Biochem. J. (2013) 455, 367–375 (Printed in Great Britain)
367
doi:10.1042/BJ20130805
Calmodulin modulates the termination threshold for cardiac ryanodine
receptor-mediated Ca2 + release
Xixi TIAN*, Yijun TANG*1 , Yingjie LIU*, Ruiwu WANG* and S. R. Wayne CHEN*†2
*Libin Cardiovascular Institute of Alberta, Department of Physiology & Pharmacology, University of Calgary, Calgary, Alberta, Canada, T2N 4N1, and †Department of Biochemistry &
Molecular Biology, University of Calgary, Calgary, Alberta, Canada, T2N 4N1
RyR2 (cardiac ryanodine receptor)-mediated Ca2 + release in
cardiomyocytes terminates when the sarcoplasmic reticulum
Ca2 + content depletes to a threshold level, known as the
termination threshold. Despite its importance, little is known
about the mechanism that regulates the termination threshold.
CaM (calmodulin), by inhibiting RyR2, has been implicated
in Ca2 + -release termination, but whether CaM modulates the
termination threshold is unknown. To this end, we monitored
the endoplasmic reticulum Ca2 + dynamics in RyR2-expressing
HEK (human embryonic kidney)-293 cells transfected with WT
(wild-type) CaM or mutants. We found that WT CaM or CaM
mutations which abolish Ca2 + binding to the N-lobe (N-terminal
lobe) of CaM increased the termination threshold (i.e. facilitated
termination), but had no effect on the activation threshold at
which spontaneous Ca2 + release occurs. On the other hand,
CaM mutations that diminish Ca2 + binding to both the N-lobe
and C-lobe (C-terminal lobe), or the C-lobe only, decreased the
termination threshold (i.e. delayed termination) with a similar
INTRODUCTION
In the heart, muscle contraction is triggered by the release of
Ca2 + from the SR (sarcoplasmic reticulum) [1,2]. Once triggered,
SR Ca2 + release is rapidly terminated to ensure proper EC
(excitation–contraction) coupling and muscle relaxation that are
fundamental to the cardiac cycle [3–6]. It is well established that
the initiation of SR Ca2 + release results from the activation of
the RyR2 (cardiac ryanodine receptor) Ca2 + -release channel via a
mechanism known as Ca2 + -induced Ca2 + release [1,2]. However,
the molecular mechanism underlying the termination of SR Ca2 +
release is largely unknown [3–5]. SR Ca2 + -release termination
probably results from the inactivation of RyR2, but how RyR2 is
inactivated is unclear. An increasing body of evidence suggests
that a decrease in SR luminal Ca2 + level following SR Ca2 +
release plays an important role in the inactivation of RyR2 and
the termination of Ca2 + release [5,7–13].
Important insights into the role of SR Ca2 + depletion in
Ca2 + -release termination have previously been revealed using
SR luminal Ca2 + -sensing dyes, such as fluo-5N [12–15]. By
directly monitoring the SR luminal Ca2 + dynamics in isolated
cardiomyocytes, Zima et al. [13] showed that Ca2 + sparks or
global Ca2 + transients terminated when the SR Ca2 + content
decreases to a threshold level, called the termination threshold.
These observations suggest that SR luminal Ca2 + depletion is a
activation threshold. Furthermore, deletion of residues 3583–
3603 or point mutations (W3587A/L3591D/F3603A, W3587A,
or L3591D) in the CaM-binding domain of RyR2 that are
known to abolish or retain CaM binding all reduced the termination threshold without having a significant impact on the activation
threshold. Interestingly, the RyR2-F3603A mutation affected
both the activation and termination threshold. Collectively, these
data indicate that CaM facilitates the termination of Ca2 + release
by increasing the termination threshold, and that this action of
CaM depends on Ca2 + binding to the C-lobe, but not to the
N-lobe, of CaM. The results of the present study also suggest that
the CaM-binding domain of RyR2 is an important determinant
of Ca2 + -release termination and activation.
