A Novel Ca2+-induced Ca2+ Release Mechanism in A7r5 Cells Regulated by Calmodulin-like Proteins*

Nael Nadif Kasri {ddagger} §, Ilse Sienaert {ddagger} , Jan B. Parys {ddagger}, Geert Callewaert {ddagger}, Ludwig Missiaen {ddagger}, Andreas Jeromin || and Humbert De Smedt {ddagger} **

From the {ddagger}Laboratorium voor Fysiologie, K.U. Leuven Campus Gasthuisberg O/N, Herestraat 49, B-3000 Leuven, Belgium and the ||Division of Neuroscience, Baylor College of Medicine, Houston, Texas 77030

Received for publication, February 26, 2003 , and in revised form, April 18, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Intracellular Ca2+ release is involved in setting up Ca2+ signals in all eukaryotic cells. Here we report that an increase in free Ca2+ concentration triggered the release of up to 41 ± 3% of the intracellular Ca2+ stores in permeabilized A7r5 (embryonic rat aorta) cells with an EC50 of 700 nM. This type of Ca2+-induced Ca2+ release (CICR) was neither mediated by inositol 1,4,5-trisphosphate receptors nor by ryanodine receptors, because it was not blocked by heparin, 2-aminoethoxydiphenyl borate, xestospongin C, ruthenium red, or ryanodine. ATP dose-dependently stimulated the CICR mechanism, whereas 10 mM MgCl2 abolished it. CICR was not affected by exogenously added calmodulin (CaM), but CaM1234, a Ca2+-insensitive CaM mutant, strongly inhibited the CICR mechanism. Other proteins of the CaM-like neuronal Ca2+-sensor protein family such as Ca2+-binding protein 1 and neuronal Ca2+ sensor-1 were equally potent for inhibiting the CICR. Removal of endogenous CaM, using a CaM-binding peptide derived from the ryanodine receptor type-1 (amino acids 3614–3643) prevented subsequent activation of the CICR mechanism. A similar CICR mechanism was also found in 16HBE14o-(human bronchial mucosa) cells. We conclude that A7r5 and 16HBE14o-cells express a novel type of CICR mechanism that is silent in normal resting conditions due to inhibition by CaM but becomes activated by a Ca2+-dependent dissociation of CaM. This CICR mechanism, which may be regulated by members of the family of neuronal Ca2+-sensor proteins, may provide an additional route for Ca2+ release that could allow amplification of small Ca2+ signals.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Changes in cytosolic-free Ca2+ concentration ([Ca2+]c)1 mediate a variety of cellular processes, ranging from fertilization to cell death (1, 2). Cells generate Ca2+ signals through both intracellular (mainly the endoplasmic/sarcoplasmic reticulum) and extracellular Ca2+ sources. Regulation of these Ca2+ signals via a variety of Ca2+ channels, expressed either in the plasma membrane or in the membranes of intracellular stores, is thereby essential. Ca2+ fluxes from extracellular and intracellular Ca2+ sources do not occur independently of each other. For example, the intracellular Ca2+ store content regulates Ca2+ entry from the extracellular medium via capacitative Ca2+ entry (3, 4), whereas Ca2+ released by one channel can alter the activity of other channels. These are all well documented mechanisms whereby Ca2+ can exert important effects on its own activity. The most important type of regulation is represented by the various mechanisms that may lead to the characteristic bell-shaped dependence of intracellular Ca2+ channels on Ca2+ itself (59). This may in principle be due to direct interaction with Ca2+ or indirectly via Ca2+-sensor proteins such as calmodulin (CaM). The inositol 1,4,5-trisphosphate receptor (IP3R) and the ryanodine receptor (RyR) are the two major families of intracellular Ca2+ release channels that have been characterized. Both types of intracellular channels are regulated in a complex way by Ca2+ and CaM. CaM has been demonstrated to affect the activity of RyRs in both a stimulatory and an inhibitory manner (10, 11) but not by the same mechanism for all three RyR isoforms. For the IP3R, CaM clearly exerts an inhibitory effect, but the precise mechanism is not yet understood (12, 13). RyRs and IP3Rs are stimulated by small increases in [Ca2+]c and inhibited at higher [Ca2+]c (1420). Stimulation is important for the mechanism of Ca2+-induced Ca2+ release (CICR), which allows amplification and regenerative propagation of intracellular Ca2+ signals. CICR seems to be an operational mode of both IP3Rs and RyRs, and it is clearly a key feature of intracellular Ca2+ signaling (21). Recent studies have emphasized the role of novel types of intracellular Ca2+ release channels possibly playing an important role in intracellular Ca2+ signaling (2228). Wissing et al. (26) identified a novel CICR mechanism in permeabilized hepatocytes that responded to modest increases in [Ca2+]c. Polycystin-2, the product of the gene mutated in type-2 autosomal dominant polycystic kidney disease and a prototypical member of a subfamily of the transient receptor potential channel superfamily (TRP), is expressed abundantly in the endoplasmic reticulum (ER) (24). It was shown recently that polycystin-2 expressed in the ER of epithelial cells is a Ca2+-activated channel that is permeable for divalent cations. Increased levels of intracellular Ca2+ activated polycystin-2-mediated release of Ca2+ from intracellular stores. Recent data also suggested that activation of the ER-associated vanilloid receptor 1 (VR1), a member of the TRP family, by capsaicin binding resulted in Ca2+ mobilization from intracellular stores. This raises the possibility that VR1 may also function as an intracellular Ca2+ release channel (27, 28).

