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J. Biol. Chem., Vol. 283, Issue 8, 4622-4631, February 22, 2008

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Local Ca²⁺ Influx through Ca²⁺ Release-activated Ca²⁺ (CRAC) Channels Stimulates Production of an Intracellular Messenger and an Intercellular Pro-inflammatory Signal^*

From the Department of Physiology, Anatomy and Genetics, Sherrington Building, University of Oxford, Parks Road, Oxford OX1 3PT, United Kingdom

Received for publication, June 18, 2007 , and in revised form, December 20, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Ca²⁺ entry through store-operated Ca²⁺ channels drives the production of the pro-inflammatory molecule leukotriene C₄ (LTC₄) from mast cells through a pathway involving Ca²⁺-dependent protein kinase C, mitogen-activated protein kinases ERK1/2, phospholipase A₂, and 5-lipoxygenase. Here we examine whether local Ca²⁺ influx through store-operated Ca²⁺ release-activated Ca²⁺ (CRAC) channels in the plasma membrane stimulates this signaling pathway. Manipulating the amplitude and spatial extent of Ca²⁺ entry by altering chemical and electrical gradients for Ca²⁺ influx or changing the Ca²⁺ buffering of the cytoplasm all impacted on protein kinase C and ERK activation, generation of arachidonic acid and LTC₄ secretion, with little change in the bulk cytoplasmic Ca²⁺ rise. Similar bulk cytoplasmic Ca²⁺ concentrations were achieved when CRAC channels were activated in 0.25 mM external Ca²⁺ versus 2 mM Ca²⁺ and 100 nM La³⁺, an inhibitor of CRAC channels. However, despite similar bulk cytoplasmic Ca²⁺, protein kinase C activation and LTC₄ secretion were larger in 2 mM Ca²⁺ and La³⁺ than in 0.25 mM Ca²⁺, consistent with the central involvement of a subplasmalemmal Ca²⁺ rise. The nonreceptor tyrosine kinase Syk coupled CRAC channel opening to protein kinase C and ERK activation. Recombinant TRPC3 channels also activated protein kinase C, suggesting that subplasmalemmal Ca²⁺ rather than a microdomain exclusive to CRAC channels is the trigger. Hence a subplasmalemmal Ca²⁺ increase in mast cells is highly versatile in that it triggers cytoplasmic responses through generation of intracellular messengers as well as long distance changes through increased secretion of paracrine signals.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
One of the most fundamental questions in cellular signaling is how a relatively small complement of second messenger pathways can provide specificity to the enormous number of hormones, neurotransmitters, and other signaling molecules that impinge on cell-surface receptors. Valuable insight into this issue has been gleaned from studies on a ubiquitous second messenger, the Ca²⁺ ion. An increase in cytoplasmic Ca²⁺ concentration activates a variety of key cellular responses, including neurotransmitter release, muscle contraction, gene transcription, and cell growth and proliferation (1, 2).

The use of such a promiscuous messenger raises the question of specificity. Following cell stimulation, why are certain mechanisms activated by Ca²⁺ and others not? Growing evidence suggests that information is contained in the temporal and spatial profile of the Ca²⁺ signal as well as its amplitude, and these discrete components can be deciphered by the cell and translated into distinct responses (2).

The simplest form of a spatially restricted Ca²⁺ signal is the Ca²⁺ microdomain, which arises from the diffusion of Ca²⁺ through an open Ca²⁺ channel in either the plasma membrane or an intracellular organelle. Depending on the type of Ca²⁺ channel, the Ca²⁺ concentration within a microdomain can reach tens of micromolar, severalfold higher than the bulk cytoplasmic Ca²⁺ rise (3). Ca²⁺ microdomains associated with voltage-gated Ca²⁺ channels activate neurotransmitter release (3), open co-localized Ca²⁺-dependent K⁺ channels (4), and trigger gene transcription (5), whereas microdomains arising from open inositol 1,4,5-trisphosphate receptors stimulate juxtaposed mitochondria to generate ATP (6). Local Ca²⁺ entry through store-operated channels, which are activated by emptying intracellular Ca²⁺ stores (7), regulates specific Ca²⁺-dependent enzymes anchored at the plasma membrane, including the Ca²⁺-ATPase pump (8), adenylate cyclase (9), and endothelial NO synthase (10). These enzymes are thought to be tethered at the plasma membrane close to CRAC² channels, thus facilitating their activation by local Ca²⁺ gradients. An interesting complication arises when the Ca²⁺-dependent enzyme is located in the cytoplasm, some distance from the membrane. For example, Ca²⁺-dependent phospholipase A₂ (cPLA₂) and 5-lipoxygenase are two cytoplasmic enzymes that bind Ca²⁺ and translocate to the nuclear membrane upon stimulation, resulting in the generation of arachidonic acid and the proinflammatory cysteinyl leukotriene LTC₄, which is secreted from the cell (11). Ca²⁺ entry through CRAC channels stimulates both enzymes, through recruitment of Ca²⁺-dependent protein kinase C and ERK1/2 (11). Importantly, Ca²⁺ release from the stores fails to activate this signaling pathway, despite raising bulk cytoplasmic Ca²⁺ to levels only slightly lower than those attained by Ca²⁺ entry (12). Activation of these enzymes might therefore be critically dependent on the local subplasmalemmal Ca²⁺ rise accompanying CRAC channel opening. Alternatively, a bulk cytoplasmic Ca²⁺ signal might be the trigger, but the Ca²⁺ has to reach a certain level to stimulate the enzymes. The molecular constraints enforced by these mechanisms are entirely different; in the bulk cytoplasmic Ca²⁺ model, it would be sufficient for Ca²⁺ to activate protein kinase C directly as this enzyme is cytoplasmic at rest. In the subplasmalemmal Ca²⁺ model, a Ca²⁺ sensor is required to relay the local Ca²⁺ rise to protein kinase C in the cytoplasm.

