A Retrograde Signal from Calsequestrin for the
Regulation of Store-operated Ca2+ Entry in Skeletal
Muscle*
Dong Wook
Shin
§,
Zui
Pan§,
Eun Kyung
Kim
,
Jae Man
Lee
,
Manjunatha B.
Bhat¶,
Jerome
Parness§
,
Do Han
Kim
**, and
Jianjie
Ma§
From the
Department of Life Science, Kwangju
Institute of Science and Technology, Kwangju, 500-712, Korea,
Departments of § Physiology and Biophysics and
Anesthesia, UMDNJ-Robert Wood Johnson Medical School,
Piscataway, New Jersey 08854, and ¶ Center for Anesthesiology
Research, Cleveland Clinic Foundation, Cleveland, Ohio 44195
Received for publication, September 4, 2002, and in revised form, October 30, 2002
 |
ABSTRACT |
Calsequestrin (CSQ) is a high capacity
Ca2+-binding protein present in the lumen of
sarcoplasmic reticulum (SR) in striated muscle cells and has been shown
to regulate the ryanodine receptor Ca2+ release
channel activity through interaction with other proteins present in the
SR. Here we show that overexpression of wild-type CSQ or a CSQ mutant
lacking the junction binding region (amino acids 86-191;
junc-CSQ) in mouse skeletal C2C12 myotube enhanced caffeine- and voltage-induced Ca2+ release by increasing
the Ca2+ load in SR, whereas overexpression of a mutant CSQ
lacking a Ca2+ binding, aspartate-rich domain (amino acids
352-367;
asp-CSQ) showed the opposite effects.
Depletion of SR Ca2+ by thapsigargin initiated
store-operated Ca2+ entry (SOCE) in C2C12 myotubes. A large
component of SOCE was inhibited by overexpression of wild-type CSQ or
junc-CSQ, whereas myotubes transfected with
asp-CSQ exhibited normal function of SOCE. These results
indicate that the aspartate-rich segment of CSQ, under conditions of
overexpression, can sustain structural interactions that interfere with
the SOCE mechanism. Such retrograde activation mechanisms are possibly
taking place at the junctional site of the SR.
 |
INTRODUCTION |
Calsequestrin (CSQ)1 is
a sarcoplasmic reticulum (SR) resident protein in muscle cells whose
primary known function is to buffer Ca2+ in the lumen of
SR. It binds Ca2+ with high capacity (40-50
Ca2+/CSQ) and moderate affinity (Kd ~1
mM) (1). Recent studies have shown, however, that CSQ
participates in the active Ca2+ release process from SR not
simply by being a passive Ca2+ storage protein but also by
actively modulating the function of the ryanodine receptor (RyR), the
primary SR Ca2+ release channel involved in
excitation-contraction coupling (2-6). The carboxyl terminus of CSQ
contains an aspartate-rich region (amino acids 354-367) (7, 8), which
functions as a major Ca2+ binding motif (9) and also
interacts with triadin or junctin, proteins of the SR membrane
complexed to RyR with unclear roles in the operation of
excitation-contraction coupling. A different region of CSQ (amino acids
86-191) has been suggested previously to bind to junctin and triadin
(6, 17). The functional significance of these CSQ regions in muscle
Ca2+ signaling has not been examined.
The internal Ca2+ store of muscle cells, located in the SR,
has a limited capacity; it must be replenished regularly through the
entry of Ca2+ from the external environment. Depletion of
SR Ca2+ stores, following activation of RyR or other
Ca2+ release mechanisms, triggers Ca2+ entry
from the external environment through a process known as capacitative
Ca2+ entry via activation of store-operated
Ca2+ channels (SOC) located in the cell surface membrane
(10, 13, 14). Research into the molecular and cellular function
of store-operated Ca2+ entry (SOCE) has been carried out
primarily in non-excitable cells (i.e. lymphocytes, mast
cells, etc.) and to some extent in smooth muscle cells (11, 12).
Recently, Kurebayashi and Ogawa (13) presented the first functional
evidence for the existence of SOC in skeletal muscle. We have extended
their observations and shown that activation of SOC in skeletal muscle
is coupled to retrograde signaling via conformational changes in the
RyR (14).
