Intracellular stores maintain stable cytosolic Ca gradients in epithelial cells by active Ca redistribution

A stable localized region of high calcium concentration near the plasma membrane has been postulated to exist as an outcome of prolonged calcium influx and to play a crucial role in regulation of cellular life. However, the mechanism supporting this phenomenon is a perplexing problem. We show here that a sustained localized region of high cytosolic Ca(2+) concentration is formed near the plasma membrane. Calcium influx, calcium uptake by intracellular stores and calcium release from the stores are essential for this phenomenon. Our results strongly suggest that the mechanism of formation of stable calcium gradient near the plasma membrane involves a process of active redistribution-uptake of entering calcium into intracellular stores and its release from the stores toward the plasma membrane.     

The regulatory role of mitochondria in capacitative calcium entry

Capacitative regulation of calcium entry is a major mechanism of Ca2+ influx into electrically non-excitable cells, but it also operates in some excitable ones. It participates in the refilling of intracellular calcium stores and in the generation of Ca2+ signals in excited cells. The mechanism which couples depletion of intracellular calcium stores located in the endoplasmic reticulum with opening of store-operated calcium channels in the plasma membrane is not clearly understood. Mitochondria located in close proximity to Ca2+ channels are exposed to high Ca2+ concentration, and therefore, they are able to accumulate this cation effectively. This decreases local Ca2+ concentration and thereby affects calcium-dependent processes, such as depletion and refilling of the intracellular calcium stores and opening of the store-operated channels. Finally, mitochondria modulate the intensity and the duration of calcium signals induced by extracellular stimuli. Ca2+ uptake by mitochondria requires these organelles to be in the energized state. On the other hand, Ca2+ flux into mitochondria stimulates energy metabolism. To sum up, mitochondria couple cellular metabolism with calcium homeostasis and signaling.     

Suppression of TRPC3 leads to disappearance of store-operated channels and formation of a new type of store-independent channels in A431 cells

In most non-excitable cells, calcium (Ca(2+)) release from the inositol 1,4,5-trisphosphate (InsP(3))-sensitive intracellular Ca(2+) stores is coupled to Ca(2+) influx through the plasma membrane Ca(2+) channels whose molecular composition is poorly understood. Several members of mammalian TRP-related protein family have been implicated to both receptor- and store-operated Ca(2+) influx. Here we investigated the role of the native transient receptor potential 3 (TRPC3) homologue in mediating the store- and receptor-operated calcium entry in A431 cells. We show that suppression of TRPC3 protein levels by small interfering RNA (siRNA) leads to a significant reduction in store-operated calcium influx without affecting the receptor-operated calcium influx. With single-channel analysis, we further demonstrate that reduction of TRPC3 levels results in suppression of specific subtype of store-operated calcium channels and activation of store-independent channels. Our data suggest that TRPC3 is required for the formation of functional store-operated channels in A431 cells.     

Capacitative calcium entry in the nervous system

Capacitative calcium entry is a process whereby the depletion of Ca(2+) from intracellular stores (likely endoplasmic or sarcoplasmic reticulum) activates plasma membrane Ca(2+) channels. Current research has focused on identification of capacitative calcium entry channels and the mechanism by which Ca(2+) store depletion activates the channels. Leading candidates for the channels are members of the transient receptor potential (TRP) superfamily, although no single gene or gene product has been definitively proven to mediate capacitative calcium entry. The mechanism for activation of the channels is not known; proposals fall into two general categories, either a diffusible signal released from the Ca(2+) stores when their Ca(2+) levels become depleted, or a more direct protein-protein interaction between constituents of the endoplasmic reticulum and the plasma membrane channels. Capacitative calcium entry is a major mechanism for regulated Ca(2+) influx in non-excitable cells, but recent research has indicated that this pathway plays an important role in the function of neuronal cells, and may be important in a number of neuropathological conditions. This review will summarize some of these more recent findings regarding the role of capacitative calcium entry in normal and pathological processes in the nervous system.     