Key words: calmodulin, Ca2 + -release termination, intracellular
Ca2 + release, luminal Ca2 + , ryanodine receptor, sarcoplasmic
reticulum.
major signal for Ca2 + -release termination [5]. Interestingly, the
termination threshold for Ca2 + sparks or global Ca2 + transients
(∼60 % of the resting SR Ca2 + level) was found to be independent
of the initial SR Ca2 + load, the magnitude of Ca2 + -release flux
or the level of cytosolic Ca2 + [13]. We have also shown that
spontaneous Ca2 + release in HEK (human embryonic kidney)293 cells expressing RyR2 displays a termination threshold of
∼57 % [16]. Hence, the phenomenon of Ca2 + -release termination
is not unique to cardiac cells. It probably reflects the intrinsic
properties of RyR2.
As with the activation of SR Ca2 + release, the termination of SR
Ca2 + release is likely to be subjected to modulation by various
conditions and factors. Indeed, the threshold for Ca2 + -release
termination is altered in disease states. For instance, in failing
cardiomyocytes, the termination threshold was reduced, leading to
delayed termination of Ca2 + release [17]. We have recently shown
that RyR2 mutations associated with cardiomyopathies altered
the termination threshold [16]. The termination threshold is also
modulated by physiological and pharmacological ligands. It has
recently been shown that isoproterenol increases the termination
threshold (i.e. facilitates the termination of Ca2 + release) [18].
On the other hand, caffeine was found to reduce the termination
threshold, resulting in delayed Ca2 + -release termination [17,19].
RyR2 is regulated by a number of protein effectors such as
CaM (calmodulin) [20–22]. Whether RyR2-interacting proteins
Abbreviations used: CaM, calmodulin; C-lobe, C-terminal lobe; CPVT, catecholaminergic polymorphic ventricular tachycardia; ER, endoplasmic
reticulum; fura 2/AM, fura 2 acetoxymethyl ester; HEK, human embryonic kidney; KRH, Krebs–Ringer–Hepes; N-Lobe, N-terminal lobe; RyR2, cardiac
ryanodine receptor; SOICR, store-overload-induced Ca2 + release; SR, sarcoplasmic reticulum; WT, wild-type.
1
Present address: Taihe Hospital Affiliated Hosptial of Hubei University of Medicine, Hubei, People’s Republic of China
2
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2013 Biochemical Society
368
X. Tian and others
also modulate the threshold for Ca2 + -release termination has yet
to be determined. CaM has been shown to inhibit the Ca2 + dependent activation of single RyR2 channels [22]. CaM binds
to a region in RyR2 encompassing residues 3583–3603. Deletion
of these residues abolishes CaM binding to RyR2 and the CaMdependent inhibition of single RyR2 channels [21]. Interestingly,
point mutations in this region either abolish both the binding
and inhibitory action of CaM or only eliminate the inhibitory
action of CaM without affecting CaM binding [21]. Importantly,
a triple mutation in this region (W3587A/L3591D/F3603A)
which abolishes CaM binding to RyR2 causes severe heart
failure, cardiomyopathies and early death in mice [23]. Neonatal
cardiomyocytes isolated from the RyR2 triple-mutant mice
exhibited prolonged Ca2 + transients, suggesting altered Ca2 + release termination [23]. However, whether CaM modulates the
termination threshold for Ca2 + release is unknown.
It is therefore of interest and importance to assess the role of
CaM in the termination of spontaneous Ca2 + release. To this end,
in the present study, we directly tested whether CaM affects the
termination and activation thresholds for SOICR (store-overloadinduced Ca2 + release) by monitoring the ER (endoplasmic
reticulum) luminal Ca2 + dynamics in HEK-293 cells expressing
RyR2 with or without CaM using an ER luminal Ca2 + -sensing
protein, D1ER [24]. We found that WT (wild-type) CaM increased
the termination threshold (i.e. facilitated termination), whereas
Ca2 + -binding-deficient CaM mutants decreased the termination
threshold, resulting in delayed Ca2 + -release termination. Thus
CaM-facilitated termination of Ca2 + release requires Ca2 +
binding to CaM. On the other hand, CaM WT or mutations had no
significant effect on the activation threshold for SOICR. Furthermore, deletion of the CaM-binding site or mutations that abolish
CaM binding to RyR2 also reduced the termination threshold.