In the present study we have identified a novel CICR mechanism in permeabilized A7r5 cells, a permanent cell line derived from embryonic rat aorta. We identified a CICR mechanism that was mediated by neither the IP3R nor the RyR. Moreover, we found that this CICR mechanism could be inhibited by CaM1234, a Ca2+-insensitive CaM mutant, and by different members of the superfamily of CaM-like Ca2+-binding proteins. Our data suggest that the CICR mechanism described here may represent a novel type of release channel, which is silent at low [Ca2+]c due to inhibition by bound apoCaM and which becomes activated by the Ca2+-dependent dissociation of CaM. This CICR mechanism may provide an additional pathway for intracellular Ca2+ release and could play an important role in amplifying Ca2+ signals generated by other Ca2+ release channels.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
45Ca2+ Fluxes—A7r5 cells, which are derived from embryonic rat aorta, were obtained from the American Tissue Type Culture Collection CRL 1444 (Bethesda, MD). Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 3.8 mM L-glutamine, 0.9% (v/v) non-essential amino acids, 85 IU/ml penicillin, 85 µg/ml streptomycin, and 20 mM HEPES (pH 7.4). For 16HBE14o-(human bronchial mucosa) and mouse embryonal fibroblast cells a mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium was used and for LLC-PK1 cells minimal essential medium {alpha} was used. 45Ca2+ fluxes were performed on saponin-permeabilized cells. The cells were seeded in 12-well clusters (Costar, MA) at a density of ~4 x 104 cm2. Experiments were carried out on confluent monolayers of cells (3 x 105 cells/well) between the 7th and 9th days after plating. Cells were permeabilized by incubating them for 10 min with a solution containing 120 mM KCl, 30 mM imidazole-HCl (pH 6.8), 2 mM MgCl2, 1 mM ATP, 1 mM EGTA, and 20 µg ml1 saponin at 25 °C. The non-mitochondrial Ca2+ stores were loaded for 45 min at 37 °C in 120 mM KCl, 30 mM imidazole-HCl (pH 6.8), 5 mM MgCl2, 5 mM ATP, 0.44 mM EGTA, 10 mM NaN3, and 150 nM free 45Ca2+ (28 µCi ml1). The cells were then washed twice with 1 ml of efflux medium containing 120 mM KCl, 30 mM imidazole-HCl (pH 6.8), 1 mM EGTA, and 10 µM thapsigargin. Thapsigargin was added to block the ER Ca2+ pumps during subsequent additions of Ca2+. The efflux medium was replaced every 2 min during 18 min, and the efflux was performed at 37 °C. The additions of 40Ca2+ and IP3 are indicated in the legends of the figures. Free [Ca2+] was calculated by the Cabuf program (available at ftp.cc.kuleuven.ac.be/pub/droogmans/cabuf.zip) and based on the stability constants given by Fabiato and Fabiato (29). At the end of the experiment the 45Ca2+ remaining in the stores was released by incubation with 1 ml of a 2% SDS solution for 30 min. Ca2+ release is plotted as the fractional loss, i.e. the amount of Ca2+ released in 2 min divided by the total store Ca2+ content at that time. The latter value was calculated by summing in retrograde order the amount of tracer remaining in the cells at the end of the efflux and the amounts of tracer collected during the successive time intervals. In experiments performed to exclude 40Ca2+/45Ca2+ exchange in Fig. 2, cells were loaded during 45 min in loading buffer, containing 4 mM EGTA and 680 µM total CaCl2, resulting in 285 nM free [Ca2+] and a specific activity for Ca2+ of 28 µCi ml1. After 45 min, the loading buffer was replaced for 2 min by a loading buffer with an EGTA concentration of 0.76 mM and supplemented with thapsigargin, to maintain the same 40Ca2+/45Ca2+ ratio but resulting in an increase in free [Ca2+] to 10 µM. Efflux was then further performed in Ca2+-free efflux buffer.



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FIG. 2.
Efflux in conditions that exclude net 45Ca2+/40Ca2+ exchange. Efflux of cells loaded during 45 min in loading buffer, at a free [Ca2+] of 285 nM. After 45 min, loading buffer was replaced for 2 min by loading buffer supplemented with 10 µM thapsigargin alone (squares), with thapsigargin together with a lowered EGTA concentration, maintaining the same 45Ca2+/40Ca2+ but resulting in 10 µM free [Ca2+] (triangles) or with 5 µM A23187 [GenBank] (circles). From time 0 onward, cells were incubated in a Ca2+-free efflux medium, and their Ca2+ content was plotted as a function of time. Results represent the means ± S.E. of three independent experiments each performed twice.

 

Peptide Synthesis—RyR1 peptide (amino acids 3614–3643) (30) was synthesized by Eurogentec S.A. (Herstal, Belgium).