Calreticulin couples calcium release and calcium influx in integrin-mediated calcium signaling

The engagement of integrin alpha7 in E63 skeletal muscle cells by laminin or anti-alpha7 antibodies triggered transient elevations in the intracellular free Ca(2+) concentration that resulted from both inositol triphosphate-evoked Ca(2+) release from intracellular stores and extracellular Ca(2+) influx through voltage-gated, L-type Ca(2+) channels. The extracellular domain of integrin alpha7 was found to associate with both ectocalreticulin and dihydropyridine receptor on the cell surface. Calreticulin appears to also associate with cytoplasmic domain of integrin alpha7 in a manner highly dependent on the cytosolic Ca(2+) concentration. It appeared that intracellular Ca(2+) release was a prerequisite for Ca(2+) influx and that calreticulin associated with the integrin cytoplasmic domain mediated the coupling of between the Ca(2+) release and Ca(2+) influx. These findings suggest that calreticulin serves as a cytosolic activator of integrin and a signal transducer between integrins and Ca(2+) channels on the cell surface.     

Role of proton gradients and vacuola H(+)-ATPases in the refilling of intracellular calcium stores in exocrine cells

Numerous hormones and neurotransmitters activate cells by increasing cytosolic calcium concentration ([Ca(2+)](i)), a key regulatory factor for many cellular processes. A pivotal feature of these Ca(2+) signals is the release of Ca(2+) from intracellular stores, which is followed by activation of extracellular calcium influx, allowing refilling of the stores by SERCA pumps associated with the endoplasmic reticulum. Although the mechanisms of calcium release and calcium influx have been extensively studied, the biology of the Ca(2+) stores is poorly understood. The presence of heterogeneous calcium pools in cells has been previously reported [1] [2] [3]. Although recent technical improvements have confirmed this heterogeneity [4], knowledge about the mechanisms underlying Ca(2+) transport within the stores is very scarce and rather speculative. A recent study in polarized exocrine cells [5] has revealed the existence of Ca(2+) tunneling from basolateral stores to luminal pools, where Ca(2+) is initially released upon cell activation. Here, we present evidence that, during stimulation, Ca(2+) transported into basolateral stores by SERCA pumps is conveyed toward the luminal pools driven by proton gradients generated by vacuolar H(+)-ATPases. This finding unveils a new aspect of the machinery of Ca(2+) stores.     

Calcium dynamics in the peroxisomal lumen of living cells

We here describe the generation of novel, green fluorescent protein-based Ca(2+) indicators targeted to the peroxisome lumen. We show that (i) the Ca(2+) concentration of peroxisomes in living cells at rest is similar to that of the cytosol; (ii) increases in cytosolic Ca(2+) concentration (elicited by either Ca(2+) mobilization from stores or Ca(2+) influx through plasma membrane Ca(2+) channels) are followed by a slow rise in intraperoxisomal [Ca(2+)]; (iii) Ca(2+) influx into peroxisomes is driven neither by an ATP-dependent pump nor by membrane potential nor by a H(+)(Na(+)) gradient. The peroxisomal membrane appears to play a low pass filter role, preventing the organelle from taking up shortlasting cytosolic Ca(2+) transients but allowing equilibration of the peroxisomal luminal [Ca(2+)] with that of the cytosol during prolonged Ca(2+) increases. Thus, peroxisomes appear to be an additional cytosolic Ca(2+) buffer, but their influx and efflux mechanisms are unlike those of any other cellular organelle.     

Respiring mitochondria determine the pattern of activation and inactivation of the store-operated Ca(2+) current I(CRAC)

In eukaryotic cells, hormones and neurotransmitters that engage the phosphoinositide pathway evoke a biphasic increase in intracellular free Ca(2+) concentration: an initial transient release of Ca(2+) from intracellular stores is followed by a sustained phase of Ca(2+) influx. This influx is generally store dependent. Most attention has focused on the link between the endoplasmic reticulum and store-operated Ca(2+) channels in the plasma membrane. Here, we describe that respiring mitochondria are also essential for the activation of macroscopic store-operated Ca(2+) currents under physiological conditions of weak intracellular Ca(2+) buffering. We further show that Ca(2+)-dependent slow inactivation of Ca(2+) influx, a widespread but poorly understood phenomenon, is regulated by mitochondrial buffering of cytosolic Ca(2+). Thus, by enabling macroscopic store-operated Ca(2+) current to activate, and then by controlling its extent and duration, mitochondria play a crucial role in all stages of store-operated Ca(2+) influx. Store-operated Ca(2+) entry reflects a dynamic interplay between endoplasmic reticulum, mitochondria and plasma membrane.     