Interestingly, mutations that only affect the inhibitory action of
CaM, but not the binding of CaM, also reduced the termination
threshold. This suggests that the CaM-binding domain of RyR2
plays an important role in Ca2 + -release termination.
cells were co-transfected with the inducible expression vector
pcDNA5/FRT/TO containing the mutant cDNAs and the pOG44
vector encoding the Flp recombinase at a ratio of 1:5 using the
Ca2 + phosphate precipitation method. The transfected cells were
washed with PBS 24 h after transfection followed by a change into
fresh medium for 24 h. The cells were then washed again with
PBS, harvested and plated on to new dishes. After the cells had
attached (∼4 h), the growth medium was replaced with a selection
medium containing 200 μg/ml hygromycin (Invitrogen). The
selection medium was changed every 3–4 days until the desired
number of cells was grown. The hygromycin-resistant cells were
pooled, divided into aliquots and stored at − 80 ◦ C. These positive
cells are believed to be isogenic, because the integration of RyR2
cDNA is mediated by the Flp recombinase at a single FRT site.
Single-cell cytosolic Ca2 + imaging of HEK-293 cells
Cytosolic Ca2 + levels in stable inducible HEK-293 cells
expressing RyR2 WT or mutant channels were monitored using
single-cell Ca2 + imaging and the fluorescent Ca2 + indicator dye
fura 2/AM (fura 2 acetoxymethyl ester) as described previously
[27,28]. Briefly, cells grown on glass coverslips for 18–22 h after
induction by 1 μg/ml tetracycline were loaded with 5 μM fura
2/AM in KRH (Krebs–Ringer–Hepes) buffer [125 mM NaCl,
5 mM KCl, 1.2 mM KH2 PO4 , 6 mM glucose, 1.2 mM MgCl2
and 25 mM Hepes (pH 7.4)] plus 0.02 % pluronic F-127 and
0.1 mg/ml BSA for 20 min at room temperature (23 ◦ C). The
coverslips were then mounted in a perfusion chamber (Warner
Instruments) on an inverted microscope (Nikon TE2000- S). The
cells were continuously perfused with KRH buffer containing
increasing extracellular Ca2 + concentrations (0, 0.1, 0.2, 0.3, 0.5,
1.0 and 2.0 mM). Caffeine (10 mM) was applied at the end of each
experiment to confirm the expression of active RyR2 channels.
Time-lapse images (0.25 frame/s) were captured and analysed
with the Compix Simple PCI 6 software. Fluorescence intensities
were measured from regions of interest centred on individual cells.
Only cells that responded to caffeine were analysed.
MATERIALS AND METHODS
Single-cell luminal Ca2 + imaging (D1ER assay)
Site-directed mutagenesis
Luminal Ca2 + in HEK-293 cells expressing RyR2 WT or
mutations were measured using single-cell Ca2 + imaging and the
Ca2 + -sensitive FRET-based cameleon protein D1ER as described
previously [29]. The cells were grown to 95 % confluency in
a 75 cm2 flask, split with PBS, and plated on to 100-mmdiameter tissue culture dishes at ∼10 % confluence 18–20 h
before transfection with D1ER or co-transfection with D1ER
and CaM(WT), CaM(1, 2), CaM(3, 4) or CaM(1–4) cDNA.
At 24 h after transfection, an induction medium containing
1 μg/ml tetracycline (Sigma) was applied to induce the expression
of RyR2 in these cells. After induction for ∼22 h, the cells
were perfused continuously with KRH buffer containing various
concentrations of CaCl2 (0–2 mM) and tetracaine (1 mM) or
caffeine (20 mM) at room temperature. Images were captured by
a QuantEM 512SC camera every 2 s using the NIS-Elements AR
software. Cells were excited at 430 nm, and the emission was split
into 465- and 535-nm beams by a dual-view device (Photometrics)
placed in a Nikon eclipse Ti microscope. D1ER signals were
determined from the ratios of the emissions at 535 +
− 30 nm (YFP)
and 465 +
− 30 nm (CFP).
The cloning and construction of the 15-kb full-length cDNA
encoding the mouse cardiac RyR2 has been described previously
[25]. The RyR2 deletion, Del-3583–3603, and the point mutations
W3587A, L3591D, F3603A and W3587A/L3591D/F3603A were
constructed according to the previously described PCR-mediated
overlap extension method [26]. A fragment containing Del-3583–
3603, W3587A, L3591D, F3603A or W3587A/L3591D/F3603A
was produced by PCR and subcloned into BsiWI (8864)-NotI
(vector) RyR2 WT DNA fragment using AgeI (10370) and SacII
(11202). The fragment containing the deletion or mutations was
then subcloned to the full-length RyR2 in pcDNA3 or pcDNA5
using BsiWI (8864) and NotI (vector). Ca2 + -binding-deficient
CaM mutations, CaM(1, 2), CaM(3, 4) or CaM(1–4), in which
two or all of the asparagine residues (residues 20, 56, 93 and 129)
were mutated to alanine, were also generated using the PCRmediated overlap extension method. The sequences of all PCR
fragments were verified by DNA sequencing analysis.