Here we have designed experiments to address whether subplasmalemmal or bulk cytoplasmic Ca²⁺ signals activate these critical metabolic enzymes. We find that a subplasmalemmal Ca²⁺ rise is the key trigger in generating the cPLA₂/5-lipoxygenase/leukotriene C₄ cascade. Furthermore, a subplasmalemmal Ca²⁺ increase seems to activate protein kinase C by recruiting the nonreceptor tyrosine kinase Syk. Hence, a subplasmalemmal Ca²⁺ rise is highly versatile in that it triggers cytoplasmic responses through generation of the intracellular messenger arachidonic acid as well as long distance changes through generation of paracrine signals. Furthermore, by stimulating the secretion of leukotrienes that stimulate cells some distance away, subplasmalemmal Ca²⁺ operating over distances of a few nanometers can result in transferral of information over distances of up to several centimeters.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cell Culture—RBL-1 cells were bought from the ATCC. Cells were cultured (37 °C, 5% CO₂) in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 2 mM L-glutamine, and penicillin/streptomycin, as described previously (11). For Ca²⁺ imaging and patch clamp experiments, cells were passaged (using trypsin) onto glass coverslips and used 24–48 h after plating.

Cell Transfection—Cells were cotransfected with cDNA encoding the human TRPC3 channel (kindly provided by Prof. Putney) plus cDNA encoding an enhanced yellow fluorescent protein using the Lipofectamine method. For Ca²⁺ imaging experiments, cells were passaged onto glass coverslips and used 24–48 h after plating. Transfected cells were identified by expression of yellow fluorescent protein.

Ca²⁺ Imaging—Ca²⁺ imaging experiments were carried out using the IMAGO CCD camera-based system from TILL Photonics, as described previously (13). Cells were alternately excited at 356 and 380 nm (20-ms exposures; 0.5 Hz) using a Polychrome monochromator. Images were analyzed offline using IGOR Pro. Cells were loaded with Fura 2-AM or Fura 5F-AM (2 µM) for 40 min at room temperature in the dark and then washed in standard external solution of composition (in mM) NaCl 145, KCl 2.8, CaCl₂ 2, MgCl₂ 2, D-glucose 10, HEPES 10, pH 7.4, with NaOH. Ca²⁺ signals are presented as the change in ratio (356:380) relative to the resting level (R). In some figures, we have compared the initial rate of rise of the Ca²⁺ signal for different conditions, compared with that seen in 2 mM Ca²⁺. In vitro calibrations showed that the calculated cytoplasmic Ca²⁺ concentration (following readmission of 2 mM Ca²⁺ to cells treated with thapsigargin in Ca²⁺-free solution) peaked at 560 nM, only slightly above the K_D value of Fura 5F for Ca²⁺ (440 nM). Hence most recordings were obtained during the linear part of the fluorescence-Ca²⁺ curve. Moreover, R in 2 mM Ca²⁺ (following readmission of 2 mM Ca²⁺ to cells treated with thapsigargin in Ca²⁺-free solution) was 0.6 that of R_max.

EGTA-AM Loading—Cells were incubated in EGTA-AM or BAPTA-AM (20 µM each) for 45 min at room temperature.

Patch Clamp Recordings—Whole cell patch clamp recordings were carried out as described (13). Sylgard-coated, fire-polished patch pipettes were filled with a solution that contained 145 mM cesium glutamate, 8 mM NaCl, 1 mM MgCl₂, 2 mM Mg-ATP, 10 mM HEPES, 10 mM EGTA, 2 µM thapsigargin, pH 7.2, with CsOH. Pipette resistance was 5 megohms when placed in an external divalent-free solution containing 145 mM NaCl, 2.8 mM KCl, 10 mM CsCl, 10 mM D-glucose, 10 mM HEPES, 2 EDTA, pH 7.4, with NaOH. A correction of +10 mV was applied for the subsequent liquid junction potential that arose from the glutamate-based pipette solution. Na⁺ current through CRAC channels was measured by stepping to –80 mV for 250 ms from a holding potential of 0 mV, applied at 0.5 Hz. Currents were filtered using an 8-pole Bessel filter at 2.5 kHz and digitized at 100 µs. Variance was computed over 200-ms periods during each step.

Immunofluorescence—RBL cells were fixed in 4% paraformaldehyde in phosphate buffer for 30 min at room temperature, as described (11). Protein kinase C was visualized using a monoclonal mouse IgG1 antibody from BD Biosciences. The anti-protein kinase C was used at a concentration of 1:1000 in carrier (0.2% bovine serum albumin, 1% goat serum) and left overnight at 4 °C. The secondary anti-mouse IgG was a HandL chain-specific (goat) fluorescein conjugate (excitation at 495 nm, emission at 515 nm), used at 1:1000.

Preparation of Cell Lysates and Western Blotting—Cell lysate preparation and Western blotting were exactly described (12). Total cell lysates (40 µg) were mixed with 5 ml of glycerol, 0.1% bromphenol blue, and analyzed by SDS-PAGE on a 10% gel. Anti-phospho-ERK antibody, which recognizes dual phosphorylated (i.e. active) ERK, was from New England Biolabs and used at 1:1000 dilution.

[³H]Arachidonic Acid Release—Cells were prelabeled with 0.25 µCi/ml [³H]arachidonic acid in Dulbecco's modified Eagle's medium for 1.5 h at 37 °C, as described previously (12). After stimulation, radioactivity in the supernatant was measured. The amount of [³H]arachidonic acid released into the medium was expressed as a percentage of the total [³H]arachidonic acid uptake.

LTC₄ Measurements—Following stimulation with thapsigargin, the supernatant was collected, and LTC₄ levels were measured by enzyme immunoassay (Cayman Chemicals, Ann Arbor, MI) as described previously (12). Results have been normalized to basal (control) levels. Statistical significance was considered as p < 0.01, using Student's t test, and is denoted by asterisks in the figures.