Cloning of sCaBp1 and lCaBp1—Mouse CaBP1 cDNA was cloned from mouse cerebellum RNA. Poly(A)+ RNA from mouse cerebellum was prepared using the Micro-FastTrack kit (Invitrogen, CA). Random primed first strand cDNA was synthesized from 1 µg of RNA using avian myeloblastosis virus reverse transcriptase. Reverse transcription-PCR was performed with forward primer 5'-GCCAGCCATATGGGCAACTGCGTCAAGTCGCC-3' and reverse primer 5'-GCGGGCAGCCTCGAGGCGAGACATCATCCGGAC-3'. The forward primer contained the site for NdeI (CATATG), and the reverse primer contained the site for XhoI (CTCGAG). PCR fragments of both isoforms, the short (sCaBp1) and long (lCaBp1) form, were then cloned into the NdeI-XhoI site of the pET21b/+ vector (Novagen), yielding an expression vector for a His6-tagged sCaBp1 and lCaBp1.

Expression and Purification of Recombinant Proteins—pET-sCaBP1 and pET-lCaBP1 were transformed in BL21 Escherichia coli cells, grown to mid-exponential phase, and induced with 0.75 mM isopropyl-1-thiol-{beta}-D-galactopyranoside for4hat28 °C. Cells were centrifuged for 10 min, and pellets were then resuspended in lysis buffer containing 50 mM NaH2PO4, pH 7.0, 300 mM NaCl, 10 mM imidazole, 1 mM {beta}-mercaptoethanol, 0.8 mM benzamidine, 0.2 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin, 1 µM pepstatin A, and 75 nM aprotinin. This cell suspension was then lysed by sonication at 20 kHz, nine times for 10 s using a probe sonicator (MSE Ltd., Crawley, Surrey, UK). 1 ml of 50% nickel-nitrilotriacetic acid (Qiagen) slurry was added to 4 ml of cleared lysate and gently mixed by shaking at 4 °C for 60 min. The lysate-nickel-nitrilotriacetic acid mixture was loaded on a column and washed with 2 volumes of lysis buffer supplemented with 10 mM imidazole. Finally, the recombinant protein was eluted with four times 0.5-ml elution buffer (lysis buffer containing 250 mM imidazole). The protein was eluted in the second and third elution fractions. Recombinant CaM and CaM1234 were expressed and purified as described in a previous study (31). Recombinant CaM1 was expressed and purified by phenyl-Sepharose chromatography in the same way as CaM. GST-NCS-1 and GST-NCS-1E120Q were expressed and purified as described previously (32).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Increase in [Ca2+]c Stimulates Ca2+ Release from Intracellular Stores—In A7r5 cells (embryonic smooth muscle) Ca2+ release from internal stores, mainly from the ER, occurs to a large extent via production of the second messenger IP3. In this permeabilized cell system a maximal effective dose of IP3 can release about 95% of the intracellular Ca2+ content (33). Here, the non-mitochondrial stores of permeabilized A7r5 cells were loaded to steady state with 45Ca2+ and then incubated in a non-labeled efflux medium containing 10 µM thapsigargin. The loss of Ca2+ from the stores under these conditions is plotted as the fractional loss in function of time (Fig. 1). After 10 min the cells were challenged with 1 µM IP3 (circles), as indicated by the bar. As previously documented in detail, using the same 45Ca2+ flux technique (17, 3437), IP3 increased the rate of Ca2+ release (Fig. 1). In the same assay, cells challenged with 3 µM free 40Ca2+ (squares) also showed an increase in the rate of Ca2+ release. 3 µM free 40Ca2+ was able to release 25 ± 2% of the stored Ca2+. The total amount of releasable Ca2+ was measured by treating the cells with 5 µM ionophore A23187 [GenBank] (triangles). This activation of Ca2+ release upon elevation of the cytosolic [Ca2+]c has also previously been observed by others (15, 38), but it could not be excluded that it reflected 45Ca2+/40Ca2+ exchange without net transport (38).



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FIG. 1.
Effect of IP3 and 40Ca2+ on the fractional loss of 45Ca2+ in Ca2+-free medium. After loading of permeabilized A7r5 cells during 45 min in 150 nM 45Ca2+, efflux was started. 1 µM IP3 (circles), 3 µM 40Ca2+ (squares), or 5 µM A23187 [GenBank] (triangles) were added for a 2-min period (black bar), 8 min after starting the efflux. Fractional loss is defined as the amount of 45Ca2+ released in 2 min, divided by the total amount of 45Ca2+ stored at that moment. Each curve represents the means ± S.E. for three wells.