In this study, we test the hypothesis that the RyR might receive
information on the state of SR Ca2+ depletion via a direct
retrograde signal from CSQ and thereby modulate both RyR-mediated
Ca2+ release and RyR-mediated SOCE. We found that
overexpression of CSQ not only enhances active Ca2+ release
through the RyR but also suppresses SOCE. Deletion of the
Ca2+ binding, aspartate-rich region of CSQ in these
overexpression experiments resulted in reversal of the suppression of
SOCE by wt-CSQ. Our data suggest that modulation of the RyR complex by CSQ from the luminal side of the SR could play a major role in regulating Ca2+ homeostasis in muscle cells and begin to
define regions of CSQ that differentially interact with the RyR complex.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
C2C12 myoblasts derived from mouse skeletal
muscle were grown in DMEM supplemented with 10% fetal bovine serum and
1% penicillin/streptomycin, as described by Shin et al.
(15). Differentiation of myoblasts into myotubes was induced by
changing the culture medium to DMEM supplemented with 2% horse serum
(HS) and 1% penicillin/streptomycin. Experiments were performed on
C2C12 myotubes expressing RyR, i.e. from the fifth day of
culture in HS-DMEM, when it was possible to select myotubes having
mature skeletal-type excitation-contraction coupling.
Cloning and Gene Transfection--
The wt-CSQ cDNA from
rabbit skeletal muscle and two deletion mutants of CSQ,
junc-CSQ and
asp-CSQ, were originally
cloned into the pCDNA-HA 3.1 vector. For functional studies with
C2C12 cells, the CSQ cDNAs were subcloned from pcDNA3.1-HA to
pCMS-EGFP to create pCMS-EGFP(wt-CSQ),
pCMS-EGFP(
asp-CSQ), and
pCMS-EGFP(
junc-CSQ). The pCMS-EGFP plasmid contains two
separate promoters that drive the transcription of green fluorescent
protein (GFP, under the SV40 promoter) and the gene of interest
(i.e. wt-CSQ or its mutants, under the CMV promoter) (16),
thereby providing a convenient way of selecting transfected cells using
GFP fluorescence. pCMS-EGFP vector alone or vector containing wt-CSQ,
asp-CSQ, or
junc-CSQ cDNAs were
transfected into proliferating myoblasts using LipofectAMINE plusTM reagent according to the manufacturer's
instructions. The culture medium was changed to HS-DMEM to allow
differentiation of myoblasts into myotubes 12 h after transfection.
Immunocytochemistry--
Five days after culturing in HS-DMEM
medium, the C2C12 myotubes growing on coverslips and transfected with
pcDNA3.1-HA plasmids containing wt-CSQ,
asp-CSQ, or
junc-CSQ were fixed with 4% paraformaldehyde and
permeabilized with 0.5% Triton X-100 in phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM
Na2HPO4, 2 mM
KH2PO4, pH 7.2) for 5 min. The cells were then
incubated for 30 min with primary monoclonal antibody against HA or
polyclonal antibody against skeletal CSQ for detecting exogenous HA-CSQ
fusion proteins or endogenous CSQ protein. The cells were washed four
times with 0.1% Triton X-100, followed by incubation with
rhodamine-conjugated secondary antibody for 30 min in
phosphate-buffered saline containing 1% bovine serum albumin. For
detection of endogenous RyR, the cells were incubated for 30 min with
primary polyclonal antibody against RyR and treated with
fluorescein-conjugated secondary antibody. The coverslips were then
mounted with 90% glycerol and 0.1% O-phenylenediamine in
phosphate-buffered saline. Immunofluorescence was analyzed under a
Leica DMRBE microscope (Heidelberg, Germany) equipped with a ×100
objective and filters for epifluorescence. Wild-type and CSQ mutant
protein expression was demonstrated by Western blot following transient
transfection in Chinese hamster ovary (CHO) cells, rather than C2C12
cells, because of the low efficiency of transfection in the latter (see
Fig. 1b). The expressed CSQ protein was probed with
polyclonal anti-CSQ antibody. The protein-antibody complex was blotted
with a horseradish peroxidase-linked secondary antibody, and the signal
was detected on Eastman Kodak Co. films using a chemiluminescent kit
(Pierce, Rockford, IL).
Single Cell Ca2+ Measurement--
The detailed
procedure has been described elsewhere (15). Briefly, C2C12 myotubes
were loaded with Fura-2/AM fluorescent Ca2+ indicator.
Individual myotubes expressing exogenous CSQ were selected by the
presence of GFP fluorescence, as described above. The changes in
intracellular Ca2+ in single live cells was measured
following exposure to 10 mM caffeine or 1 µM
thapsigargin (Tg), with no [Ca2+]o
present in the bath solution (Ca2+-free balanced salt
solution containing 140 mM NaCl, 2.8 mM KCl, 2 mM MgCl2, 10 mM HEPES, pH 7.2, 0.5 mM EGTA).