View larger version (21K): [in this window] [in a new window]   FIGURE 1. Effects of Ca2+ release, store-operated Ca2+ entry, and mitochondrial Ca2+ removal on arachidonic acid production and LTC4 secretion. A, panel i, comparison of the time course of an intracellular Ca2+ signal following stimulation with thapsigargin (Thap.) (4 µM) in the absence (0 Ca2+; n = 107 cells) and presence (+Ca2+; n = 113 cells) of 2 mM external Ca2+. Data is summarized as mean ± S.E. A, panels ii and iii, arachidonic acid production (panel ii) and LTC4 secretion (panel iii) are compared between control (nonstimulated) cells, cells stimulated for 4 min in the presence of external Ca2+ (+Ca2+), and cells stimulated for 4 min but in the absence of external Ca2+ (–Ca2+). B, panel i, time course of the Ca2+ signal is compared between cells exposed to thapsigargin in Ca2+-free solution in the absence (control; n = 88 cells) and presence of depolarized mitochondria (following a 15-min pretreatment with antimycin A (10 µg/ml) and oligomycin (oligo)(1 µg/ml); n = 79 cells). B, panels ii and iii compare arachidonic acid release (B, panel ii) and LTC4 secretion (B, panel iii) for the two conditions. For comparison, arachidonic acid production and LTC4 secretion following stimulation with thapsigargin in the presence of external Ca2+ (labeled +Ca2+) are included.

	RESULTS

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One interpretation of these findings is that the spatial profile of the Ca²⁺ signal is key, with the subplasmalemmal Ca²⁺ rise emanating from Ca²⁺ influx exclusively activating the ERK/cPLA₂ cascade. Alternatively, the inability of Ca²⁺ release (i.e. thapsigargin in Ca²⁺-free external solution) to recruit the ERK/cPLA₂ pathway might simply reflect the smaller amplitude and/or more transient nature of the bulk cytoplasmic Ca²⁺ signal. If this latter scenario is the case, then increasing the amplitude and duration of the cytoplasmic Ca²⁺ signal that arises from Ca²⁺ release should activate ERK/cPLA₂. The decline of the cytoplasmic Ca²⁺ signal following stimulation with thapsigargin in Ca²⁺-free solution in Fig. 1A, panel i, reflects Ca²⁺ clearance by both mitochondria and plasma membrane Ca²⁺ pumps (14). Inhibition of Ca²⁺ clearance during the Ca²⁺ release phase would result in a larger bulk cytoplasmic Ca²⁺ rise, enabling us to discriminate between the two hypotheses described above. Impairing mitochondrial Ca²⁺ uptake by exposing cells to antimycin A (a complex III inhibitor) together with oligomycin modestly increased the size of the Ca²⁺ signal evoked by thapsigargin in Ca²⁺-free solution (Fig. 1B, panel i). However, this still failed to activate cPLA₂ (Fig. 1B, panel ii) or LTC₄ secretion (Fig. 1B, panel iii). Inhibition of the plasma membrane Ca²⁺ pump with 1 mM La³⁺ (15, 16) dramatically increased the size of the Ca²⁺ signal evoked by thapsigargin in Ca²⁺-free solution (R 2.6) and substantially prolonged its time course (data not shown). Because it is possible that Fura 2 is close to saturation following such a large Ca²⁺ rise, we repeated these experiments but with the lower affinity dye Fura 5F instead (17). Again, a significant increase in the amplitude of the Ca²⁺ signal was seen as well as a substantial slowing in the rate of recovery (Fig. 2A). We compared the amplitude of the cytoplasmic Ca²⁺ signal arising from readmission of external Ca²⁺ for 4 min to cells treated with thapsigargin in Ca²⁺-free solution with that obtained after 4 min of stimulation of cells with thapsigargin in Ca²⁺-free solution but in the presence of La³⁺ (Fig. 2B). These responses were quite similar in overall extent (Fig. 2C). Nevertheless, the large and sustained Ca²⁺ rise seen with thapsigargin in Ca²⁺-free solution in the presence of La³⁺ was significantly less effective in activating ERK1/2 (Fig. 2D), cPLA₂ (Fig. 2E), and LTC₄ secretion (Fig. 2F), when compared with corresponding responses due to store-operated Ca²⁺ influx.

We were concerned that La³⁺ might have been transported across the plasma membrane and subsequently interfered with cytoplasmic cPLA₂/5-lipoxygenase pathways. Although La³⁺ can be transported into cells, this is prominent in those cell types that express Na⁺-Ca²⁺ exchange (18). RBL-1 cells do not express this transporter (14), at least at a functional level. Nevertheless, because La³⁺ binds to Fura 2, we monitored La³⁺ influx in cells loaded with Fura 2 and exposed to Ca²⁺-free external solution containing 1 mM La³⁺. No detectable La³⁺ influx occurred over 800 s (Fig. 2A), in agreement with a previous report from Chinese hamster ovary cells lacking the Na⁺-Ca²⁺ exchanger (18). Collectively, these results demonstrate that store-operated Ca²⁺ entry is much more effective in activating cPLA₂ and LTC₄ secretion than a similar bulk cytoplasmic Ca²⁺ elevation, establishing that the subplasmalemmal Ca²⁺ rise is key in stimulating these enzymes. Although the present findings have utilized thapsigargin, we have previously reported that Ca²⁺ release following activation of muscarinic receptors in RBL cells was entirely ineffective in stimulating ERK, arachidonic acid generation, and LTC₄ secretion, whereas subsequent Ca²⁺ influx robustly activated these processes (19). Similarly, activation of FCRI receptors with antigen triggered ERK activation and LTC₄ secretion through a mechanism indistinguishable from that utilized by thapsigargin (11).

