Synchronous Ca(2+) oscillation emerges from voltage fluctuations of Ca(2+) stores

Synchronous Ca(2+) oscillation occurs in various cell types to regulate cellular functions. However, the mechanism for synchronization of Ca(2+) increases between cells remains unclear. Recently, synchronous oscillatory changes in the membrane potential of internal Ca(2+) stores were recorded using an organelle-specific voltage-sensitive dye [Yamashita et al. (2006) FEBS J273, 3585-3597], and an electrical coupling model of the synchronization of store potentials and Ca(2+) releases has been proposed [Yamashita (2006) FEBS Lett580, 4979-4983]. This model is based on capacitative coupling, by which transient voltage changes can be synchronized, but oscillatory slow potentials cannot be communicated. Another candidate mechanism is synchronization of action potentials and ensuing Ca(2+) influx through voltage-dependent Ca channels. The present study addresses the question of whether Ca(2+) increases are synchronized by action potentials, and how oscillatory store potentials are synchronized across the cells. Electrophysiological and Ca(2+)-sensitive fluorescence measurements in early embryonic chick retina showed that synchronous Ca(2+) oscillation was caused by releases of Ca(2+) from Ca(2+) stores without any evidence of action potentials in retinal neuroepithelial cells or newborn neurons. High-speed fluorescence measurement of store membrane potential surprisingly revealed that the synchronous oscillatory changes in the store potential were periodic repeats of a burst of high-frequency voltage fluctuations. The burst coincided with a Ca(2+) increase. The present study suggests that synchronization of Ca(2+) release is mediated by the high-frequency fluctuation in the store potential. Close apposition of the store membrane and plasma membrane in an epithelial structure would allow capacitative coupling across the cells.     

Role of elementary Ca(2+) puffs in generating repetitive Ca(2+) oscillations

Inositol (1,4,5)-trisphosphate (IP(3)) liberates intracellular Ca(2+) both as localized 'puffs' and as repetitive waves that encode information in a frequency-dependent manner. Using video-rate confocal imaging, together with photorelease of IP(3) in Xenopus oocytes, we investigated the roles of puffs in determining the periodicity of global Ca(2+) waves. Wave frequency is not delimited solely by cyclical recovery of the cell's ability to support wave propagation, but further involves sensitization of Ca(2+)-induced Ca(2+) release by progressive increases in puff frequency and amplitude at numerous sites during the interwave period, and accumulation of pacemaker Ca(2+), allowing a puff at a 'focal' site to trigger a subsequent wave. These specific 'focal' sites, distinguished by their higher sensitivity to IP(3) and close apposition to neighboring puff sites, preferentially entrain both the temporal frequency and spatial directionality of Ca(2+) waves. Although summation of activity from many stochastic puff sites promotes the generation of regularly periodic global Ca(2+) signals, the properties of individual Ca(2+) puffs control the kinetics of Ca(2+) spiking and the (higher) frequency of subcellular spikes in their local microdomain.     

Role of endothelial Ni(2+)-sensitive Ca(2+) entry pathway in regulation of EDHF in porcine coronary artery

Elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) in endothelial cells is proposed to be required for generation of vascular actions of endothelium-derived hyperpolarizing factor (EDHF). This study was designed to determine the endothelial Ca(2+) source that is important in development of EDHF-mediated vascular actions. In porcine coronary artery precontracted with U-46619, bradykinin (BK) and cyclopiazonic acid (CPA) caused endothelium-dependent relaxations in the presence of N(G)-nitro-L-arginine (L-NNA). The L-NNA-resistant relaxant responses were inhibited by high K(+), indicating an involvement of EDHF. In the presence of Ni(2+), which inhibits Ca(2+) influx through nonselective cation channels, the BK-induced EDHF relaxant response was greatly diminished and the CPA-induced response was abolished. BK and CPA elicited membrane hyperpolarization of smooth muscle cells of porcine coronary artery. Ni(2+) suppressed the hyperpolarizing responses in a manner analogous to removal of extracellular Ca(2+). EDHF-mediated relaxations and hyperpolarizations evoked by BK and CPA in porcine coronary artery showed a temporal correlation with the increases in [Ca(2+)](i) in porcine aortic endothelial cells. The extracellular Ca(2+)-dependent rises in [Ca(2+)](i) in endothelial cells stimulated with BK and CPA were completely blocked by Ni(2+). These results suggest that Ca(2+) influx into endothelial cells through nonselective cation channels plays a crucial role in the regulation of EDHF.     