Pharmacology of capacitative calcium entry

The versatility of Ca(2+) as a messenger in multiple signaling events requires that the concentration of calcium ions within the cytoplasm be highly regulated. In particular, the release of calcium from intracellular stores must often be linked to calcium influx across the cell membrane. Capacitative calcium entry, whereby the depletion of intracellular Ca(2+) stores induces the influx of extracellular calcium, is a crucial element of concerted calcium signaling. Investigations into the phenomenon are contributing to a new appreciation for the organized cytoplasmic framework that supports calcium signaling.     

Functional properties of endogenous receptor- and store-operated calcium influx channels in HEK293 cells

Activation of phospholipase C (PLC)-mediated signaling pathways in non-excitable cells causes the release of calcium (Ca2+) from inositol 1,4,5-trisphosphate (InsP3)-sensitive intracellular Ca2+ stores and activation of Ca2+ influx via plasma membrane Ca2+ channels. The properties and molecular identity of plasma membrane Ca2+ influx channels in non-excitable cells is a focus of intense investigation. In the previous studies we used patch clamp electrophysiology to describe the properties of Ca2+ influx channels in human carcinoma A431 cell lines. Now we extend our studies to human embryonic kidney HEK293 cells. By using a combination of Ca2+ imaging and whole cell and single channel patch clamp recordings we discovered that: 1) HEK293 cells contain four types of plasma membrane Ca2+ influx channels: I(CRAC), Imin, Imax, and I(NS); 2) I(CRAC) channels are highly Ca2+-selective (P(Ca/Cs)>1000) and I(CRAC) single channel conductance is too small for single channel analysis; 3) Imin channels in HEK293 cells display functional properties identical to Imin channels in A431 cells, with single channel conductance of 1.2 pS for divalent cations, 10 pS for monovalent cations, and divalent cation selectivity P(Ba/K)=20; 4) Imin channels in HEK293 cells are activated by InsP3 and inhibited by phosphatidylinositol 4,5-bisphosphate, but store-independent; 5) when compared with Imin, Imax channels have higher conductance for divalent (17 pS) and monovalent (33 pS) cations, but less selective for divalent cations (P(Ba/K)=4), 6) Imax channels in HEK293 cells can be activated by InsP3 or by Ca2+ store depletion; 7) I(NS) channels are non-selective (P(Ba/K)=0.4) and display a single channel conductance of 5 pS; and 8) I(NS) channels are not gated by InsP3 but activated by depletion of intracellular Ca2+ stores. Our findings provide novel information about endogenous Ca2+ channels supporting receptor-operated and store-operated Ca2+ influx pathways in HEK293 cells.     

Properties of a native cation channel activated by Ca2+ store depletion in vascular smooth muscle cells

Depletion of intracellular Ca(2+) stores activates capacitative Ca(2+) influx in smooth muscle cells, but the native store-operated channels that mediate such influx remain unidentified. Recently we demonstrated that calcium influx factor produced by yeast and human platelets with depleted Ca(2+) stores activates small conductance cation channels in excised membrane patches from vascular smooth muscle cells (SMC). Here we characterize these channels in intact cells and present evidence that they belong to the class of store-operated channels, which are activated upon passive depletion of Ca(2+) stores. Application of thapsigargin (TG), an inhibitor of sarco-endoplasmic reticulum Ca(2+) ATPase, to individual SMC activated single 3-pS cation channels in cell-attached membrane patches. Channels remained active when inside-out membrane patches were excised from the cells. Excision of membrane patches from resting SMC did not by itself activate the channels. Loading SMC with BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid), which slowly depletes Ca(2+) stores without a rise in intracellular Ca(2+), activated the same 3-pS channels in cell-attached membrane patches as well as whole cell nonselective cation currents in SMC. TG- and BAPTA-activated 3-pS channels were cation-selective but poorly discriminated among Ca(2+), Sr(2+), Ba(2+), Na(+), K(+), and Cs(+). Open channel probability did not change at negative membrane potentials but increased significantly at high positive potentials. Activation of 3-pS channels did not depend on intracellular Ca(2+) concentration. Neither TG nor a variety of second messengers (including Ca(2+), InsP3, InsP4, GTPgammaS, cyclic AMP, cyclic GMP, ATP, and ADP) activated 3-pS channels in inside-out membrane patches. Thus, 3-pS nonselective cation channels are present and activated by TG or BAPTA-induced depletion of intracellular Ca(2+) stores in intact SMC. These native store-operated cation channels can account for capacitative Ca(2+) influx in SMC and can play an important role in regulation of vascular tone.     