View larger version (27K): [in this window] [in a new window]   FIGURE 2. A bulk cytosolic Ca2+ rise is not as effective as local Ca2+ influx in stimulating cPLA2 or LTC4 secretion. A, time course of the Ca2+ signal is compared between cells exposed to thapsigargin (Thap.) in Ca2+-free solution in the absence (control; n = 71 cells) and presence of La3+ (1 mM; n = 74 cells). Cells exposed to La3+ (in Ca2+-free solution) are also included, demonstrating no detectable La3+ influx over this time scale. B, cytoplasmic Ca2+ rise following stimulation with thapsigargin in Ca2+-free solution followed by readdition of 2 mM Ca2+ (continuous line) is compared with that seen following stimulation with thapsigargin in Ca2+-free solution in the presence of La3+ (dotted line). C, compares the bulk cytosolic Ca2+ amplitude for the three different conditions indicated. Data have been normalized to the response obtained after 4 min of exposure to 2 mM Ca2+ after pretreatment with thapsigargin in Ca2+-free solution for 4 min. 0Ca2+ then Ca2+ refers to Ca2+ readmission after thapsigargin treatment in 0 Ca2+ solution. D, Western blots comparing ERK activation (ERK-P) for the different conditions (shown below histogram). Total ERK, to ensure similar protein loading in each well, is also shown. The histogram compares aggregate data obtained from three independent experiments. ERK activation is normalized to control levels (absence of stimulation). E and F depict the release of arachidonic acid (E) and LTC4 (F) for control (nonstimulated) cells, for cells exposed to thapsigargin in Ca2+-free solution for 8 min (labeled 0 Ca2+), for cells exposed to thapsigargin in Ca2+-free solution for 8 min but with impaired plasma membrane Ca2+-ATPase (labeled 0Ca2+ + La3+) and after stimulation with thapsigargin in Ca2+-free solution for 4 min, followed by readmission of external Ca2+ for 4 min (labeled 2Ca2+). * denotes p < 0.01. E and F, arachidonic acid release and LTC4 secretion have been normalized to control levels. **, p < 0.001.

Effect of Varying the Concentration Gradient for Ca²⁺ Entry—We varied the concentration gradient for Ca²⁺ influx by readmitting different external Ca²⁺ concentrations (0.25–2 mM) to cells pretreated with thapsigargin in Ca²⁺-free solution. The subsequent cytoplasmic Ca²⁺ increases, measured with Fura 2, are shown in Fig. 3A. The rate of rise was slightly slower in 0.5 mM than 2 mM Ca²⁺, but the bulk cytoplasmic Ca²⁺ level was similar. This is surprising because Ca²⁺ flux through I_CRAC is considerably smaller in 0.5 mM than in 2 mM Ca²⁺ (20). We considered that Fura 2 might be close to saturation under these conditions, and hence the dye underestimated the true response to 2 mM Ca²⁺. We therefore repeated these experiments using the lower affinity dye Fura 5F. Results are shown in Fig. 3B. A clearer difference in amplitude between 0.25, 0.5, and 2 mM Ca²⁺ was apparent. The rate of rise of the Ca²⁺ signal as well as the bulk cytoplasmic Ca²⁺ amplitude after 4 min of exposure to Ca²⁺ (the time when we measured ERK and cPLA₂ activity and LTC₄ secretion) are compared in Fig. 3, C and D (both normalized to the response in 2 mM Ca²⁺). Based on the relationship between external Ca²⁺ concentration and amplitude of I_CRAC in RBL-1 cells, raising external Ca²⁺ from 0.5 to 2 mM should increase Ca²⁺ influx rate by 2.4-fold (20). The rate of rise of the Ca²⁺ signal increased 2.15-fold (Fig. 3B) when external Ca²⁺ was raised from 0.5 to 2 mM, whereas the bulk cytosolic response increased only 1.20-fold (Fig. 3D). Hence the rate of rise of the Ca²⁺ signal is a more reliable indicator of Ca²⁺ flux through CRAC channels. The ability of 0.25–2 mM Ca²⁺ to activate ERK is shown in Fig. 3, E and F, and to evoke LTC₄ secretion is shown in Fig. 3G. Although the bulk cytoplasmic Ca²⁺ signal was only slightly smaller in 0.5 mM Ca²⁺ than 2 mM Ca²⁺ (Fig. 3D), the former was considerably less effective in activating ERK (Fig. 3, E and F) or triggering LTC₄ secretion (Fig. 3G). In 0.25 mM Ca²⁺, LTC₄ secretion was 15% that seen in 2 mM Ca²⁺ (Fig. 3G) yet the bulk cytoplasmic averaged Ca²⁺ was 53.0% of that seen in 2 mM Ca²⁺ (Fig. 3D). Collectively, these findings demonstrate that ERK activation and LTC₄ secretion correlate poorly with the bulk cytoplasmic Ca²⁺ signal, consistent with a major role for a subplasmalemmal Ca²⁺ rise in driving these responses.

View larger version (29K): [in this window] [in a new window]   FIGURE 3. Effect of varying the chemical gradient for Ca2+ influx on cytoplasmic Ca2+, ERK activation, and LTC4 secretion. A, compares the cytoplasmic Ca2+ signal in cells loaded with Fura 2 and pre-exposed to thapsigargin in Ca2+-free solution. At time 0, Ca2+ was readmitted at the indicated concentrations. B, same as A but cells were loaded with Fura 5F instead. C plots the rate of rise of the Ca2+ signal for the different external Ca2+ concentrations; D compares the bulk cytoplasmic averaged Ca2+. Data for C and D were obtained using Fura 5F, and between 105 and 130 cells for each point. E, upper gel, Western blot comparing ERK phosphorylation following stimulation with thapsigargin in Ca2+-free solution (4 min) followed by readmission of the indicated external Ca2+ concentrations for 4 min; lower gel, total ERK. F, histogram depicts aggregate data from three independent experiments measuring ERK-P. G plots the extent of LTC4 secretion following stimulation with thapsigargin as in E. G also plots the extent of LTC4 secretion against the rate of Ca2+ influx (measured in 0.25, 0.5, and 2 mM external Ca2+). Data have been normalized to the responses obtained in 2 mM Ca2+.

View larger version (19K): [in this window] [in a new window]   FIGURE 4. Reducing the electrical gradient for Ca2+ influx through CRAC channels reduces LTC4 secretion but only weakly affects bulk cytosolic Ca2+. A compares the cytoplasmic Ca2+ signal (measured with Fura 5F) in control cells and in those pre-exposed to 10 mM Cs+, which depolarizes the membrane potential. Thapsigargin was applied in Ca2+-free solution and Ca2+ readmitted after 4 min. Only the Ca2+ influx-driven signal is shown. B compares the rate of rise of Ca2+ from experiments carried out as in A. C compares LTC4 secretion between control cells and those exposed to Cs+.