 

It is indeed important to emphasize that in this type of experiment the challenge by 3 µM 40Ca2+ could have caused an exchange of 45Ca2+ for 40Ca2+. To exclude the contribution of 45Ca2+/40Ca2+ exchange we maintained the same 45Ca2+/40Ca2+ ratio during the loading and efflux phases and we changed the [EGTA] to alter the free [Ca2+] (Fig. 2). After incubation in loading buffer during 45 min, the cells were incubated for 2 min in the same loading buffer supplemented with thapsigargin and lowered [EGTA]. This resulted in a complete inhibition of the Ca2+ uptake via the sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase (SERCA) and in a rise of the free [Ca2+] up to 10 µM, while maintaining the 45Ca2+/40Ca2+ ratio constant. Subsequently, the cells were incubated in Ca2+-free efflux medium. The traces in Fig. 2 illustrate how the Ca2+ content of the stores decreased during the 10-min incubation in the Ca2+-free efflux medium and show that the initial Ca2+ content was decreased by the rise in free [Ca2+] during the first 2 min subsequent to the loading. Cells incubated during 2 min in 10 µM free [Ca2+] medium showed a decrease in Ca2+ content of 27 ± 6% compared with cells that were not subjected to a [Ca2+] rise. This finding demonstrates that a decrease in the Ca2+ content was induced by 10 µM free Ca2+ without a change in the 45Ca2+/40Ca2+ ratio thereby excluding passive 45Ca2+/40Ca2+ exchange.

CICR Is Neither IP3R- nor RyR-mediated—The two major classes of intracellular Ca2+ release channels are the IP3Rs and the RyRs. In A7r5 cells both IP3R1 (73%) and IP3R3 (26%) are expressed (39). No evidence has been found for a functional role of the RyR in A7r5 cells (33, 40). IP3R1 and IP3R3 are both known to be regulated by increases in [Ca2+]c (9, 1416). We therefore investigated whether the CICR described here originated from the IP3-sensitive stores. Permeabilized cells were loaded with 45Ca2+ in the presence or absence of a saturating dose of IP3 (300 µM). Efflux was then performed in medium without added Ca2+. After 10 min cells were incubated for 2 min with 10 µM free 40Ca2+. No CICR was observed in cells that were loaded in the presence of IP3 (data not shown). This finding suggested that the CICR mechanism only occurred from the IP3-sensitive stores. Furthermore we looked whether this CICR mechanism was also restricted to the thapsigargin-sensitive stores. In permeabilized A7r5 cells 92% of the total Ca2+ uptake involved a thapsigargin-sensitive SERCA pump, and 8% was mediated by a thapsigargin-insensitive Ca2+-uptake mechanism (41). Cells that were loaded in the presence of 10 µM thapsigargin were challenged with 10 µM free 40Ca2+. Also in this condition no CICR mechanism was observed (data not shown). Taken together, these results suggest that this CICR mode is only occurring from the thapsigargin and IP3-sensitive compartments of the ER.

Heparin, 2-aminoethoxydiphenyl borate (2-APB) and xestospongin C (XeC) are the most used antagonists of the IP3R. In Fig. 3 it is shown that none of these components affected the fractional loss induced by 10 µM free 40Ca2+, revealing that the IP3R was not involved in this mechanism. Although there is no evidence for a functional RyR in A7r5 cells, we also used antagonists of the RyR to exclude any role of the RyR in this CICR mechanism. Fig. 3 illustrates that neither ruthenium red (RuRed) (100 µM) nor ryanodine (5 µM) had any effect on the fractional loss induced by 10 µM free 40Ca2+.



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FIG. 3.
Effects of IP3R and RyR antagonists on CICR. Cells were incubated for a 2-min period with 10 µM 40Ca2+ in the presence of IP3R or RyR antagonists: heparin (1 mg/ml), 2-APB (100 µM), XeC (5 µM), RuRed (100 µM), and ryanodine (5 µM). Fractional loss was measured and compared with fractional loss induced with 10 µM 40Ca2+ alone (100%). None of the antagonists significantly altered the fractional loss. Results represent the means ± S.E. of three independent experiments each performed twice.

 

Ca2+ release stimulated by sphingosine 1-phosphate (42) and NAADP (43) has been observed in a number of cell types. However, it is unlikely that one of these mechanisms mediated CICR in A7r5 cells, because NAADP-stimulated Ca2+ release was not modulated by Ca2+ (44) and no sphingosine 1-phosphate or NAADP-stimulated Ca2+ release was observed in A7r5 cells under our assay conditions (data not shown).

Characteristics of the Observed CICR—To further characterize the CICR mechanism in A7r5 cells, we measured its [Ca2+] dependence. The Ca2+ release as a function of increasing free [40Ca2+] was plotted in Fig. 4a. A maximally effective free [40Ca2+] of 10 µM stimulated release of 27 ± 4% of the stored 45Ca2+. The activation by Ca2+ occurred with an EC50 of 700 ± 30 nM and had a positive cooperativity, with a Hill coefficient of 1.9 ± 0.2. This means that a steep activation occurs within the physiological range of cytosolic Ca2+ levels (0.1–10 µM). Fig. 4b illustrates that the CICR mechanism was controlled by the level of store loading. Ca2+ stores from permeabilized A7r5 cells loaded to steady state with 45Ca2+ were incubated in Ca2+-free efflux medium, and their Ca2+ content was plotted as a function of time. 40Ca2+ (10 µM) was added either after 2 min (circles, full stores) or after 20 min (triangles, less filled stores). Application of 10 µM free 40Ca2+ was clearly less efficient to release 45Ca2+ from less filled stores. These results indicate that the CICR mechanism was controlled by the luminal [Ca2+]. In this respect the CICR mechanism shows the same dependence on the luminal Ca2+ content as described for IP3-induced Ca2+ release in those cells (34).