Signaling between intracellular Ca2+ stores and depletion-activated Ca2+ channels generates [Ca2+]i oscillations in T lymphocytes

Stimulation through the antigen receptor (TCR) of T lymphocytes triggers cytosolic calcium ([Ca2+]i) oscillations that are critically dependent on Ca2+ entry across the plasma membrane. We have investigated the roles of Ca2+ influx and depletion of intracellular Ca2+ stores in the oscillation mechanism, using single-cell Ca2+ imaging techniques and agents that deplete the stores. Thapsigargin (TG; 5-25 nM), cyclopiazonic acid (CPA; 5-20 microM), and tert- butylhydroquinone (tBHQ; 80-200 microM), inhibitors of endoplasmic reticulum Ca(2+)-ATPases, as well as the Ca2+ ionophore ionomycin (5-40 nM), elicit [Ca2+]i oscillations in human T cells. The oscillation frequency is approximately 5 mHz (for ATPase inhibitors) to approximately 10 mHz (for ionomycin) at 22-24 degrees C. The [Ca2+]i oscillations resemble those evoked by TCR ligation in terms of their shape, amplitude, and an absolute dependence on Ca2+ influx. Ca(2+)- ATPase inhibitors and ionomycin induce oscillations only within a narrow range of drug concentrations that are expected to cause partial depletion of intracellular stores. Ca(2+)-induced Ca2+ release does not appear to be significantly involved, as rapid removal of extracellular Ca2+ elicits the same rate of [Ca2+]i decline during the rising and falling phases of the oscillation cycle. Both transmembrane Ca2+ influx and the content of ionomycin-releasable Ca2+ pools fluctuate in oscillating cells. From these data, we propose a model in which [Ca2+]i oscillations in T cells result from the interaction between intracellular Ca2+ stores and depletion-activated Ca2+ channels in the plasma membrane.     

Sequence, cloning, expression and characterisation of the 81-kDa surface membrane protein (P80) of Mycoplasma agalactiae

In response to heat-stable enterotoxin of Vibrio cholerae non-O1, the initial rise of cytosolic Ca(2+) occurred with activation of IP(3). Chelation of extracellular Ca(2+) with EGTA and suspension of cells in Ca(2+) free buffer both demonstrated the involvement of internal stores in the rise of [Ca(2+)]i. Cells pretreated with dantrolene resulted in decrease of [Ca(2+)]i response which suggested that the rise of intracellular level of Ca(2+) was mostly due to the mobilization from IP(3) sensitive stores. When the cytosolic Ca(2+) was chelated by loading the cells with BAPTA, NAG-ST could not induce Ca(2+) entry to the cell as assessed by Mn(2+) quenching of fura-2 fluorescence which suggested that calcium influx across the plasma membrane depends upon initial rise of this bivalent cation that maintained the sustained phase of [Ca(2+)]i response. Addition of toxin to the fura-2-loaded cells, preincubated with lanthanum chloride, resulted in reduction of [Ca(2+)]i level with a short duration of irregular sustained phase further suggesting that the influx of Ca(2+) across the plasma membrane might be through the calcium channel.     