Mn2+ Quenching Assay of Store-operated
Ca2+ Entry--
The detailed procedure has been described
elsewhere (14). Briefly, to measure Mn2+ influx rate
through SOC, 0.5 mM Mn2+ was added to the
extracellular medium after Tg-induced SR Ca2+ depletion
with or without the buffering of cytosolic Ca2+ by 50 µM BAPTA-AM. The Mn2+ quenching of
Fura-2 fluorescence was measured at the Ca2+-independent
wavelength of Fura-2 excitation (360 nm). The decay of Fura-2
fluorescence upon Mn2+ addition was expressed as percent
decrease in Fura-2 fluorescence per unit time (initial
fluorescence = 100%).
Statistical Analysis--
Values are means ± S.E.
Significance was determined by Student's t test or analysis
of variance. A value of p < 0.05 was used as criterion
for statistical significance.
 |
RESULTS |
Localization of Exogenous Wild-type and Mutant CSQ in SR of C2C12
Cells--
CSQ contains a putative junctin-binding region (amino acids
86-191; junc), as well as the Ca2+-binding
aspartate-rich region (amino acids 354-367; asp) (7, 17).
To examine the function of junc and asp regions
of CSQ, two deletion mutants,
junc-CSQ and
asp-CSQ, were generated using the PCR-based method for
expression and functional studies in C2C12 cells (9). To distinguish
the subcellular distribution of endogenous CSQ from expressed exogenous
CSQ, wt and the CSQ mutants were expressed as HA-CSQ fusion
proteins in differentiated C2C12 myotubes. Subcellular localization of
HA-tagged proteins was performed by immunostaining with monoclonal
antibody against HA. These experiment revealed a perinuclear
distribution of HA-
asp-CSQ expressed in C2C12 myotubes
(Fig. 1b), in a pattern that
is indistinguishable from that of endogenous CSQ present in the SR
detected by polyclonal anti-CSQ (Fig. 1a). The subcellular
distributions of HA-wt-CSQ and HA-
junc-CSQ were similar
to that of endogenous CSQ, indicating that both exogenously expressed
proteins were also localized to the SR (data not shown). This was
further confirmed by co-localization studies with polyclonal anti-RyR,
as shown in the lower panels of Fig. 1, a and
b. Clearly, the patterns of RyR distribution are virtually
identical to those of wt and mutant CSQ expressed in C2C12 cells.

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Fig. 1.
Localization of CSQ mutants overexpressed in
SR of C2C12 myotubes. a and b,
Co-localization of endogenous CSQ and exogenous
HA- asp-CSQ expressed in C2C12 myotubes. Upper
panels, monoclonal anti-HA antibody (b) or polyclonal
anti-CSQ antibody (a) was used to label CSQ proteins. Their
subcellular localization was visualized by red fluorescence of
rhodamine-conjugated secondary antibody. Lower panels,
endogenous RyR was labeled with polyclonal anti-RyR1 antibody and
visualized by the green fluorescence of fluorescein-conjugated
secondary antibody. The similar patterns of subcellular distribution
observed with RyR and HA- asp-CSQ expressed in C2C12 cells
indicate the SR localization of exogenously expressed CSQ. Exogenously
expressed HA-wt-CSQ, HA- junc-CSQ, also showed the similar
patterns to that of endogenously expressed CSQ (a) (data not
shown). c, Western blot of CSQ expressed in CHO cells. The
various CSQ cDNAs were introduced into CHO cells using
LipofectAMINE. Total cell lysates (30 µg, 12 h after gene
transfection) were separated on a 10% SDS-polyacrylamide gel and
subjected to Western blot analysis with polyclonal anti-CSQ. Lane
1, mock-transfected cells; lane 2, cells transfected
with wt-CSQ; lane 3, cells transfected with
asp-CSQ; lane 4, cells transfected with
junc-CSQ.
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To confirm that the translational products of the various CSQ cDNAs
were indeed CSQ, Western blots were performed on SDS-PAGE separated
proteins derived from CHO cells transiently transfected with the
wt-CSQ,
asp-CSQ, and
junc-CSQ cDNAs.
With LipofectAMINE-mediated gene transfection, CHO cells have higher
transfection efficiency than C2C12 cells (30-60% for CHO
versus 3-6% for C2C12), making it easier to detect
expressed CSQ proteins. As shown in Fig. 1c, proteins of the
predicted molecular masses are identified by anti-CSQ antibody. As with
the C2C12 cells, immunostaining studies of these CHO cells also
indicated that the expressed CSQ proteins were localized in the ER (not shown).
