid arrangement that is slightly tilted. A realistic model for the mechanism of the exchanger's allosteric regulation should not only address this property, but also it should explain the distinctive behavior of Drosophila melanogaster's sodium/calcium exchanger, CALX, for which Ca²⁺‐binding to CBD1 inhibits Ca²⁺ exchange. Here, NMR spin relaxation and residual dipolar couplings were used to show that Ca²⁺ modulates CBD1 and CBD2 interdomain flexibility of CALX in an analogous way as for NCX. A mechanistic model for the allosteric Ca²⁺ regulation of the Na⁺/Ca²⁺ exchanger is proposed. In this model, the intracellular loop acts as an entropic spring whose strength is modulated by Ca²⁺‐binding to CBD1 controlling ion transport across the plasma membrane. Proteins 2016; 84:580–590. © 2016 Wiley Periodicals, Inc.     

Spatiotemporal Correlations between Cytosolic and Mitochondrial Ca2+ Signals Using a Novel Red-Shifted Mitochondrial Targeted Cameleon

The transfer of Ca²⁺ from the cytosol into the lumen of mitochondria is a crucial process that impacts cell signaling in multiple ways. Cytosolic Ca²⁺ ([Ca²⁺]_cyto) can be excellently quantified with the ratiometric Ca²⁺ probe fura-2, while genetically encoded Förster resonance energy transfer (FRET)-based fluorescent Ca²⁺ sensors, the cameleons, are efficiently used to specifically measure Ca²⁺ within organelles. However, because of a significant overlap of the fura-2 emission with the spectra of the cyan and yellow fluorescent protein of most of the existing cameleons, the measurement of fura-2 and cameleons within one given cell is a complex task. In this study, we introduce a novel approach to simultaneously assess [Ca²⁺]_cyto and mitochondrial Ca²⁺ ([Ca²⁺]_mito) signals at the single cell level. In order to eliminate the spectral overlap we developed a novel red-shifted cameleon, D1GO-Cam, in which the green and orange fluorescent proteins were used as the FRET pair. This ratiometric Ca²⁺ probe could be successfully targeted to mitochondria and was suitable to be used simultaneously with fura-2 to correlate [Ca²⁺]_cyto and [Ca²⁺]_mito within same individual cells. Our data indicate that depending on the kinetics of [Ca²⁺]_cyto rises there is a significant lag between onset of [Ca²⁺]_cyto and [Ca²⁺]_mito signals, pointing to a certain threshold of [Ca²⁺]_cyto necessary to activate mitochondrial Ca²⁺ uptake. The temporal correlation between [Ca²⁺]_mito and [Ca²⁺]_cyto as well as the efficiency of the transfer of Ca²⁺ from the cytosol into mitochondria varies between different cell types. Moreover, slow mitochondrial Ca²⁺ extrusion and a desensitization of mitochondrial Ca²⁺ uptake cause a clear difference in patterns of mitochondrial and cytosolic Ca²⁺ oscillations of pancreatic beta-cells in response to D-glucose.     

Activation of endoplasmic reticulum calcium leak by 2-APB depends on the luminal calcium concentration

It has been shown that 2-APB is a nonspecific modulator of ion channel activity, while most of the channels are inhibited by this compound, there are few examples of channels that are activated by 2-APB. Additionally, it has been shown that, 2-APB leads to a reduction in the luminal endoplasmic reticulum Ca²⁺ level ([Ca²⁺]_ER) and we have carried out simultaneous recordings of both [Ca²⁺]_i and the [Ca²⁺]_ER in HeLa cell suspensions to assess the mechanism involved in this effect. This approach allowed us to determine that 2-APB induces a reduction in the [Ca²⁺]_ER by activating an ER-resident Ca²⁺ permeable channel more than by inhibiting the activity of SERCA pumps. Interestingly, this effect of 2-APB of reducing the [Ca²⁺]_ER is auto-limited because depends on a replete ER Ca²⁺ store; a condition that thapsigargin does not require to decrease the [Ca²⁺]_ER. Additionally, our data indicate that the ER Ca²⁺ permeable channel activated by 2-APB does not seem to participate in the ER Ca²⁺ leak revealed by inhibiting SERCA pump with thapsigargin. This work suggests that, prolonged incubations with even low concentrations of 2-APB (5 μM) would lead to the reduction in the [Ca²⁺]_ER that might explain the inhibitory effect of this compound on those signals that require Ca²⁺ release from the ER store.