Calcium signalling in secretory cells

Stimulation of secretory cells with muscarinic agonists leads to an increase in the intracellular Ca (2+)concentration ([Ca (2+)]( i)), which activates protein secretion through exocytosis and causes closure of gap junctions between adjacent cells. In addition, the increase in [Ca (2+)](i) activates three different kinds of ion channels: large K(+) channels, Cl(-) channels and non-specific cation channels. The opening of those channels leads to an increase of [Na(+ )] and a decrease of [Cl(-)] and [K(+) ] in the cell. The two components that contribute to the increase in [Ca (2+)]( i) are calcium release from intracellular stores, localised in the endoplasmic reticulum and calcium influx through the plasma membrane. Several models for the regulation of [Ca (2+)](i) have been proposed, including a recently suggested model whereby a distinct pathway involving arachidonic acid is added to the well-established capacitative model. Different hypotheses concerning coupling between the intra-cellular calcium stores and membrane channels co-exist. In addition to a historical overview, recent developments and future challenges are discussed in this review.     

Ca2+-calmodulin-dependent facilitation and Ca2+ inactivation of Ca2+ release-activated Ca2+ channels

In non-excitable cells, one major route for Ca2+ influx is through store-operated Ca2+ channels in the plasma membrane. These channels are activated by the emptying of intracellular Ca2+ stores, and in some cell types store-operated influx occurs through Ca2+ release-activated Ca2+ (CRAC) channels. Here, we report that intracellular Ca2+ modulates CRAC channel activity through both positive and negative feedback steps in RBL-1 cells. Under conditions in which cytoplasmic Ca2+ concentration can fluctuate freely, we find that store-operated Ca2+ entry is impaired either following overexpression of a dominant negative calmodulin mutant or following whole-cell dialysis with a calmodulin inhibitory peptide. The peptide had no inhibitory effect when intracellular Ca2+ was buffered strongly at low levels. Hence, Ca2+-calmodulin is not required for the activation of CRAC channels per se but is an important regulator under physiological conditions. We also find that the plasma membrane Ca2+ATPase is the dominant Ca2+ efflux pathway in these cells. Although the activity of the Ca2+ pump is regulated by calmodulin, the store-operated Ca2+ entry is more sensitive to inhibition by the calmodulin mutant than by Ca2+ extrusion. Hence, these two plasmalemmal Ca2+ transport systems may differ in their sensitivities to endogenous calmodulin. Following the activation of Ca2+ entry, the rise in intracellular Ca2+ subsequently feeds back to further inhibit Ca2+ influx. This slow inactivation can be activated by a relatively brief Ca2+ influx (30-60 s); it reverses slowly and is not altered by overexpression of the calmodulin mutant. Hence, the same messenger, intracellular Ca2+, can both facilitate and inactivate Ca2+ entry through store-operated CRAC channels and through different mechanisms.     

A realistic model of biphasic calcium transients in electrically nonexcitable cells.

In many electrically nonexcitable cells, the release of calcium from internal stores is followed by a much slower phase in which the intracellular calcium concentration decreases gradually to a sustained value higher than the concentration before stimulation. This elevated calcium plateau has been shown to be the result of calcium influx. The model presented in this work describes a system consisting of a cytoplasmic calcium store and a plasma membrane calcium channel, both excitable by a membrane receptor; a fast cytoplasmic calcium buffer; and calcium pumps in both the calcium store and cellular membranes. Inherent difficulties in the numerical evaluation of the model, caused by very large calcium fluxes across the store membrane, were overcome by analytically separating the fast processes of calcium release from the slower processes of calcium cycling across the plasma membrane. This enabled the simulation of realistic biphasic calcium transients similar to those observed experimentally. The model predicted 1) a strong correlation between the rate of calcium cycling across the plasma membrane and the rate of calcium decay; and 2) a dependence on the level of cell excitation of the maximum rise in cytoplasmic calcium concentration, the level of the elevated calcium plateau, and the rate of calcium decay. Using the model, we simulated the washout of agonist from the bathing solution and the depletion of the calcium store by a pharmacological agent (such as thapsigargin) under several experimental conditions.     