Ca(2+)-ionophore A23187 and the Ca(2+)-mobilizing hormones serotonin, vasopressin, and bradykinin increase mitochondrially bound hexokinase in muscle.

We show that a rise in cytosolic-free Ca2+ in muscle, induced by Ca(2+)-ionophore A23187 or by the Ca(2+)-mobilizing hormones serotonin, vasopressin, and bradykinin, increases the binding of hexokinase to mitochondria in muscle. This increase could be prevented by treatment with the calmodulin antagonists trifluoperazine or CGS 9343B (a novel, potent, and selective inhibitor of calmodulin activity) which strongly suggests that calmodulin is involved in the Ca(2+)-induced binding of the enzyme to muscle mitochondria.     

Mitochondrial Ca(2+)-induced Ca(2+) release mediated by the Ca(2+) uniporter

We have reported that a population of chromaffin cell mitochondria takes up large amounts of Ca(2+) during cell stimulation. The present study focuses on the pathways for mitochondrial Ca(2+) efflux. Treatment with protonophores before cell stimulation abolished mitochondrial Ca(2+) uptake and increased the cytosolic [Ca(2+)] ([Ca(2+)](c)) peak induced by the stimulus. Instead, when protonophores were added after cell stimulation, they did not modify [Ca(2+)](c) kinetics and inhibited Ca(2+) release from Ca(2+)-loaded mitochondria. This effect was due to inhibition of mitochondrial Na(+)/Ca(2+) exchange, because blocking this system with CGP37157 produced no further effect. Increasing extramitochondrial [Ca(2+)](c) triggered fast Ca(2+) release from these depolarized Ca(2+)-loaded mitochondria, both in intact or permeabilized cells. These effects of protonophores were mimicked by valinomycin, but not by nigericin. The observed mitochondrial Ca(2+)-induced Ca(2+) release response was insensitive to cyclosporin A and CGP37157 but fully blocked by ruthenium red, suggesting that it may be mediated by reversal of the Ca(2+) uniporter. This novel kind of mitochondrial Ca(2+)-induced Ca(2+) release might contribute to Ca(2+) clearance from mitochondria that become depolarized during Ca(2+) overload.     

Role of endogenous regucalcin in the regulation of Ca(2+)-ATPase activity in rat liver nuclei

The role of endogenous regucalcin in the regulation of Ca(2+)-ATPase, a Ca(2+) sequestrating enzyme, in rat liver nuclei was investigated. Nuclear Ca(2+)-ATPase activity was significantly reduced by the addition of regucalcin (0.1-0.5 microM) into the enzyme reaction mixture. The presence of anti-regucalcin monoclonal antibody (25 or 50 ng/ml) caused a significant elevation of Ca(2+)-ATPase activity; this effect was completely abolished by the addition of regucalcin (0.1 microM). The effect of anti-regucalcin antibody (50 ng/ml) in increasing Ca(2+)-ATPase activity was completely prevented by the presence of thapsigargin (10(-6) M), an inhibitor of Ca(2+) sequestrating enzyme, N-ethylmaleimide (1 mM), a modifying reagent of thiol groups, or vanadate (10(-5) M), an inhibitor of phosphorylation of the enzyme by ATP, which revealed an inhibitory effect on nuclear Ca(2+)-ATPase activity. Meanwhile, the effect of anti-regucalcin antibody (50 ng/ml) was significantly enhanced by the addition of calmodulin (5 microg/ml), which could increase nuclear Ca(2+)-ATPase activity. In addition, the effect of antibody (50 ng/ml) was significantly reduced by the presence of trifluoperazine (20 microM), an antagonist of calmodulin. These results suggest that the endogenous regucalcin in liver nuclei has a suppressive effect on nuclear Ca(2+)-ATPase activity, and that regucalcin can inhibit an activating effect of calmodulin on the enzyme.     