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FIG. 4.
[Ca2+] dependence of CICR. a, after loading of permeabilized A7r5 cells during 45 min in 150 nM 45Ca2+, efflux was started. After 10 min of efflux a challenge with 40Ca2+ was given for a time period of 2 min. The increase in fractional loss provoked by the 40Ca2+ challenge was plotted as a function of the applied [40Ca2+]. The Ca2+ release was normalized to the total releasable fraction by 5 µM A23187 [GenBank] , which was taken as 100%. Results represent the means ± S.E. of three independent experiments each performed twice. b, loading dependence of the CICR mechanism. The stores were loaded for 45 min at 150 nM free 45Ca2+ and from time 0 onwards incubated in efflux medium. The traces illustrate how the 45Ca2+ content of the stores decreased during the efflux (squares) and how this Ca2+ content was affected by a 2-min application of 10 µM free 40Ca2+ after 2 min (circles) or after 20 min (triangles). Results represent the means ± S.E. for three wells.

 

Fig. 5a illustrates that Mg2+ dose-dependently blocked the CICR in A7r5 cells. The EC50 for this inhibition was 0.59 ± 0.04 mM. The inhibitory effect of Mg2+ was not due to the increase in osmolarity of the medium, because a similar increase in osmolarity by addition of 15 mM KCl instead of 10 mM MgCl2, did not inhibit the CICR (data not shown).



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FIG. 5.
Mg2+ and ATP dependence of CICR. a, Ca2+-induced Ca2+ release was measured in the presence of an increasing [MgCl2]. 100% equals the release by 10 µM free 40Ca2+ without added MgCl2. b, Ca2+-induced Ca2+ release was measured in the presence of an increasing [ATP]. 100% equals the release by 10 µM free 40Ca2+ without added ATP. Results represent the means ± S.E. of three independent experiments each performed twice.

 

Furthermore, CICR was stimulated by increasing the [ATP] in the absence of Mg2+ (Fig. 5b). Stimulation occurred with an EC50 of 320 ± 23 µM. By adding 1 mM ATP together with 10 µM free 40Ca2+, maximal Ca2+ release was increased from 27 ± 4% to 41 ± 3%. This indicates that in physiological conditions this CICR can release a significant fraction of the intracellular stores.

Regulation of CICR by CaM-like Proteins—CaM is a ubiquitous regulator of most if not all types of Ca2+ channels, including the intracellular Ca2+ release channels. We therefore investigated the effect of CaM and CaM mutants on the CICR mechanism in A7r5 cells. CaM1234, which is CaM rendered Ca2+-insensitive by point mutations (45), has the ability to associate with apoCaM-binding sites on Ca2+ release channels (46). In this way CaM1234 can prevent access to Ca2+/CaM effector sites, thereby eliminating Ca2+ regulation via CaM as the Ca2+ sensor. Recombinant CaM (10 µM)orCaM1234 (10 µM) was added together with 3 µM free 40Ca2+ to permeabilized cells loaded with 45Ca2+. Fig. 6a shows that exogenously added CaM had no effect on the Ca2+ release induced by 3 µM free 40Ca2+, whereas CaM1234 almost completely inhibited the CICR. CaM1234 inhibited the CICR with micromolar affinity (Fig. 6b). These data obtained with CaM and CaM1234 led us to hypothesize that Ca2+-free CaM (apoCaM) in resting conditions may be tethered to the protein responsible for the CICR. Binding of Ca2+ to the tethered CaM could then dissociate or dislocate CaM, which could provoke a conformational change thereby activating Ca2+ release from the intracellular stores. CaM1234 would render the system insensitive to activation by Ca2+. To know whether CaM needed to be mutated in all four EF-hands to fulfill its inhibitory role on this CICR mechanism, we also tested CaM mutated in only one EF-hand. CaM1 is mutated in the first EF-hand (Fig. 7a). Recombinant CaM1 (10 µM) was added together with 3 µM free 40Ca2+ to permeabilized cells loaded with 45Ca2+ (Fig. 6a). CaM1 was also able to inhibit the CICR, although not to the same extent as CaM1234. The EC50 for CaM1 inhibition was lower and inhibition was not complete (Fig. 6b). These data suggest that only wild type CaM is capable to fulfill the activation of the CICR by sensing the increase in free Ca2+, whereas mutated CaMs act as inhibitors of this mechanism.



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FIG. 6.
Effects of CaM, CaM1, and CaM1234 on CICR. a, Ca2+ release from permeabilized cells incubated during 45 min in loading buffer, expressed as fractional loss. After incubation during 8 min in Ca2+-free efflux buffer, cells were challenged with 3 µM free Ca2+ (squares), 3 µM free Ca2+ and 10 µM CaM (circles), 3 µM free Ca2+ and 10 µM CaM1 (diamonds), or 3 µM free Ca2+ and 10 µM CaM1234 (triangles) during 2 min as indicated by the bar. Results represent the means ± S.E. for three wells. b, Ca2+ release induced by 10 µM Ca2+ was measured in the presence of increasing concentrations of CaM (circles), CaM1 (diamonds), or CaM1234 (triangles). 100% represents the release induced by 10 µM Ca2+ alone. Results represent the means ± S.E. of three independent experiments each performed twice.