Reducing the Electrical Gradient for Ca²⁺ Influx Impairs LTC₄ Secretion—Depolarization of the membrane potential reduces the electrical driving force for Ca²⁺ flux through open CRAC channels and therefore reduces the amplitude and spatial extent of the Ca²⁺ signal below the plasma membrane. The main determinant of the resting membrane potential in RBL-1 cells is the inwardly rectifying K⁺ channel, which can be inhibited by external Cs⁺ (21, 22). Current clamp recordings revealed that application of 10 mM Cs⁺, a concentration that fully blocks the inward rectifier in RBL cells, depolarized the resting membrane potential from –80 ± 4 mV to –43 ± 6 mV (data not shown). Pretreatment with Cs⁺ significantly slowed the rate of rise of the Ca²⁺ signal that occurred upon readmission of 2 mM Ca²⁺ to thapsigargin-treated cells (Fig. 4, A and B), but the bulk cytoplasmic response was only slightly reduced (Fig. 4A). LTC₄ secretion was significantly reduced by Cs⁺ (Fig. 4C). As with varying external Ca²⁺ (described above), the inhibition of LTC₄ generation by Cs⁺ correlated better with the substantial reduction in the rate of rise of the Ca²⁺ signal rather than with the modest fall in the bulk cytoplasmic Ca²⁺ concentration.

The inhibitory effects of Cs⁺ were mimicked by raising external K⁺ to 40 mM (data not shown), a concentration that depolarizes the membrane potential to a similar extent. Collectively, these results are consistent with the idea that a subplasmalemmal Ca²⁺ increase arising from open CRAC channels and not a bulk cytoplasmic Ca²⁺ rise drives ERK activation and subsequent LTC₄ secretion.

Effects of the Slow Ca²⁺ Chelator EGTA on Protein Kinase C Translocation, Arachidonic Acid Release, and LTC₄ Secretion—One key determinant of the spatial extent of Ca²⁺ entry is subplasmalemmal Ca²⁺ buffering in the vicinity of the Ca²⁺ channels. To see how changes in buffering affected activation of cPLA₂ and 5-lipoxygenase, we investigated the effects of the slow chelator EGTA on CRAC channel-driven protein kinase C translocation to the plasma membrane, a step critical for its activation, arachidonic acid production, and LTC₄ generation. Loading cells with Fura 2 together with EGTA resulted in a slower bulk cytoplasmic Ca²⁺ rise following stimulation with thapsigargin (Fig. 5A). Ca²⁺ entry through CRAC channels stimulates the movement of protein kinase C and βI from the cytoplasm to the plasma membrane, a critical early step in ERK activation (11). Compared with the cytoplasmic distribution of protein kinase C in nonstimulated cells (Fig. 5B). Stimulation of EGTA-loaded cells with thapsigargin in 2 mM Ca²⁺ resulted in robust migration of protein kinase C to the plasma membrane (Fig. 5C).

View larger version (21K): [in this window] [in a new window]   FIGURE 5. The slow Ca2+ chelator EGTA has little effect on protein kinase C translocation and cPLA2 activation. A, cytoplasmic Ca2+ signals are compared between control (non-chelator-loaded) cells and those loaded with EGTA. Cells were stimulated with thapsigargin in 2 mM Ca2+-containing solution. B and C, immunocytochemical analyses of protein kinase C distribution in resting cells (B) and in those pretreated with EGTA-AM followed by stimulation with thapsigargin (Thap.) in 2 mM Ca2+ (C) are compared. Experiments were repeated three times on three different RBL-1 cell preparations. D and E, effects of EGTA on arachidonic acid release (D) and LTC4 secretion (E) are shown.

We also examined the effects of loading cells with the faster Ca²⁺ chelator BAPTA. BAPTA prevented translocation of protein kinase C to the plasma membrane and suppressed the generation of both arachidonic acid and LTC₄ secretion following CRAC channel activation with thapsigargin (supplemental Fig. 1). However, BAPTA also prevented the Ca²⁺-independent translocation of protein kinase C by phorbol ester (supplemental Fig. 1), suggesting an action independent of Ca²⁺ buffering and which may reflect its ability to block protein kinase C directly.

Effects of Partially Blocking CRAC Channels—To provide further evidence for the importance of subplasmalemmal Ca²⁺ rather than a bulk cytoplasmic Ca²⁺ rise, we blocked CRAC channels partially with a low concentration of La³⁺ (100 nM) such that the bulk cytoplasmic Ca²⁺ rise following stimulation with thapsigargin in 2 mM Ca²⁺ was similar to that achieved with thapsigargin in 0.25 mM Ca²⁺ in the absence of La³⁺ (Fig. 6A). Such low concentrations do not affect plasma membrane Ca²⁺-ATPAse activity (23) or the recovery of a Ca²⁺ transient (data not shown). As argued by Bautista and Lewis (8), La³⁺ likely blocks CRAC channels through a mechanism similar to that through which it blocks voltage-gated L-type Ca²⁺ channels, considering the similarities in ion selectivity and permeation between the two types of channel. La³⁺ blocks L-type Ca²⁺ channels by slow permeation, resulting in brief bursts of channel openings (24). Importantly, La³⁺ block of Ca²⁺ channels does not reduce the electrochemical driving force for Ca²⁺ influx; hence the amplitude of Ca²⁺ microdomains would be larger in 2 mM Ca²⁺ plus 100 nM La³⁺ than in 0.25 mM Ca²⁺. If a local Ca²⁺ rise indeed drives protein kinase C translocation and LTC₄ secretion, larger responses should be obtained in 2 mM Ca²⁺ and La²⁺ than in 0.25 mM Ca²⁺, despite the similar bulk cytoplasmic Ca²⁺ rise. Fig. 6B shows that protein kinase C translocated to the plasma membrane more effectively in cells challenged with thapsigargin in 2 mM Ca²⁺ and La³⁺ than was the case with 0.25 mM Ca²⁺. This was also reflected in more LTC₄ secretion (Fig. 6C). Hence, it is a subplasmalemmal Ca²⁺ elevation that drives these cellular responses.