Glucose 6-phosphate and hexokinase can be used as an ATP-regenerating system by the Ca(2+)-ATPase of sarcoplasmic reticulum.

In the presence of hexokinase, vesicles derived from the sarcoplasmic reticulum of skeletal muscle are able to accumulate Ca2+ in a medium containing ADP and glucose 6-phosphate. No significant Ca2+ uptake is observed if one of these components is omitted from the assay medium. Due to its high affinity for ATP, the Ca(2+)-ATPase can use the very low concentrations of ATP formed from glucose 6-phosphate and ADP to form a Ca2+ gradient. This finding indicates that glucose 6-phosphate and hexokinase can be used as an ATP-regenerating system. The Ca2+ uptake supported by glucose 6-phosphate and ADP is inhibited by glucose and D-xylose. Half-maximal inhibition is observed in the presence of 0.4 mM glucose and 100 mM D-xylose. The transport ratio (Ca2+ transported:substrate utilized) is the same for glucose 6-phosphate and ATP. The Ca2+ gradient formed when glucose 6-phosphate and ADP are the substrates can be used to synthesize ATP from ADP and Pi. The concentration of ATP formed after reversal of the Ca2+ pump is much higher than that expected from direct equilibration of the reaction between glucose 6-phosphate and ADP.     

Ca(2+)-calmodulin regulates receptor-operated Ca(2+) entry activity of TRPC6 in HEK-293 cells

Mammalian homologues of the Drosophila transient receptor potential channel (TRPC) are involved in Ca(2+) entry following agonist stimulation of nonexcitable cells. Seven mammalian TRPCs have been cloned but their mechanisms of activation and/or regulation are still the subject of intense research efforts. It has already been shown that calmodulin (CaM) can regulate the activity of Drosophila TRP and TRPL and, more recently, CaM has been shown to interact with mammalian TRPCs. In this study, TRPC6 stably transfected into HEK-293 cells was used to investigate the possible influence of CaM on TRPC6-dependent Ca(2+) entry. Overexpression of TRPC6 in mammalian cells is known to enhance agonist-induced Ca(2+) entry, but not thapsigargin-induced Ca(2+) entry. Here, we show that CaM inhibitors (calmidazolium and trifluoperazine) abolish receptor-operated Ca(2+) entry (ROCE) without affecting thapsigargin-operated Ca(2+) entry and that the activity of CaM is dependent on complexation with Ca(2+). We also show that Ca(2+)-CaM binds to TRPC6 and that the binding can be abolished by CaM inhibitors. These results indicate that CaM is involved in the modulation of ROCE.     

Mitochondria fine-tune the slow Ca(2+) transients induced by electrical stimulation of skeletal myotubes

Mitochondria sense cytoplasmic Ca(2+) signals in many cell types. In mammalian skeletal myotubes, depolarizing stimuli induce two independent cytoplasmic Ca(2+) signals: a fast signal associated with contraction and a slow signal that propagates to the nucleus and regulates gene expression. How mitochondria sense and possibly affect these cytoplasmic Ca(2+) signals has not been reported. We investigated here (a) the emergence of mitochondrial Ca(2+) signals in response to electrical stimulation of myotubes, (b) the contribution of mitochondrial Ca(2+) transients to ATP generation and (c) the influence of mitochondria as modulators of cytoplasmic and nuclear Ca(2+) signals. Rhod2 and Fluo3 fluorescence determinations revealed composite Ca(2+) signals associated to the mitochondrial compartment in electrically stimulated (400 pulses, 45 Hz) skeletal myotubes. Similar Ca(2+) signals were detected when using a mitochondria-targeted pericam. The fast mitochondrial Ca(2+) rise induced by stimulation was inhibited by pre-incubation with ryanodine, whereas the phospholipase C inhibitor U73122 blocked the slow mitochondrial Ca(2+) signal, showing that mitochondria sense the two cytoplasmic Ca(2+) signal components. The fast but not the slow Ca(2+) transient enhanced mitochondrial ATP production. Inhibition of the mitochondrial Ca(2+) uniporter prevented the emergence of mitochondrial Ca(2+) transients and significantly increased the magnitude of slow cytoplasmic Ca(2+) signals after stimulation. Precluding mitochondrial Ca(2+) extrusion with the Na(+)/Ca(2+) exchanger inhibitor CGP37157 decreased mitochondrial potential, increased the magnitude of the slow cytoplasmic Ca(2+) signal and decreased the rate of Ca(2+) signal propagation from one nucleus to the next. Over expression of the mitochondrial fission protein Drp-1 decreased mitochondrial size and the slow Ca(2+) transient in mitochondria, but enhanced cytoplasmic and nuclear slow transients. The present results indicate that mitochondria play a central role in the regulation of Ca(2+) signals involved in gene expression in myotubes.     