Differential Effects of wt-CSQ and
asp-CSQ on Intracellular
Ca2+ Release in Skeletal Muscle--
Insertion of the
various CSQ cDNAs into another eukaryotic expression vector,
pCMS-EGFP, enabled selection of significantly transfected C2C12 cells
using GFP fluorescence. The pCMS-EGFP plasmid expresses 1:1 ratio of
GFP and CSQ under the control of two independent promoters (15, 16).
Individual C2C12 myotubes exhibiting similar levels of GFP
fluorescence, and therefore most likely similar level of exogenous CSQ
proteins, were selected for functional studies with caffeine-induced
Ca2+ release measurements. As shown in Fig.
2a, application of 10 mM caffeine resulted in Ca2+ release from SR in
myotubes transfected with GFP alone (control). The peak amplitude of
caffeine-induced Ca2+ release in myotubes transfected with
wt-CSQ was ~1.7-fold higher than cells transfected with GFP alone
(
F340/F380 = 0.80 ± 0.02, n = 13, GFP; 1.38 ± 0.03, n = 11, wt-CSQ) (Fig. 2c). In contrast, expression of
asp-CSQ in C2C12 cells significantly
reduced caffeine-induced Ca2+ release (0.41 ± 0.03, n = 14). Myotubes transfected with
junc-CSQ, on the other hand, showed similar enhancement
of the amplitude of the caffeine-induced Ca2+ release
transient (1.23 ± 0.03, n = 14) as wt-CSQ (Fig.
2c).

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Fig. 2.
Intracellular Ca2+ release in
C2C12 myotubes transfected with wt-CSQ,
asp-CSQ, and
junc-CSQ. a, C2C12 myotubes
transfected with pCMS-EGFP vector containing cDNA for wt-CSQ,
asp-CSQ, or junc-CSQ were loaded with 5 µM Fura-2-AM. Myotubes showing green fluorescence on the
fifth day of differentiation were used for intracellular
Ca2+ measurements in a bath solution containing no
[Ca2+]o. Addition of 10 mM caffeine induced Ca2+ release detected as
the changes in the Fura-2 fluorescence ratio at excitation wavelengths
of 340 and 380 nm
(F340/F380).
b, depolarization of the cell surface membrane via addition
of 10 mM KCl to the bath solution initiates voltage-induced
Ca2+ release in all cell preparations. Averaged data from
multiple experiments are presented in c with
caffeine-induced Ca2+ release and in d with
voltage-induced Ca2+ release. e, A23187 was used
to release the total intracellular Ca2+ store in individual
C2C12 myotubes bathed in no Ca2+ balanced salt solution,
and the released Ca2+ was assayed by changes in Fura-2
fluorescence. The data are presented as mean ± S.E. from multiple
experiments (n, number of cells).
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A simple explanation for the enhancement of caffeine-induced
Ca2+ release in C2C12 myotubes overexpressing wt-CSQ is
that this phenomenon likely reflects release from a concomitantly
increased SR Ca2+ store. To determine whether the SR
Ca2+ store was indeed increased, we treated cells with
A23187, a Ca2+ ionophore that will release the entire
intracellular Ca2+ store and allow its quantitation (18).
As shown in Fig. 2e, the A23187-releasable Ca2+
pool was significantly larger in myotubes transfected with wt-CSQ and
junc-CSQ than those transfected with GFP alone (0.87 ± 0.12, n = 6, GFP; 1.43 ± 0.13, n = 8, wt-CSQ; 1.35 ± 0.11, n = 6,
junc-CSQ), whereas cells transfected with
asp-CSQ contained an A23187-releasable Ca2+
pool that was statistically identical to GFP controls (0.83 ± 0.15, n = 6) (Fig. 2e).
We then endeavored to determine whether the effects of transfected CSQ
proteins on depolarization-induced Ca2+ release would
parallel the results seen with caffeine-induced Ca2+
release. Changing the extracellular KCl concentration from 2.8 to 10 mM led to depolarization of the cell surface membrane and induced the release of Ca2+ from the SR in C2C12 cells. As
shown in Fig. 2, b and d, the peak amplitude of
depolarization-induced Ca2+ release in cells overexpressing
wt-CSQ and
junc-CSQ was again ~1.4-1.6-fold higher
than that of GFP controls (0.74 ± 0.01, n = 10, GFP; 1.20 ± 0.03, n = 8, wt-CSQ; 1.02 ± 0.01, n = 10,
junc-CSQ), whereas
overexpression of
asp-CSQ led to a significantly
decreased depolarization-induced Ca2+ release (0.46 ± 0.03, n = 11). These results parallel the aggregate caffeine-induced Ca2+ release data shown in Fig.