TRPC3 Channels and Protein Kinase C Activation—To see whether other plasma membrane Ca²⁺-permeable channels could mimic CRAC channels in recruiting the protein kinase C/ERK/LTC₄ pathway, we overexpressed TRPC3 channels, which are permeable to Ca²⁺ (25, 26), in RBL-1 cells and then activated them with OAG (100 µM). OAG was applied in Ca²⁺-free solution and then external Ca²⁺ was readmitted. OAG failed to trigger any detectable Ca²⁺ release when applied in Ca²⁺-free solution, but readmission of external Ca²⁺ resulted in a prominent Ca²⁺ rise (Fig. 6D, upper panel; aggregate data summarized in lower panel). OAG failed to evoke a cytoplasmic Ca²⁺ rise when applied to nontransfected cells (Fig. 6D). Similarly, no detectable Ca²⁺ rise was seen when TRPC3-expressing cells were exposed to Ca²⁺-free solution followed by Ca²⁺ readmission in the absence of OAG (data not shown). The Ca²⁺ rise induced by Ca²⁺ readmission to cells treated with OAG in Ca²⁺-free solution was similar in extent to that evoked following store-operated entry (Fig. 6D).

View larger version (39K): [in this window] [in a new window]   FIGURE 6. Partial block of CRAC channels by La3+ supports protein kinase C translocation and LTC4 secretion. A, readmission of 2 mM Ca2+ in the presence of 100 nM La3+ to thapsigargin (Thap.)-treated cells resulted in a cytoplasmic Ca2+ rise that was slightly smaller than that seen when 0.25 mM Ca2+ was readmitted (no La3+). B, protein kinase C translocation to the plasma membrane was considerably more pronounced when cells were stimulated with thapsigargin in 2 mM Ca2+ and La3+ than in 0.25 mM Ca2+, despite similar bulk cytoplasmic Ca2+ increases. C, LTC4 secretion was larger in cells stimulated with thapsigargin in 2 mM Ca2+ and La3+ than in 0.25 mM Ca2+. D, upper panel, Ca2+ influx signals in response to OAG are shown for wild type cells and for those expressing recombinant human TRPC3 channels. Lower panel, aggregate data from several cells are summarized. The size of the response following application of Ca2+ to cells exposed to OAG and Ca2+-free solution in nontransfected cells is compared with that seen in cells expressing human TRPC3. The response following activation of CRAC channels (thapsigargin in Ca2+-free solution followed by Ca2+ readmission) is also included. E, protein kinase C translocation to the plasma membrane is increased by Ca2+ entry through TRPC3 channels. Control denotes resting cells, and OAG denotes cells exposed to OAG in Ca2+-free solution followed by readmission of external Ca2+ for 4 min. F, data from experiments as in E were analyzed and are plotted for the various conditions. Note that application of OAG in Ca2+-free solution failed to induce any significant protein kinase C translocation, whereas it did when external Ca2+ was readmitted. Images were analyzed in Image J by drawing several small regions of interest around the plasma membrane, and the non-nuclear cytoplasm and the fluorescences were then computed. Translocation of protein kinase C was quantified as the plasma membrane to cytosolic ratio.

Central Role for the Tyrosine Kinase Syk—Because Ca²⁺-dependent protein kinase C isozymes are generally located in the cytoplasm in resting cells but translocate to the plasma membrane upon stimulation, we reasoned that they would be unlikely to monitor the subplasmalemmal Ca²⁺ concentration directly. In some systems, including mast cells, the nonreceptor tyrosine kinase Syk phosphorylates protein kinase C (27), and Syk can co-localize with ion channels in the plasma membrane (28). The Syk inhibitor 3-(1-methyl-1H-indol-3-yl-methylene)-2-oxo-2,3-dihydro-1H-indole-5-sulfonamide prevented ERK phosphorylation (Fig. 7A), arachidonic acid release (Fig. 7B), and LTC₄ secretion (Fig. 7C) following stimulation with thapsigargin. Importantly, these inhibitory effects could be overcome fully by subsequent stimulation of protein kinase C with the phorbol ester PMA (Fig. 7, A–C). Hence, Syk is upstream of protein kinase C in the signaling cascade. The effects of the Syk inhibitor were concentration-dependent (Fig. 7D) and were not mimicked by inhibition of the closely related tyrosine kinase Src with the antagonist 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine or by blocking phosphatidylinositol 3,4,5-trisphosphate kinase with either LY294002 (20 µM) or wortmannin (0.2–2 µM) (Fig. 7D). Syk inhibition had no effect on the thapsigargin-evoked Ca²⁺ signal (data not shown) or on the extent of I_CRAC (Fig. 7E). Collectively, these results demonstrate that Ca²⁺ entry through CRAC channels recruits protein kinase C through the nonreceptor tyrosine kinase Syk.

View larger version (27K): [in this window] [in a new window]   FIGURE 7. CRAC channels activate ERK through the nonreceptor tyrosine kinase Syk. A, Western blot showing that thapsigargin-evoked phosphorylation of ERK is suppressed by the Syk inhibitor (5 µM, 20 min pretreatment). ERK phosphorylation is rescued, in the presence of Syk inhibitor, by subsequent stimulation of protein kinase C with 1 µM PMA. B and C, thapsigargin-evoked arachidonic acid release (B) and LTC4 secretion are both suppressed by the Syk inhibitor but can be rescued by PMA. D, arachidonic acid released by thapsigargin is inhibited, in a concentration-dependent manner, by the Syk inhibitor but not by inhibiting the tyrosine kinase Src with 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PPI) or phosphatidylinositol 3,4,5-trisphosphate kinase with either LY294002 or wortmannin. Cells were pretreated with each inhibitor for 20–30 min prior to stimulation. E, extent of activation of ICRAC is not impaired by the Syk inhibitor.