 


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FIG. 7.
Effects of EF-hand containing Ca2+-binding proteins on CICR. a, schematic representation of the Ca2+-binding proteins used in this study. Dark bars represent functionally active EF-hands, and white bars represent inactive EF-hands. Circles represent the myristoylation sites at the N terminus. Two splice variants of CaBP1, both long (lCaBP1) and short (sCaBP1) are represented. b, inhibitory effects of sCaBP1 (squares) and lCaBP1 (triangles) on Ca2+ release induced by 10 µM free 40Ca2+. 100% represents the Ca2+ release in the absence of sCaBP1 or lCaBP1. c, inhibitory effects of GST-NCS-1 (squares) and GST-NCS-1E120Q (triangles) on Ca2+ release induced by 10 µM free Ca2+. 100% represents the Ca2+ release in the absence of GST-NCS-1 or GST-NCS-1E120Q. Results represent the means ± S.E. of three independent experiments each performed twice.

 

CaM is the most ubiquitous mediator of cellular Ca2+ functions, but it has also become clear in recent studies that there is a large number of other EF-hand-containing Ca2+-binding proteins belonging to the CaM superfamily. Particularly the Ca2+-binding protein (CaBP) subfamily and the neuronal Ca2+-sensor (NCS-1) subfamilies that are primarily expressed in neurons may be important for Ca2+ signaling.

Both members of the CaM superfamily are small proteins (about 20 kDa) that share with CaM the basic structure of two N-terminal and two C-terminal EF-hands. However, only three of their EF-hands can bind Ca2+ (Fig. 7a). Hence, we investigated whether two of these Ca2+-binding proteins, CaBP1 and NCS-1 protein, could also alter the activity of this CICR mechanism in A7r5 cells, because they both have one inactive EF-hand. CICR was measured as described above. For CaBP1, both short (sCaBP1) or long (lCaBP1) isoforms were added for 2 min together with a maximally effective free [40Ca2+] of 10 µM. 10 µM of sCaBP1 or lCaBP1 inhibited the CICR by more than 80% (Fig. 7b). Under the same conditions 10 µM GST-NCS-1 equally inhibited the CICR mechanism (Fig. 7c). GST (10 µM) by itself, however, did not affect the CICR mechanism in our system, indicating a specific effect of NCS-1 (data not shown). To exclude that the remaining Ca2+-binding sites of NCS-1 could contribute to the inhibitory effect on CICR through simple Ca2+ chelation, the same experiments were conducted using a mutant of NCS-1. NCS-1E120Q, with its third EF-hand disrupted, showed impaired Ca2+-dependent conformational changes (47). This mutant was still able to inhibit the CICR mechanism to the same extent as wild type NCS-1 (Fig. 7c), thereby excluding a Ca2+ chelation effect.

To test the hypothesis that the CICR mechanism is activated by a Ca2+-dependent dissociation or dislocation from an apoCaM-binding site, we performed experiments in which we trapped the endogenous CaM with a high affinity CaM-binding peptide derived from the RyR1 (amino acids 3614–3643) (30). Fig. 8a shows that in cells incubated during the loading phase with 10 µM of the CaM-binding peptide the CICR mechanism was nearly abolished. However, the RyR1 peptide had no effect on the extent of 45Ca2+ loading of the cells (data not shown). To strengthen the argument regarding the specific effects of the RyR1 peptide, CaM and CaM1234 were re-added for a 2-min period after stripping the cells with the RyR1 peptide. Readdition of 10 µM CaM almost completely restored CICR activation by 3 µM free 40Ca2+, whereas 10 µM CaM1234 was unable to restore CICR activation (Fig. 8b). Therefore, it is likely that, in permeabilized A7r5 cells, Ca2+ activates a Ca2+ release mechanism by binding to endogenously bound apoCaM.



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FIG. 8.
Effect of RyR1 (amino acids 3614–3643) derived peptide on CICR. a, permeabilized A7r5 cells were loaded for 45 min in the absence (squares) or presence of 10 µM RyR1 peptide (3614–3643) (triangles). After a 6-min incubation in Ca2+-free efflux buffer, cells were challenged with 3 µM free Ca2+ during a 2-min time period as indicated by the bar. In cells preincubated with the peptide CICR was abolished. Results represent the means ± S.E. for three wells. b, permeabilized A7r5 cells were loaded for 45 min in the presence of 10 µM RyR1 peptide (3614–3643). Before challenging the cells with 3 µM free Ca2+ during a 2-min time period as indicated by the dark bar, cells were incubated with 10 µM CaM (squares) or 10 µM CaM1234 (triangles) during a 2-min period as indicated by the white bar. Cells that were incubated with CaM responded to 3 µM free Ca2+, whereas cells incubated with CaM1234 did not. Results represent the means ± S.E. for three wells.