Generation of stable inducible HEK-293 cell lines
Stable inducible HEK-293 cell lines expressing RyR2 WT,
deletion and mutations were generated using the Flp-In TREx Core Kit from Invitrogen. Briefly, Flp-In T-REx-293
c The Authors Journal compilation c 2013 Biochemical Society
Statistical analysis
All values shown are means +
− S.E.M. unless indicated otherwise.
To test for differences between groups, we used Student’s t test
CaM modulates the termination threshold
369
(two-tailed) or one-way ANOVA with post-hoc test. A P value of
<0.05 was considered to be statistically significant.
RESULTS
CaM increases the luminal Ca2 + threshold level at which Ca2 +
release terminates
To assess the effect of CaM on the termination of Ca2 + release,
we co-transfected HEK-293 cells expressing WT RyR2 with CaM
and a FRET-based ER luminal Ca2 + -sensing protein D1ER [24].
The ER luminal Ca2 + dynamics in transfected HEK-293 cells
was then monitored using single-cell FRET imaging as described
previously [16,19,29,30]. As shown in Figure 1(A), elevating
extracellular Ca2 + from 0 to 2 mM induced spontaneous Ca2 +
oscillations in HEK-293 cells expressing WT RyR2, also known
as SOICR [27,28] (observed as downward deflections of the FRET
signal). SOICR occurred when the ER luminal Ca2 + increased to
a threshold level (the SOICR activation threshold, FSOICR ), and
terminated when the ER luminal Ca2 + fell to another threshold
level (the SOICR termination threshold, Ftermi ) (Figure 1A).
This SOICR in WT RyR2-expressing HEK-293 cells displayed
an activation threshold of 93 +
− 0.5 % store capacity and a
termination threshold of 58 +
− 0.7 % store capacity. Interestingly, a
termination threshold of ∼60 % was observed in cardiomyocytes
[13]. Thus Ca2 + release in both cardiomyocytes and HEK-293
cells terminates before the store is completely depleted. It should
be noted that SOICR did not occur in control HEK-293 cells
expressing no RyR2, and that SOICR was not affected by the IP3 R
(inositol 1,4,5-trisphosphate receptor) inhibitor xestospongin C
[16], indicating that SOICR in RyR2-expressing HEK-293 cells
is mediated by RyR2.
The effect of CaM on SOICR is shown in Figure 1(B). Coexpression of CaM in RyR2-expressing HEK-293 cells had no
significant effect on the activation threshold (Figure 1D), but
it significantly increased the termination threshold (65 +
− 1.1 %)
as compared with the control (P < 0.001) (Figure 1E). As a
result, the fractional Ca2 + release during SOICR (activation
threshold − termination threshold) was significantly reduced in
HEK-293 cells transfected with CaM (29 +
− 0.7 %) than in
control cells (36 +
− 0.9 %) (P < 0.001) (Figure 1F). There were
no significant differences in the store capacity (Fmax − Fmin )
between the control and the CaM-transfected cells (Figure 1G).
The frequency of Ca2 + oscillation in CaM-transfected cells
(0.89 +
− 0.02 event/min) is slightly higher than that in control cells
(0.78 +
− 0.04 event/min), but the significance of this difference is
marginal (P = 0.05). It is important to note that the frequency of
Ca2 + oscillation depends not only on the activity of RyR2, but
also on store Ca2 + refilling, which is influenced by a number of
factors, such as Ca2 + influx, Ca2 + uptake or Ca2 + buffering. It is
also important to note that the D1ER probe was not saturated in
HEK-293 cells (results not shown), consistent with that reported
previously [31]. Thus the effect of CaM is to terminate Ca2 +
release at a higher ER luminal Ca2 + concentration or with a
lesser ER Ca2 + depletion. In other words, CaM facilitates the
termination of Ca2 + release.