Particulars on the regulation of Ca(2+) in the cell nucleus

The data of literature on the structural and functional features of nuclear pore complexes, the role of receptor-activated Ca(2+)-channels and Ca(2+)-ATPase of nuclear membranes are presented. The possible ways of Ca(2+)-signal transmission into the nucleus and the role of nuclear Ca2+ in apoptosis are discussed.     

Dynamic regulation of sarcoplasmic reticulum Ca(2+) content and release by luminal Ca(2+)-sensitive leak in rat ventricular myocytes.

In cardiac muscle, excitation-contraction (E-C) coupling is determined by the ability of the sarcoplasmic reticulum (SR) to store and release Ca(2+). It has been hypothesized that the Ca(2+) sequestration and release mechanisms might be functionally linked to optimize the E-C coupling process. To explore the relationships between the loading status of the SR and functional state of the Ca(2+) release mechanism, we examined the effects of changes in SR Ca(2+) content on spontaneous Ca(2+) sparks in saponin-permeabilized and patch-clamped rat ventricular myocytes. SR Ca(2+) content was manipulated by pharmacologically altering the capacities of either Ca(2+) uptake or leak. Ca(2+) sparks were recorded using a confocal microscope and Fluo-3 and were quantified considering missed events. SR Ca(2+) content was assessed by application of caffeine. Exposure of permeabilized cells to anti-phospholamban antibodies elevated the SR Ca(2+) content and increased the frequency of sparks. Suppression of the SR Ca(2+) pump by thapsigargin lowered [Ca(2+)](SR) and reduced the frequency of sparks. The ryanodine receptor (RyR) blockers tetracaine and Mg(2+) transiently suppressed the frequency of sparks. Upon washout of the drugs, sparking activity transiently overshot control levels. Low doses of caffeine transiently potentiated sparking activity upon application and transiently depressed the sparks upon removal. In patch-clamped cardiac myocytes, exposure to caffeine produced only a transient increase in the probability of sparks induced by depolarization. We interpret these results in terms of a novel dynamic control scheme for SR Ca(2+) cycling. A central element of this scheme is a luminal Ca(2+) sensor that links the functional activity of RyRs to the loading state of the SR, allowing cells to auto-regulate the size and functional state of their SR Ca(2+) pool. These results are important for understanding the regulation of intracellular Ca(2+) release and contractility in cardiac muscle.     

Ca(2+) sensors of L-type Ca(2+) channel

Ca(2+)-induced inactivation of L-type Ca(2+) is differentially mediated by two C-terminal motifs of the alpha(1C) subunit, L (1572-1587) and K (1599-1651) implicated for calmodulin binding. We found that motif L is composed of a highly selective Ca(2+) sensor and an adjacent Ca(2+)-independent tethering site for calmodulin. The Ca(2+) sensor contributes to higher Ca(2+) sensitivity of the motif L complex with calmodulin. Since only combined mutation of both sites removes Ca(2+)-dependent current decay, the two-site modulation by Ca(2+) and calmodulin may underlie Ca(2+)-induced inactivation of the channel.     