 

CICR in Different Cell Types—To verify whether a similar CICR mechanism is also expressed in other cell types we have also screened 16HBE14o-(human bronchial mucosa), LLC-PK1 (porcine kidney cell line), COS-1, and mouse embryonal fibroblast cells in the same conditions as described above for A7r5 cells. Permeabilized cells loaded with 45Ca2+ were challenged with 10 µM free 40Ca2+. A significant CICR response was only found in 16HBE14o-cells, although the fraction of released Ca2+ was smaller (15 ± 3%) than for A7r5 cells. This response in 16HBE14o-cells was also inhibited by CaM1234, sCaBP1, lCaBP1, and NCS-1 (data not shown), suggesting that this same CICR mechanism is not only expressed in A7r5 cells but that it could be more widespread.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ca2+ release from the intracellular stores can be triggered by either IP3 or by a CICR mechanism. Here we report that in permeabilized A7r5 cells an increase in the free [Ca2+]c stimulated a Ca2+ release of up to 41% of the intracellular stores with an EC50 of 700 nM and a Hill coefficient of about 2. This type of CICR mechanism was neither mediated by IP3Rs nor by RyRs, because it was not blocked by ruthenium red, ryanodine, heparin, 2-APB, or xestospongin C. ATP dose-dependently stimulated the CICR mechanism, whereas 10 mM MgCl2 completely abolished it. All these results suggested a novel type of CICR from the non-mitochondrial intracellular stores in permeabilized A7r5 cells. This CICR mechanism did not simply reflect passive 45Ca2+/40Ca2+ exchange and did not result from the SERCA pumps running in reverse, because thapsigargin was present during the efflux phase.

Recently a similar CICR pathway was identified in hepatocytes (26) suggesting that it may be more ubiquitously expressed in different cell types. We identified a similar type of CICR pathway in 16HBE14o-cells, confirming this idea. Although these CICR pathways appear to be quite similar, there are also striking differences between the observations made in the present study as compared with these described for hepatocytes. The CICR mechanism in hepatocytes appeared to be more sensitive, with an EC of 170 nM compared with 700 nM in the present study and was reported to be ATP-independent. This may suggest different types of transporters or at least differences in their regulation.

Polycystin-2 was recently identified as a new Ca2+ release channel. Polycystin-2 is a member of the TRP channel superfamily. Polycystin-2 behaved as a Ca2+-activated, high conductance ER channel that is permeable to divalent ions and exhibited channel behavior reminiscent of RyRs and IP3Rs (2224). It remains to be established if the CICR mechanism described in our study could be related to polycystin-2. The observation that LLC-PK1 cells that endogenously express polycystin-2 did not show the CICR mechanism, however, seems to disprove this hypothesis. Another member of the TRP family, the VR1, was also recently found to act as an intracellular Ca2+ release channel. Capsaicin binding to the VR1 resulted in Ca2+ mobilization from the intracellular Ca2+ stores, and it was found to localize with the ER (27, 28). These data suggest that different members of the TRP family can act as intracellular Ca2+ release channels.

The presence of a CICR mechanism could be important for the propagation and amplification of Ca2+ signals initiated by other Ca2+ release channels. Indeed, CICR mediated by RyRs and IP3Rs plays a crucial role in amplifying the Ca2+ signals provided by Ca2+ entry in cells such as cardiac myocytes (48), neurons (49, 50), astrocytes (51), and pancreatic {beta}-cells (52). For example the nature of long-term changes in synaptic activity in the hippocampus depends on whether Ca2+ entry triggers CICR via RyRs or IP3Rs (50). It became clear that CICR is an important feature of intracellular signaling. The available data strongly suggest the presence of additional CICR pathways different from the well documented IP3R and RyR.