CaM-facilitated termination of Ca2 + release requires Ca2 + binding
to the C-lobe (C-terminal lobe) of CaM
CaM contains four EF-hand Ca2 + -binding sites. To determine
whether Ca2 + binding to these sites is required for its facilitation
of Ca2 + -release termination, we used a Ca2 + -binding mutant of
CaM, CaM(1–4), in which all four Ca2 + -binding sites have been
Figure 1
SOICR
Effect of WT CaM and a Ca2 + -binding-deficient CaM mutant on
A stable inducible HEK-293 cell line expressing WT RyR2 was co-transfected with the FRET-based
ER luminal Ca2 + -sensing protein D1ER and CaM(WT) or a Ca2 + -binding-deficient CaM
mutant, CaM(1–4), 48 h before single-cell FRET imaging. The expression of WT RyR2 was
induced 24 h before imaging. The cells were perfused with KRH buffer containing increasing
levels of extracellular Ca2 + (0–2 mM) to induce SOICR. This was followed by the addition of
1.0 mM tetracaine to inhibit SOICR, and then 20 mM caffeine to deplete the ER Ca2 + stores.
Single-cell luminal Ca2 + dynamics (FRET recordings) from representative WT RyR2 cells
transfected with no CaM (control) (176 cells) (A), CaM(WT) (88 cells) (B) or the CaM(1–4)
mutant (95 cells) (C) are shown. The activation threshold (D) and termination threshold (E) were
determined using the equations shown in (A). FSOICR indicates the FRET level at which SOICR
occurs, whereas Ftermi represents the FRET level at which SOICR terminates. The fractional
Ca2 + release (F) was calculated by subtracting the termination threshold from the activation
threshold. The maximum FRET signal Fmax is defined as the FRET level after tetracaine treatment.
The minimum FRET signal Fmin is defined as the FRET level after caffeine treatment. The store
capacity (F) was calculated by subtracting Fmin from Fmax . Data shown are means +
− S.E.M.
(n = 4–13) (*P < 0.001 compared with the control).
c The Authors Journal compilation c 2013 Biochemical Society
370
X. Tian and others
mutated [32]. As shown in Figure 1(C), CaM(1–4) significantly
reduced the termination threshold (49 +
− 2.4 %) as compared with
the control (P < 0.001) (Figure 1E), but it had no effect on
the activation threshold (Figure 1D). As a result, the fractional
Ca2 + release was significantly increased in HEK-293 cells
transfected with CaM(1–4) (44 +
− 2.7 %) (P < 0.001) (Figure 1F).
Thus in contrast with CaM(WT), the Ca2 + -binding deficient
CaM(1–4) mutant inhibited Ca2 + -release termination, probably
by suppressing the effect of endogenous CaM. It should be noted
that both apoCaM and Ca2 + –CaM are able to bind RyR2 [22].
Thus these observations suggest that Ca2 + binding to CaM is
required for its action on Ca2 + -release termination.
To further dissect the importance of Ca2 + binding to the N-lobe
(N-terminal lobe) and C-lobe of CaM in mediating the effects
of CaM on Ca2 + -release termination, we took advantage of the
Ca2 + -binding mutants of CaM, CaM(1, 2), in which the two Ca2 + binding sites in the N-lobe were mutated, and CaM(3, 4), in
which the two Ca2 + -binding sites in the C-lobe were mutated. As
shown in Figure 2, CaM(1, 2) increased the termination threshold
and fractional Ca2 + release in HEK-293 cells (Figures 2A and
2D), an effect similar to that of CaM(WT) (Figure 1B). On the
other hand, CaM(3, 4) reduced the termination threshold and
fractional Ca2 + release (Figures 2D and 2E), an effect similar to
that of CaM(1–4) (Figure 1C). Neither CaM(1, 2) nor CaM(3, 4)
altered the activation threshold (Figure 2C), similar to CaM(WT)
or CaM(1–4). Collectively, these data suggest that Ca2 + binding
to the C-lobe, but not to the N-lobe, is critical for CaM-facilitated
termination of Ca2 + release.
To maximize the co-expression of RyR2, CaM and the luminal
Ca2 + sensor protein D1ER, we used an inducible stable HEK293 cell line that expresses RyR2. We transfected these RyR2expressing cells with D1ER and CaM. Only HEK-293 cells that
were both fluorescently labelled (i.e. transfected with D1ER)
and responded to caffeine (i.e. expressing RyR2) were used for
analysis. Thus all cells analysed expressed both RyR2 and D1ER.