A dual role for Ca(2+) in autophagy regulation

Autophagy is a cellular process responsible for delivery of proteins or organelles to lysosomes. It participates not only in maintaining cellular homeostasis, but also in promoting survival during cellular stress situations. It is now well established that intracellular Ca²⁺ is one of the regulators of autophagy. However, this control of autophagy by intracellular Ca²⁺ signaling is the subject of two opposite views. On the one hand, the available evidence indicates that intracellular Ca²⁺ signals, and mainly inositol 1,4,5-trisphosphate receptors (IP₃Rs), suppress autophagy. On the other hand, elevated cytosolic Ca²⁺ concentrations ([Ca²⁺]_cyt) were also shown to promote the autophagic process. Here, we will provide a critical overview of the literature and discuss both hypotheses. Moreover, we will suggest a model explaining how changes in intracellular Ca²⁺ signaling can lead to opposite outcomes, depending on the cellular state.     

Role of the beta1 subunit in large-conductance Ca(2+)-activated K(+) channel gating energetics. Mechanisms of enhanced Ca(2+) sensitivity

Over the past few years, it has become clear that an important mechanism by which large-conductance Ca²⁺-activated K⁺ channel (BK_Ca) activity is regulated is the tissue-specific expression of auxiliary β subunits. The first of these to be identified, β1, is expressed predominately in smooth muscle and causes dramatic effects, increasing the apparent affinity of the channel for Ca²⁺ 10-fold at 0 mV, and shifting the range of voltages over which the channel activates −80 mV at 9.1 μM Ca²⁺. With this study, we address the question: which aspects of BK_Ca gating are altered by β1 to bring about these effects: Ca²⁺ binding, voltage sensing, or the intrinsic energetics of channel opening? The approach we have taken is to express the β1 subunit together with the BK_Ca α subunit in Xenopus oocytes, and then to compare β1''s steady state effects over a wide range of Ca²⁺ concentrations and membrane voltages to those predicted by allosteric models whose parameters have been altered to mimic changes in the aspects of gating listed above. The results of our analysis suggest that much of β1''s steady state effects can be accounted for by a reduction in the intrinsic energy the channel must overcome to open and a decrease in its voltage sensitivity, with little change in the affinity of the channel for Ca²⁺ when it is either open or closed. Interestingly, however, the small changes in Ca²⁺ binding affinity suggested by our analysis (K_c 7.4 μM → 9.6 μM; K_o = 0.80 μM → 0.65 μM) do appear to be functionally important. We also show that β1 affects the mSlo conductance–voltage relation in the essential absence of Ca²⁺, shifting it +20 mV and reducing its apparent gating charge 38%, and we develop methods for distinguishing between alterations in Ca²⁺ binding and other aspects of BK_Ca channel gating that may be of general use.     

Role of Ca(2+) channels in short-term synaptic plasticity

Repetitive nerve activity induces various forms of short-term synaptic plasticity that have important computational roles in neuronal networks. Several forms of short-term plasticity are caused largely by changes in transmitter release, but the mechanisms that underlie these changes in the release process have been difficult to address. Recent studies of a giant synapse - the calyx of Held - have shed new light on this issue. Recordings of Ca(2+) currents or Ca(2+) concentrations at nerve terminals reveal that regulation of presynaptic Ca(2+) channels has a significant role in three important forms of short-term plasticity: short-term depression, facilitation and post-tetanic potentiation.     

Phospholamban regulation of cardiac sarcoplasmic reticulum (Ca(2+)-Mg2+)-ATPase. Mechanism of regulation and site of monoclonal antibody interaction.

Monoclonal antibodies raised against canine cardiac sarcoplasmic reticulum phospholamban were used to study the structure-function relationship between phospholamban and the sarcoplasmic reticulum (SR) (Ca(2+)-Mg2+)-ATPase (Suzuki, T., and Wang, J. H. (1986) J. Biol. Chem. 261, 7018-7023). Additional monoclonal antibodies are characterized further. When five of these monoclonal antibodies were assessed for their ability to affect SR Ca2+ uptake three of these antibodies had no effect on SR Ca2+ uptake, whereas the other two monoclonals were able to stimulate SR Ca2+ uptake to levels similar to those caused by phosphorylation of phospholamban at different calcium concentrations. Using synthetic peptides corresponding to various portions of phospholamban in a competitive binding assay, it was possible to map the epitope site of monoclonals which stimulate the (Ca(2+)-Mg2+)-ATPase activity to phospholamban residues 7-16. These results implicate phospholamban residues 7-16 in the regulation of the (Ca(2+)-Mg2+)-ATPase.