A Novel Ca2+-induced Ca2+ Release Mechanism in A7r5 Cells Regulated by Calmodulin-like Proteins*
Nael Nadif Kasri
,
Ilse Sienaert
¶,
Jan B. Parys
,
Geert Callewaert
,
Ludwig Missiaen
,
Andreas Jeromin || and
Humbert De Smedt
**
From the
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.
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ABSTRACT
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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 36143643) 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.
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INTRODUCTION
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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.
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EXPERIMENTAL PROCEDURES
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45Ca2+ FluxesA7r5 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
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.
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Peptide SynthesisRyR1 peptide (amino acids 36143643)
(30) was synthesized by
Eurogentec S.A. (Herstal, Belgium).
Cloning of sCaBp1 and lCaBp1Mouse 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
ProteinspET-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-
-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
-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).
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RESULTS
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Increase in [Ca2+]c Stimulates
Ca2+ Release from Intracellular StoresIn
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.
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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-mediatedThe 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.
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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 CICRTo 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
(0.110 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.
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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.
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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 ProteinsCaM 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.
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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 36143643)
(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.

View larger version (8K):
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|
FIG. 8. Effect of RyR1 (amino acids 36143643) derived peptide on
CICR. a, permeabilized A7r5 cells were loaded for 45 min in the
absence (squares) or presence of 10 µM RyR1 peptide
(36143643) (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 (36143643). 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 TypesTo 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
|
---|
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
-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
-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. 
¶ A postdoctoral fellow of the Fund for Scientific Research Flanders
(Belgium). 
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. 
 |
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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