2c. These data suggest that
asp-CSQ may either
directly suppress RyR channel activity or reduce the efficiency of
signal transduction from the dihydropyridine receptor to the RyR.
Theoretically,
asp-CSQ could suppress SR Ca2+
release by reducing the Ca2+ buffering capacity of the SR.
The aggregate Ca2+ store data presented in Fig.
2e, however, demonstrate that the Ca2+ store of
the GFP control cells and the
asp-CSQ cells are equivalent.
Overexpression of functional CSQ, wt or mutant, in C2C12 cells should
inevitably increase the Ca2+ buffering capacity of the SR
and thereby alter the duration of passive Ca2+ movement
across the SR membrane through as yet undescribed leak pathways. The
kinetics of decay of this passive myoplasmic Ca2+ signal
reflects a competition between continuing Ca2+ leak from
the SR store and the removal of myoplasmic Ca2+ to the
external environment by various plasma membrane-based mechanisms. One
would expect that cells containing an elevated SR Ca2+
store would have a longer kinetic decay of the myoplasmic
Ca2+ signal. To test this possibility, Tg, a potent
inhibitor of SR Ca2+-ATPase (19), was used to block the
Ca2+ uptake function of the SR membrane, allowing for
depletion of the luminal Ca2+ store via SR Ca2+
leak pathways. Our results, shown in Fig.
3A, demonstrate that the peak
amplitudes of Tg-induced increases in myoplasmic
[Ca2+]i were comparable among the
GFP control and those overexpressing wt-CSQ,
asp-CSQ, and
junc-CSQ. As predicted, however, the decay phase of
Ca2+ transients in myotubes overexpressing wt-CSQ or
junc-CSQ, shown in Fig. 2e to contain greater
Ca2+ stores than control or
asp-CSQ cells,
were significantly longer (t1/2 = 167 ± 11 s, n = 9, GFP; 332 ± 11 s,
n = 10, wt-CSQ; 295 ± 28 s,
n = 8,
junc-CSQ) (Fig. 3b). Importantly, the decay pattern of Tg-induced Ca2+
transients in cells overexpressing
asp-CSQ was similar to
the GFP control (t1/2 = 161 ± 8 s,
n = 9). These results suggest that removal of the
asp-rich region significantly reduces Ca2+
buffering capacity of endogenous CSQ, or to the exclusion of endogenous
CSQ, does not participate in Ca2+ buffering of the SR.
These results are consistent with our previous finding that the
asp-rich region contains a major Ca2+ binding
motif (9).

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Fig. 3.
Altered Ca2+ transients in C2C12
cells transfected with wt-CSQ, asp-CSQ,
and junc-CSQ. a, passive
movements of Ca2+ across the SR membrane were induced by an
application of 1 µM Tg, a SR Ca2+-ATPase
inhibitor, with no Ca2+ balanced salt solution in the
external solution. After Tg-induced depletion of SR Ca2+
stores, an addition of 2 mM Ca2+ in bath
solution induced SOCE. Changes of intracellular Ca2+ in
myotubes transfected with GFP alone (control), wt-CSQ,
asp-CSQ, or junc-CSQ were monitored. Each
trace is a representative of eight to ten independent experiments.
b, the t1/2 of Tg-induced
Ca2+ efflux from SR are summarized as means ± S.E.
c, the relative SOCE values after Tg-induced SR
Ca2+ depletion are shown as means ± S.E.
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Inhibition of Store-operated Ca2+ Entry in Skeletal
Muscle by wt-CSQ--
We have shown recently (14) that depletion of SR
Ca2+ storage leads to activation of SOCE in skeletal
muscle. The activation of SOC in skeletal muscle appears to be coupled
to conformational changes of RyR. Because our data above suggest that
the Ca2+ store, as determined by the functional
Ca2+ binding capacity of CSQ, determines the degree of
Ca2+ release via the RyR, we asked whether this
Ca2+ store could affect SOCE in this system. Sustained
treatment of C2C12 myotubes with 1 µM Tg in
Ca2+-free medium resulted in complete depletion of SR
Ca2+. Addition of 2 mM Ca2+ to the
bath solution after the myoplasmic Ca2+ signal had returned
to baseline triggered SOCE in these Ca2+-depleted cells
(Fig. 3a). The degree of SOCE in myotubes transfected with
asp-CSQ was similar to GFP control cells (Fig. 3,
a and c). Strikingly, overexpression of wt-CSQ
and
junc-CSQ in the presence of a Tg-depleted SR
Ca2+ store resulted in significant inhibition of SOCE
(1.03 ± 0.07, n = 9, GFP; 0.51 ± 0.04, n = 10, wt-CSQ; 0.56 ± 0.06, n = 8,
junc-CSQ) (Fig. 3c).