A new finding in our study is that the CICR mechanism described here was inhibited by CaM1234 and by members of the family of CaM-like Ca2+-sensor proteins. It became clear from recent work that most of the Ca2+ channels, both situated in the plasma membrane or in intracellular stores, are regulated by CaM, apoCaM, or members of the CaM superfamily. This has recently been well documented for the RyR and the IP3R. The skeletal-muscle Ca2+ release channel, RyR1, is activated by apoCaM and inhibited by Ca2+-bound CaM (10, 11, 30). For the IP3R the functional significance of CaM is not clear (12, 13). Other Ca2+ channels, like the voltage-dependent Ca2+channels (5356), as well as members of the TRP family (5761), have CaM- and apoCaM-binding sites. We found that the CICR mechanism described in this study is regulated by CaM. The CICR mechanism was not affected by CaM itself, but CaM1 and CaM1234 inhibited it. Using CaM1234 as a negative dominant already revealed the role of CaM in K+ channels (62), L-type Ca2+ channels (45, 55), P/Q-type Ca2+ channels (56), store-operated channels (63), and the RyR (10, 11, 30). Our data indicate the presence of an inhibitory CaM-binding site in the absence of Ca2+ (apoCaM-binding site). CaM tethered to this position could then act as a Ca2+ sensor, and CICR could be interpreted as a Ca2+-dependent dissociation or delocalization of CaM from its inhibitory binding site. The dominant negative effect of CaM1234 results from its inability to perform a Ca2+-dependent interaction. Further evidence supporting this hypothesis was obtained by preincubation of permeabilized cells with a high-affinity peptide for CaM, derived from RyR1. Endogenous CaM could be trapped by this peptide, and therefore the Ca2+ sensor for the CICR mechanism would be removed. In agreement with our hypothesis the preincubation with the RyR1 peptide indeed abolished a subsequent CICR mechanism. Moreover, re-addition of CaM, but not of CaM1234, could restore CICR after preincubation with the RyR1 peptide. Because preincubation with the RyR1 peptide during the loading phase did not interfere with 45Ca2+ loading of the cells, stripping of CaM per se seems not to be sufficient for CICR. The data rather support a mechanism where a Ca2+-dependent delocalization of CaM to another binding site is responsible for CICR activation. Results obtained with other members of the CaBPs can also be explained by this hypothesis. These CaM-like proteins apparently all show binding affinity in the absence of Ca2+. By binding to the apoCaM-binding site they may prevent the role of CaM as a Ca2+ sensor. The C termini of CaBPs are highly homologous to the corresponding region in CaM, whereas the N termini are longer and have more variation, including the myristoylation sites or alternative exons. CaBPs also have an extended 32-amino acid-long flexible central {alpha}-helical segment, versus 28 amino acids in CaM. These differences together with a disabled EF-hand 2 could explain the different binding characteristics as compared with CaM. Indeed, the sequential binding of the highly homologous C-terminal domain with further binding of the N-terminal domain could tether CaBPs to the effector molecules at all [Ca2+] (64). Such changes in binding properties of CaM have also been observed when EF-hand 2 was disabled by mutations (65, 66). Furthermore, NCS-1 bound in a Ca2+-independent manner to rat brain membranes (67). The more restricted expression and subcellular localization of the CaM-like Ca2+-sensor proteins could thereby provide a physiological mechanism to inhibit CICR in specific areas of neurons.

In summary, we found a novel CICR mechanism in A7r5 and 16HBE14o-cells. Although we have not yet established the molecular identity of this novel Ca2+ release pathway, we found that its activation is mediated by CaM. The data suggest that CaM tethered to an inhibitory apoCaM site may act as the Ca2+ sensor for activation of CICR. A possible candidate for this pathway could be a member of the TRP-channel superfamily, like the polycystin-2 channel, but there is as yet no evidence to support this. The apoCaM-binding property described here may offer a practical tool for the future identification of the transport protein involved. In addition, this novel CICR mechanism may provide an additional pathway in Ca2+ release and could play an important role in amplifying Ca2+ signals generated by other Ca2+ release channels.


    FOOTNOTES
 
* This work was supported in part by Grants 1.5.112.02 [EC] (to I. S.), G.0210.03 (to H. D. S. and J. B. P.), and G.O206.01 (to L. M.) from the Fund for Scientific Research Flanders (Belgium); by Grant 99/08 from the Concerted Actions of the K.U. Leuven (to L. M., H. D. S., G. C., and J. B. P.); and by the Interuniversity Poles of Attraction Program-Belgian State, Prime Minister's Office, Federal Office for Scientific, Technical, and Cultural Affairs (Grant IUAP P5/05). 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. Back

A postdoctoral fellow of the Fund for Scientific Research Flanders (Belgium). Back

§ To whom correspondence may be addressed. Tel.: 32-16-34-58-34; Fax: 32-16-34-59-91; E-mail: nael.nadifkasri{at}med.kuleuven.ac.be.

** To whom correspondence may be addressed. Tel.: 32-16-34-57-25; Fax: 32-16-34-59-91; E-mail: humbert.desmedt{at}med.kuleuven.ac.be.

1 The abbreviations used are: [Ca2+]c, cytosolic calcium concentration; apoCaM, apocalmodulin; CaM, calmodulin; CICR, calcium-induced calcium release; ER, endoplasmic reticulum; IP3, inositol 1,4,5-trisphosphate; IP3R, IP3 receptor; lCaBP, long calcium-binding protein; NAADP, nicotinic acid-adenine dinucleotide phosphate; NCS-1, neuronal calcium sensor-1; RuRed, ruthenium red; RyR, ryanodine receptor; sCaBP, short calcium-binding protein; TRP, transient receptor potential; VR1, vanilloid receptor 1; XeC, xestospongin C; 2-APB, 2-aminoethoxydiphenyl borate; SERCA, sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase; GST, glutathione S-transferase. Back


    ACKNOWLEDGMENTS
 
We thank Lea Bauwens and Marina Crabbé for their technical assistance. The mammalian CaM cDNA was kindly provided by Dr. Z. Grabarek (Boston, MA), and the rat cDNA for CaM1 and CaM1234 was kindly provided by Dr. J. Adelman (Portland, OR). We thank Dr. D. C. Gruenert (University of Vermont, Colchester, VT) for the supply of 16HBE14o-cells. We also thank Dr. B. De Strooper (Centre for Human Genetics, Flanders Interuniversity Institute for Biotechnology, K.U. Leuven).



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