Contribution of intracellular calcium stores to an increase in cytosolic calcium concentration induced by Mannheimia haemolytica leukotoxin

The contribution of intracellular calcium stores to Mannheimia haemolytica leukotoxin (LKT)-induced increase in cytosolic calcium concentration was studied by pharmacologically inhibiting transport of calcium across the plasma and endoplasmic reticulum membranes of bovine neutrophils exposed to LKT. Active intracellular storage of calcium by sarcoplasmic/endoplasmic reticulum calcium ATPase, influx of extracellular calcium across the plasma membrane, and release of stored calcium via inositol triphosphate receptors and ryanodine-sensitive calcium channels were inhibited using thapsigargin, lanthanum chloride, xestospongin C, and magnesium chloride, respectively. Pre-incubation with thapsigargin attenuated the increase in cytosolic calcium concentration produced by LKT, thus confirming the involvement of intracellular calcium stores. Inhibitory effects of lanthanum chloride, xestospongin C, and magnesium chloride indicated that the increase in cytosolic calcium concentration induced by LKT resulted from both influx of calcium across the plasma membrane and release of calcium from intracellular stores.     

Regulation of receptor-mediated calcium influx across the plasma membrane in a human leukemic T-cell line: evidence of its dependence on an initial calcium mobilization from intracellular stores

It has been repeatedly shown that stimulation of a human leukemic T-cell line, JURKAT, by lectins such as phytohaemagglutinin and anti-T3 antibody (OKT3) leads to an elevation in the concentration of cytosolic free Ca2. This Ca2+ transient results from both an intracellular mobilization and an influx of Ca2+ through specific membrane channels. The objective of this study was to investigate the mechanism by which receptor-mediated influx of Ca2+ is regulated in JURKAT cells, which demonstrably lack 'voltage-dependent calcium channels'. It was found that upon increased loading with quin2 or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA) there was a pronounced decline of both phytohaemagglutinin-stimulated and OKT3-stimulated influx of 45Ca2+. Using 15 microM quin2/AM or 30 microM BAPTA/AM, agonist-stimulated 45Ca2+ influx was almost totally abolished. At these concentrations of both quin2/AM or BAPTA/AM, phytohaemagglutinin and OKT3 could still induce a rise of cytosolic free Ca2+ above 200 nM. In the presence of La3+ (200 microM), which completely inhibited the agonist-induced 45Ca2+ influx, both phytohaemagglutinin and OKT3 were able to raise the concentrations of cytosolic free Ca2+ to well above 200 nM by merely mobilizing Ca2+ from intracellular stores alone. The data suggest that an agonist-induced increase in the concentration of cytosolic free Ca2+, due to mobilization from intracellular stores, could either directly or indirectly, initiate receptor-mediated Ca2+ influx across the plasma membrane in JURKAT cells.     

Calcium signaling in non-excitable cells: Ca2+ release and influx are independent events linked to two plasma membrane Ca2+ entry channels

The regulatory mechanism of Ca2+ influx into the cytosol from the extracellular space in non-excitable cells is not clear. The "capacitative calcium entry" (CCE) hypothesis suggested that Ca2+ influx is triggered by the IP(3)-mediated emptying of the intracellular Ca2+ stores. However, there is no clear evidence for CCE and its mechanism remains elusive. In the present work, we have provided the reported evidences to show that inhibition of IP(3)-dependent Ca2+ release does not affect Ca2+ influx, and the experimental protocols used to demonstrate CCE can stimulate Ca2+ influx by means other than emptying of the Ca2+ stores. In addition, we have presented the reports showing that IP(3)-mediated Ca2+ release is linked to a Ca2+ entry from the extracellular space, which does not increase cytosolic [Ca2+] prior to Ca2+ release. Based on these and other reports, we have provided a model of Ca2+ signaling in non-excitable cells, in which IP(3)-mediated emptying of the intracellular Ca2+ store triggers entry of Ca2+ directly into the store, through a plasma membrane TRPC channel. Thus, emptying and direct refilling of the Ca2+ stores are repeated in the presence of IP(3), giving rise to the transient phase of oscillatory Ca2+ release. Direct Ca2+ entry into the store is regulated by its filling status in a negative and positive manner through a Ca2+ -binding protein and Stim1/Orai complex, respectively. The sustained phase of Ca2+ influx is triggered by diacylglycerol (DAG) through the activation of another TRPC channel, independent of Ca2+ release. The plasma membrane IP(3) receptor (IP(3)R) plays an essential role in Ca2+ influx, by interacting with the DAG-activated TRPC, without the requirement of binding to IP(3).