View larger version (8K): [in this window] [in a new window]   FIGURE 8. Estimation of Ca2+ microdomain amplitude as a function of distance from the plasma membrane. A, current in divalent-free solution before (upper recording) and then after (lower recording) store depletion. B, variance is plotted against corresponding whole cell current for the cell shown in A. The slope yields the single channel current, assuming open probability is low. C, graph plots the computed amplitude of a Ca2+ microdomain following opening of a single CRAC channel against distance from the plasma membrane. See text for further details.

(Eq. 1)

where r is distance from the channel pore; F is Faraday's constant; and D_Ca is the Ca²⁺ diffusion coefficient (3 x 10^–6 µm² s¹). Because microdomains build up and collapse within tens to hundreds of microseconds whereas cPLA₂ activation develops over seconds, the steady-state model is applicable. Additionally, for simplicity, we have ignored the involvement of buffered diffusion of Ca²⁺ away from microdomains, which would sharpen the local Ca²⁺ gradient.

The unitary CRAC channel conductance is extremely small, well beyond current levels of resolution (30). However, in the absence of external divalent cations, CRAC channels become permeable to monovalent ions like Na⁺, resulting in a clear increase in whole cell current variance (31, 32). Using fluctuation analysis, we measured current variance following dialysis with 10 mM EGTA and thapsigargin to deplete the stores passively (Fig. 8A). Variance is plotted against whole cell current amplitude in Fig. 8B. Assuming a channel open probability of <<1, the slope yields the unitary current, in this example –24.9 fA. The unitary chord conductance from four such experiments was calculated to be 0.23 ± 0.09 pS. Previous studies assumed open probability of CRAC channels was low, but a more recent analysis exploiting Ca²⁺ block of the Na⁺ current has found that open probability is high (0.8), leading to a revised single channel conductance of 0.7 pS in divalent-free solution (33). If open probability is high, theory predicts that the slope of variance/current should initially increase as macroscopic current falls but then fall in parallel with whole cell current. Consistent with this, we found that variance increased (15%) in divalent-free solution containing 10 µM Ca²⁺ even though the macroscopic current fell (data not shown). Because our findings are in good agreement with the detailed study by Prakriya and Lewis (33), we took their estimate for the single channel current at –80 mV in 2 mM Ca²⁺ and scaled it to correct for both an open probability of 0.8 and the lower K_D for Ca²⁺ permeation in RBL-1 cells compared with T lymphocytes (0.7 mM (20) versus 2.1 mM (34), respectively). Fig. 8C plots the calculated Ca²⁺ concentration as a function of distance from the plasma membrane in 2 and 0.5 mM external Ca²⁺.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Ca²⁺-dependent generation of the pro-inflammatory cysteinyl leukotriene LTC₄ is central to mast cell function (35, 36). In this study, we have shown that a subplasmalemmal Ca²⁺ rise driven by CRAC channels in native cells, but not bulk cytoplasmic Ca²⁺ elevation, promotes the generation of the important intracellular messenger arachidonic acid, which is then metabolized to form LTC₄.

The most local form of Ca²⁺ signaling is the microdomain that results from the opening of single Ca²⁺-permeable ion channels. Detection of such microdomains is beyond the resolution of epifluorescence microscopy, and their involvement is generally inferred from examining the impact of altering Ca²⁺ flux through channels on cellular responses without changing the bulk cytoplasmic Ca²⁺ rise. Although some of our results do demonstrate that a local, and not global, Ca²⁺ rise controls the protein kinase C/ERK/cPLA2/LTC₄ signaling cascade, our study does not completely distinguish between the trigger Ca²⁺ sensor for this cascade being tightly associated with microdomains from CRAC channels or located further away from the channels but nevertheless responsive to subplasmalemmal Ca²⁺ elevation. Furthermore, although the predominant electrogenic Ca²⁺ influx pathway in mast and RBL-1 cells is the CRAC channel, Ca²⁺ influx through recombinant TRPC3 channels also links into the protein kinase C/LTC₄ cascade. Hence, this signaling pathway is not exclusively controlled by a Ca²⁺ microdomain specific to CRAC channels.

A Subplasmalemmal Ca²⁺ Rise Drives Activation of ERK and cPLA₂ Followed by LTC₄ Secretion—Several pieces of evidence suggest a subplasmalemmal Ca²⁺ rise underlies the activation of ERK, cPLA₂, and subsequent LTC₄ First, Ca²⁺ secretion. release evoked by stimulation with thapsigargin in Ca²⁺-free solution under conditions where plasma membrane Ca²⁺-ATPases were blocked generated a bulk cytoplasmic Ca²⁺ rise that was no different in amplitude to that seen following Ca²⁺ influx through CRAC channels. However, the latter was significantly more effective in activating cPLA₂ and LTC₄ secretion. Second, varying extracellular Ca²⁺ concentration following CRAC channel opening resulted in ERK activation and LTC₄ secretion that correlated only poorly with the bulk cytoplasmic Ca²⁺ concentration. Third, blockade of inwardly rectifying K⁺ channels with Cs⁺, which reduces the driving force for Ca²⁺ entry and thus the amplitude of Ca²⁺ influx that accompanies CRAC channel opening, had little impact on the bulk cytoplasmic Ca²⁺ response, yet significantly reduced LTC₄ secretion. Finally, increasing cytoplasmic Ca²⁺ buffering with the slow chelator EGTA reduced the bulk cytoplasmic Ca²⁺ signal evoked by store-operated Ca²⁺ entry but had little effect on either protein kinase C translocation to the plasma membrane or cPLA₂ activation. Although EGTA can blunt bulk cytoplasmic Ca²⁺ increases, it is too slow to prevent the local build up of subplasmalemmal Ca²⁺ arising in the vicinity of open Ca²⁺ channels (3).