However, it is possible that some of the RyR2- and D1ER-positive
cells may not have been transfected with CaM WT or mutants.
Therefore the effect of CaM WT and mutations on SOICR would
have been underestimated.
Deletion of the CaM-binding site in RyR2 abolishes CaM-facilitated
termination of Ca2 + release
CaM interacts with a large number of cellular targets, including
RyR2. The CaM-facilitated termination of Ca2 + release observed
in HEK-293 cells expressing WT RyR2 could result from RyR2independent pathways. To address this possibility, we generated
a HEK-293 cell line expressing a CaM-binding mutant of RyR2,
RyR2 Del-3583–3603, in which the CaM-binding site (amino
acids 3583–3603) in RyR2 have been deleted. It has previously
been shown that deletion of residues 3583–3603 abolishes CaM
binding to and the inhibitory effect of CaM on RyR2 [21].
We reasoned that if CaM facilitates the termination of Ca2 +
release by binding to RyR2, removal of the CaM-binding site in
RyR2 should abolish CaM-dependent facilitation of Ca2 + -release
termination. Consistent with this view, we found that deletion of
the CaM-binding site (residues 3583–3603) in RyR2 significantly
lowered the termination threshold (32 +
− 1.3 %) (P < 0.001)
(Figures 3A and 3E), but had no effect on the activation threshold
(Figures 3A and 3D). As a result, the fractional Ca2 + release
(62 +
− 1.0 %) in these cells was significantly increased (P < 0.001)
(Figure 3F). Furthermore, expression of CaM(WT) or CaM(1–4)
in HEK-293 cells expressing RyR2 Del-3583–3603 did not affect
the termination or activation threshold for Ca2 + release (Fig
c The Authors Journal compilation c 2013 Biochemical Society
Figure 2 Effect of CaM mutants deficient in Ca2 + binding to the N-lobe or
C-lobe of CaM on SOICR
Single-cell luminal Ca2 + (FRET) imaging was carried out as described in the legend to Figure 1.
Luminal Ca2 + dynamics from representative single RyR2-WT-expressing cells transfected
with the N-lobe Ca2 + -binding-deficient CaM mutant, CaM(1, 2) (116 cells) (A) or C-lobe
Ca2 + -binding-deficient CaM mutant, CaM(3, 4) (153 cells) (B) are shown. The activation
threshold (C) and termination threshold (D) were determined using the equations shown in (A).
The fractional Ca2 + release (E) was calculated by subtracting the termination threshold from
the activation threshold. The store capacity (F) was calculated by subtracting Fmin from Fmax .
Data shown are means +
− S.E.M. (n = 6–9) (*P < 0.001 compared with the control).
ures 3B–3F). Thus the termination or activation of Ca2 + release
in HEK-293 cells expressing the CaM-binding mutant of RyR2
was no longer sensitive to modulation by CaM(WT) or CaM(1–4).
These data are consistent with the notion that CaM-facilitated termination of Ca2 + release requires the CaM-binding site in RyR2.
Effect on Ca2 + release termination of mutations in the CaM-binding
site of RyR2 that exert different effects on CaM binding
Meissner and co-workers have reported that a triple mutation,
W3587A/L3591D/F3603A, in the CaM-binding domain of RyR2
CaM modulates the termination threshold
371
completely abolished CaM binding to and CaM-dependent
inhibition of RyR2, whereas single point mutations W3587A and
L3591D eliminated the inhibitory action of CaM with partial
or no effect on CaM binding [21]. To determine whether these
mutations with different effects on CaM binding affect Ca2 + release termination to different extents, we generated HEK-293
cell lines that express each of these mutations and determined their
termination and activation thresholds. As shown in Figure 4, the
triple RyR2 mutation (W3587A/L3591D/F3603A) markedly reduced the termination threshold and increased the fractional Ca2 +
release, similar to the effect of the deletion mutant RyR2 Del3583–3603. Interestingly, single RyR2 mutations (W3587A and
L3591D) were also able to reduce the termination threshold and
increase the fractional Ca2 + release to an extent similar to that seen
with the triple mutation (W3587A/L3591D/F3603A). Note that
neither of these mutations affected the activation threshold. Thus
these RyR2 mutations all significantly reduce the termination
threshold for Ca2 + release regardless of whether or not they affect
CaM binding to RyR2. These data suggest that the CaM-binding
domain of RyR2 is involved not only in binding CaM, but also in
mediating the action of CaM in the termination of Ca2 + release.