The total SOCE measured in these experiments is likely the result of a
summation of competing processes, SR Ca2+ uptake and
release and surface membrane Ca2+ extrusion and influx. To
isolate the measurement of SOC-mediated Ca2+ influx, we
used the technique of Mn2+ quenching of the Fura-2
fluorescence (14). Mn2+ is known to be able to permeate
into cells via SOC but is impervious to surface membrane extrusion
processes or SR uptake by Ca2+ pumps. Hence,
Mn2+ fluorescence quenching represents a measurement of
unidirectional Ca2+ flux into cells via SOC. Under resting
conditions (i.e. cells with an intact SR Ca2+
pool), no detectable Mn2+ quenching of Fura-2 was observed
(not shown). Myotubes with Tg-depleted SR Ca2+ stores
in a Ca2+-free medium exhibited rapid quenching of Fura-2
fluorescence upon addition of 0.5 mM Mn2+ to
the bath solution (Fig. 4a).
Surprisingly, cells overexpressing wt-CSQ and
junc-CSQ
displayed significant reduction in the rate of Fura-2 fluorescence
quenching even with a depleted SR Ca2+ store. On average,
~10-fold reduction in Mn2+ influx rate was observed in
cells overexpressing wt-CSQ and
junc-CSQ compared with
control. Consistent with the results shown in Fig. 3, overexpression of
asp-CSQ did not appear to affect the rate of
Mn2+ influx in C2C12 cells (Fig. 4a). If the
presence of exogenous CSQ is merely to increase the Ca2+
load of the SR, then the complete depletion of this load should give
equivalent activation of SOCE and resultant Mn2+
fluorescence quenching in all four of the cell preparations. Our
results imply (a) that CSQ itself initiates a signal to
SOCs, and (b) that the asp-rich region of the
protein is likely involved in this signal transmission process.

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Fig. 4.
Inhibition of store-operated Ca2+
entry into C2C12 cells by CSQ. a, the quenching of
Fura-2 fluorescence by 0.5 mM Mn2+ entry after
Tg-induced SR Ca2+ depletion was monitored in control,
wt-CSQ, asp-CSQ, and junc-CSQ transfected
myotubes, respectively (left panel). Fluorescence was
measured with excitation wavelength of 360 nm and emission wavelength
of 510 nm in an arbitrary unit, count per second (cps).
Changes in the rate of Fura-2 quenching by Mn2+ were
expressed as percent of fluorescence decrease per 100 s and
averaged from multiple experiments (right panel).
b, effects of BAPTA on intracellular Ca2+
movement in C2C12 cells. Ca2+ measurements were performed
after pre-incubation of the myotubes with 50 µM BAPTA-AM
for 30 min. Addition of 1 µM Tg did not cause detectable
changes in Fura-2 fluorescence
(F340/F380) in all four
cell preparations. 15 min after addition of Tg, 2 mM
Ca2+ was added to the bath solution, and rates of
F340/F380 increase were
observed (left panel). Averaged data from multiple
experiments are presented in the right panel. c,
similar to studies shown in a, the Mn2+
quenching measurement of Fura-2 was performed in C2C12 myotubes after
30 min of preincubation with 50 µM BAPTA-AM (left
panel). Data from multiple experiments were presented as
means ± S.E. (right panel).
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Lack of Effect of BAPTA on SOCE in Skeletal Muscle--
Studies
from other investigators (33) suggest that gating of SOC is sensitive
to the local level of [Ca2+]i. To
test the role of [Ca2+]i on the
CSQ-mediated changes in the activation of SOCE in skeletal muscle,
C2C12 cells were equilibrated with 50 µM BAPTA-AM, a
concentration sufficient to buffer the changes in
[Ca2+]i because of passive
Ca2+ movement across the SR membrane, as indicated by the
complete lack of Tg-induced changes in Fura-2 signal (Fig.