Nevertheless, we considered alternative explanations for these findings. It is possible that our Ca²⁺ measurements are confounded by dye saturation, which would underestimate the bulk cytoplasmic Ca²⁺ concentration, particularly at higher external Ca²⁺ concentrations. Although this may well be an issue with Fura 2, it clearly was not the case for those experiments where we used the lower affinity dye Fura 5F. It is also possible that activation of the Ca²⁺sensing receptor might have occurred in those experiments where external Ca²⁺ was altered. However, reducing the amplitude of local Ca²⁺ influx through CRAC channels by blocking the inward rectifier with Cs⁺ while external Ca²⁺ was kept constant clearly established that LTC₄ secretion was tightly coupled to Ca²⁺ influx and not the bulk cytoplasmic Ca²⁺ signal.

Further evidence supporting the central role played by a subplasmalemmal Ca²⁺ rise in driving these responses was obtained from partial block of Ca²⁺ entry through CRAC channels. If LTC₄ secretion is indeed driven by local Ca²⁺ influx, then one would expect conditions that promote larger, albeit fewer, Ca²⁺ microdomains from CRAC channels to be more effective than those that lead to smaller but more numerous Ca²⁺ microdomains, despite similar bulk cytoplasmic Ca²⁺ rises. We blocked CRAC channels partially by applying a low concentration of the channel blocker La³⁺ (31). La³⁺ does not affect the driving force for Ca²⁺ influx and therefore would not alter the amplitude or spatial extent of the Ca²⁺ microdomains, although they would be more transient because of the briefer channel openings that occur in the presence of La³⁺. The bulk cytoplasmic Ca²⁺ rise following readmission of 2 mM Ca²⁺ in the presence of La³⁺ to cells with depleted stores was similar to that seen following readmission of 0.25 mM Ca²⁺ in the absence of La³⁺. Nevertheless, the former was considerably more effective in promoting protein kinase C translocation to the plasma membrane and secretion of LTC₄.

Recombinant TRPC3 Channels Are as Effective as Native CRAC Channels in Stimulating Protein Kinase C Translocation—To see whether different plasmalemmal Ca²⁺ channels could activate protein kinase C translocation, a key early step in cPLA₂ activation and LTC₄ secretion, we compared the effects of recombinant TRPC3 channels with CRAC channels on protein kinase C migration. OAG activates TRPC3 channels independently of store depletion (37, 38), resulting in a bulk cytoplasmic Ca²⁺ rise that was similar to that evoked by CRAC channels. Strikingly, OAG was as effective as CRAC channels in stimulating protein kinase C translocation to the plasma membrane. TRPC3 channels are nonselective cation channels (PCa/Na of 1.65) with a single channel conductance of 23 pS (26), around 3 orders of magnitude larger than that of the CRAC channel. Although much of the single TRPC3 channel current is carried by Na⁺, the Ca²⁺ flux will nevertheless generate a sizeable Ca²⁺ microdomain with a spatial extent larger than that of a CRAC channel. The fact that a non-CRAC plasma membrane channel can link into the protein kinase C/LTC₄ pathway indicates that this signaling cascade is not exclusively coupled to CRAC channel opening, and Ca²⁺ channels with more expansive Ca²⁺ microdomains are able to recruit this pathway.

Involvement of Syk in Coupling Ca²⁺ Entry to ERK, cPLA₂, and LTC₄ Secretion—Both cPLA₂ and 5-lipoxygenase enzymes are activated by ERK1/2 (11). ERK1/2 is stimulated, in turn, by Ca²⁺-dependent protein kinase C and β isoforms. These enzymes are primarily cytoplasmic at rest and migrate to the plasma membrane following Ca²⁺ influx through CRAC channels (11), a process that is essential for protein kinase C activation. Because these isoforms are initially cytoplasmic, they are unlikely to respond to local Ca²⁺ influx directly. Instead, there needs to be a sensor that detects the subplasmalemmal Ca²⁺ rise and then activates protein kinase C. Although the molecular identity of the sensor is not known, our experiments demonstrate that the nonreceptor tyrosine kinase Syk is upstream of protein kinase C in this signaling cascade. Block of Syk, but not the closely related tyrosine kinase Src, inhibited ERK phosphorylation, cPLA₂ activation, and LTC₄ secretion but without compromising CRAC channel activity or the ensuing cytoplasmic Ca²⁺ signal. Importantly, even when Syk was blocked, ERK phosphorylation, cPLA₂ activation, and LTC₄ secretion could all be rescued by direct application of phorbol ester, demonstrating that Syk is upstream of protein kinase C. How Ca²⁺ entry activates Syk is not established yet, and we are currently investigating this. A rise in cytoplasmic Ca²⁺ can activate Syk (39, 40), although it is unclear whether this is direct or via an intermediary Ca²⁺-dependent molecule. Because nonreceptor tyrosine kinases can associate with proteins in the plasma membrane (28), it is possible that Syk relays subplasmalemmal Ca²⁺ signals directly to protein kinase C. In this regard, it is interesting to note that in mast cells Syk can directly phosphorylate protein kinase C and β1 (27), the two isozymes that we have found to be central to ERK activation and LTC₄ secretion in RBL-1 cells (11). Moreover, phosphorylation by Syk occurs in a membrane compartment (27), ostensibly the plasma membrane. Phosphorylation of protein kinase C by Syk results in the formation of a binding pocket for the Src homology 2 domain of Grb2, thereby activating Ras and ERK (27). A subplasmalemmal Ca²⁺ rise can therefore activate temporally and spatially distinct responses, increasing its versatility in driving fundamental cellular responses.

	FOOTNOTES

 
^* This work was supported by a Medical Research Council Program grant (to A. B. P.), Overseas Research Studentship, Y. Tan awards (to W.-C. C.), and a Christopher Welch scholarship (to J. D. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.

¹ To whom correspondence should be addressed. Tel.: 44-1865-272439; E-mail: anant.parekh{at}dpag.ox.ac.uk.

² The abbreviations used are: CRAC, Ca²⁺ release-activated Ca²⁺; cPLA₂, Ca²⁺-dependent phospholipase A₂; LTC₄, leukotriene C₄; ERK, extracellular signal-regulated kinase; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; OAG, 1-oleoyl-2-acetylglycerol; PMA, phorbol 12-myristate 13-acetate.

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