4b). Fifteen min after the addition of Tg, changing the bath
solution from no [Ca2+] to 2 mM
[Ca2+] resulted in measurable increases in the Fura-2
signal, indicating significant Ca2+ influx across the cell
surface membrane (Fig. 4b). Myotubes transfected with wt-CSQ
and
junc-CSQ showed slower Ca2+ entry than
GFP control and
asp-CSQ-transfected myotubes. Direct Mn2+ quenching studies of Tg-induced
Ca2+-depleted and BAPTA-buffered myotubes confirmed that
the changes in Fura-2 fluorescence above were because of activation of
SOCE (Fig. 4c). Here, a striking reduction in the rate of
Mn2+ quenching was observed in myotubes overexpressing
wt-CSQ and
junc-CSQ, but not
asp-CSQ, with
50 µM BAPTA-AM present in the cytosol. These results
indicate that buffering of [Ca2+]i
does not interfere with function of SOC in skeletal muscle and that the
wt-CSQ-mediated inhibition of SOCE in C2C12 cells is unlikely to
correlate with any changes in myoplasmic [Ca2+]i.
 |
DISCUSSION |
Until recently, CSQ has been thought of as the SR
Ca2+-binding protein whose function is simply to sequester
Ca2+ in the vicinity of the RyR/Ca2+ release
channel, to maintain a store for this ion, and to facilitate its rapid
release during excitation-contraction coupling in muscle cells (2-6).
We have shown here that overexpression of wt-CSQ enhances both
caffeine- and voltage-induced Ca2+ release in skeletal
muscle myotubes that are associated with an increased Ca2+
store in the SR. A profound reduction of SOCE was observed in cells
overexpressing wt-CSQ or
junc-CSQ, but not
asp-CSQ, in cells with depleted SR Ca2+
stores and whose myoplasmic Ca2+ concentrations were
buffered with BAPTA. Thus, the SR Ca2+ store is necessary
for RyR-dependent Ca2+ release, but
Ca2+ store per se is not the sole signal that
regulates SOCE. Rather, CSQ adds a proximal signal in the regulation of
SOCE in muscle cells. Our data suggests that the asp-rich
region of CSQ is essential for retrograde signaling in both
RyR-mediated Ca2+ release and regulation of SOCE in
skeletal muscle.
The enhancement of caffeine-induced Ca2+ release by wt-CSQ
in C2C12 myotubes is similar to that seen in cardiomyocytes isolated from transgenic mice overexpressing CSQ (20, 21). In those studies,
caffeine-induced Ca2+ release was increased by ~10-fold
in the CSQ transgenic mouse, paralleling the ~10-fold overexpression
of CSQ in the heart. Because
asp-CSQ did not change the
total SR Ca2+ store, the negative effect of this mutant on
caffeine- and voltage-induced Ca2+ release in skeletal
muscle may reflect a reduced activity of RyR or its interaction with
accessory proteins or reduced local Ca2+ in the vicinity of
RyR. Our data suggest that the asp-rich region of CSQ may
regulate the proper functioning of the RyR, either by directly
interacting with this channel or affecting other partners in the
RyR/Ca2+ release channel complex. Others have suggested
that proper formation of a quaternary molecular complex among CSQ,
triadin, junctin, and RyR plays a critical role in the active
Ca2+ release process across the SR membrane (6, 20).
Indeed, both the carboxyl-terminal-containing asp-rich and
amino-terminal regions of CSQ have been suggested as necessary for
forming this quaternary SR Ca2+ release complex (8, 22,
23). Our previous studies have shown that the asp-rich
region of CSQ binds Ca2+, and this region is also involved
in interaction with triadin (9). Thus, it is possible that
overexpression of
asp-CSQ may alter the conformation of
the quaternary complex and therefore cause inhibition of the RyR
channel function.
A surprising and critical observation of the present study is that
overexpression of wt-CSQ inhibits the function of SOCE in skeletal
muscle. The CSQ-mediated inhibition of SOCE appears to involve the
asp-rich region of CSQ, because the inhibitory effect was
only observed with wt-CSQ and
junc-CSQ but not with
asp-CSQ. Our studies provide the first direct evidence
for regulation of SOCE, a cell surface membrane function, through the
luminal side of the SR membrane. Previous studies with other cell types have suggested that the physical docking of the ER or SR with the cell
surface membrane is involved in the activation of SOC, presumably
through contact interaction between SOC and protein components in the
ER or SR (e.g. the inositol 1,4,5-trisphosphate receptor or
RyR) (10, 24-26). Alternatively, the release of as yet undefined
diffusible second messenger(s) from the intracellular organelle into
the cytosol has been proposed to serve as an activator of SOC in
response to depletion of intracellular Ca2+ stores (27,
28). Our recent studies with primary cultured skeletal muscle cells
derived from different genetically engineered mouse models suggest that
activation of SOC can be achieved in a graded fashion, depending on the
filling state of the intracellular Ca2+ stores and/or the
conformational changes of RyR (14). Although the gene(s) responsible
for SOC has yet to be identified, and the exact nature of signal
transduction involved in the activation of SOC remains largely unknown,
our data indicate that the aspartate-rich segment of calsequestrin,
under conditions of overexpression, can sustain structural interactions
that interfere with the SOCE mechanism. These interactions are possibly
taking place at the junctional site of the SR. Previous studies
suggested that cardiomyocytes overexpressing CSQ showed abnormal
enlarged junctional SR structure in triad junction, resulting in
alteration of calcium signaling in muscle cells (20, 21). It will be
interesting, therefore, to see how the absence of CSQ in a knock-out
model would affect the function of SOC in skeletal muscle.
The presence of exogenously expressed CSQ in the SR lumen adds extra
Ca2+ buffering capacity and increases the driving force for
Ca2+ movement across the SR membrane. Our experiments with
Tg-induced SR Ca2+ store depletion and myoplasmic BAPTA
Ca2+ buffering have ruled out the possibility that the
reduction of SOCE seen with overexpression of wt-CSQ and
junc-CSQ results from an incomplete depletion of SR
Ca2+ stores or because of potential changes in myoplasmic
[Ca2+]i (29, 30). A previous study
(19) in mouse fibroblast cells showed that overexpression of
calreticulin, a major Ca2+-binding protein in the ER lumen
of non-muscle cells, also inhibited SOCE through a mechanism that is
independent of its Ca2+ binding properties. Examination of
the primary amino acid sequence of calreticulin reveals that, similar
to CSQ, it too contains a highly negatively charged region at its
carboxyl terminus. We speculate that the conservation of this
negatively charged region of the carboxyl terminus of both of these
SR/ER Ca2+-binding proteins supports a significant
functional role for the protein. We further suggest that this region in
calreticulin will be involved in regulating SOCE in non-muscle cells.
Similar to the retrograde interaction between the inositol
1,4,5-trisphosphate receptor and SOC in non-excitable cells (10, 11,
30), a retrograde RyR-dihydropyridine receptor interaction exists in
the skeletal muscle, as revealed by reduced dihydropyridine receptor
function in RyR knock-outs (31, 32). Cumulative evidence also suggests
that the conformational state of the RyR can regulate the function of
SOC (10, 14). Our data reported here provide additional evidence for a
tight link between Ca2+ homeostasis in SR and
Ca2+ permeability in the cell surface membrane.
Overexpression of CSQ in skeletal muscle not only affects caffeine- and
voltage-induced Ca2+ release but also regulates SOCE. The
aspartate residues located in the carboxyl terminus of CSQ not only
constitute binding pockets for Ca2+ but also can regulate
the function of the surface membrane-located, store-operated
Ca2+ channel, likely via retrograde interaction with the
junctional protein complex in the SR.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Kevin P. Campbell for providing
cDNA encoding the rabbit CSQ and Dr. Woo Jin Park for providing
polyclonal anti-CSQ antibody. We also thank Mr. Chun Shik Park for
invaluable technical help in immunocytochemistry.
 |
FOOTNOTES |
*
This work was supported in part by grants from the Korea
Ministry of Science and Technology (Critical Technology 21, 00-J-LF-01-B-54), Korea Science and Engineering Foundation (Basic
Research Program 1999-1-20700-002-5), and the Brain Korea 21 Project
(to D. H. K.) and by National Institutes of Health Grants
RO1-AG15556, RO1-HL69000, and RO1-CA95379 (to J. M.) and R01-AR045593
(to J. P.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
To whom correspondence should be addressed. Tel.: 82-62-970-2485;
Fax: 82-62-970-3411; E-mail: dhkim@kjist.ac.kr.
Published, JBC Papers in Press, November 4, 2002, DOI 10.1074/jbc.M209045200
 |
ABBREVIATIONS |
The abbreviations used are:
CSQ, calsequestrin;
SOCE, store-operated Ca2+ entry;
RyR, ryanodine receptor;
SOC, store-operated Ca2+ channel;
SR, sarcoplasmic reticulum;
Tg, thapsigargin;
wt, wild-type;
DMEM, Dulbecco's modified Eagle's medium;
HS, horse serum;
HA, hemagglutinin;
pCMS, promoter CMV IE, MCS,
SV;
GFP, green fluorescent protein;
EGFP, enhanced GFP;
CHO, Chinese hamster ovary;
BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid tetrakis (acetoxy-methyl ester);
ER, endoplasmic reticulum.
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