Mutation F3603A in the CaM-binding region of RyR2 alters both the
activation and termination of Ca2 + release
Figure 3 CaM or CaM(1–4) mutant has no effect on SOICR in HEK-293 cells
expressing a CaM-binding-deficient RyR2 mutant
A stable inducible HEK-293 cell line expressing the RyR2 mutant, Del-3583–3603, in which
the CaM-binding site (residues 3583–3603) was deleted, was co-transfected with D1ER and
CaM(WT) or Ca2 + -binding-deficient CaM mutant, CaM(1–4). Luminal Ca2 + dynamics in the
transfected cells were monitored using single-cell FRET imaging as described in the legend to
Figure 1. Single-cell FRET recordings from representative RyR2 Del-3583–3603 cells transfected
with no CaM (control) (58 cells) (A), CaM(WT) (51 cells) (B) or CaM(1–4) mutant (81 cells) (C)
are shown. The activation threshold (D) and termination threshold (E) were determined using
the equations shown in (A). The fractional Ca2 + release (F) was calculated by subtracting the
termination threshold from the activation threshold. The store capacity (G) was calculated by
subtracting Fmin from Fmax . Data shown are means +
− S.E.M. (n = 4) (*P < 0.001 compared
with the control).
Figure 5 shows the impact of the F3603A mutation in the CaMbinding site of RyR2 on SOICR. Unlike the triple mutation
and other single mutations (W3587A and L3591D), the F3603A
mutation diminished SOICR. Elevating extracellular Ca2 + from
0 to 2 mM induced SOICR in HEK-293 cells expressing WT
RyR2 (Figure 5A), but not in HEK-293 cells expressing the
F3603A mutant (Figure 5B). However, SOICR could be induced
in these F3603A-expressing cells by adding caffeine (1 mM) to the
perfusate (Figure 5B). Since caffeine is known to induce SOICR
by decreasing the activation threshold [19], the rescue of
SOICR by caffeine in the F3603A cells suggests that the F3603A
mutation may suppress SOICR by increasing the activation
threshold. To test this possibility, we induced SOICR in both
RyR2-WT and F3603A-expressing HEK-293 cells by caffeine
(1 mM) and compared their SOICR properties under the same
conditions. In the presence of 1 mM caffeine, WT RyR2expressing cells exhibited an activation threshold of 70 +
− 2.3 %
store capacity (Figures 5A and 5C), whereas the F3603A mutantexpressing cells displayed a significantly increased activation
threshold (88 +
− 0.9 %, P < 0.01) compared with the WT cells
(Figures 5B and 5C). With an increased activation threshold, the
F3603A mutation would be expected to decrease the propensity
for SOICR. Indeed, single-cell cytosolic Ca2 + imaging revealed
that only a very small fraction of F3603A mutant-expressing
HEK-293 cells displayed Ca2 + oscillations upon elevation
of extracellular Ca2 + , which is markedly different from WT
RyR2 cells (Figure 6). Furthermore, the F3603A mutation also
significantly decreased the termination threshold (27 +
− 2.0 %
compared with 37 +
− 1.1 %2 +in WT, P < 0.01) (Figure 5D), and
increased the fractional Ca release (61 +
− 2.4 % compared with
33 +
1.3
%
in
WT,
P
<
0.01)
(Figure
5E).
Thus the F3603A
−
mutation affects not only the termination, but also the activation
of Ca2 + release, suggesting that the CaM-binding site of RyR2 is
important for both the activation and termination of Ca2 + release.
DISCUSSION
It has previously been shown that Ca2 + release in cardiac cells
terminates when the SR Ca2 + content depletes to a threshold level
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372
X. Tian and others
Figure 5 Effect of the RyR2-F3603A mutation on the activation and
termination of SOICR
Figure 4
Effect of mutations in the CaM-binding site of RyR2 on SOICR
Stable inducible HEK-293 cell lines expressing a triple RyR2 mutant, W3587A/L3591D/F3603A,
and single RyR2 mutants, W3587A and L3591D, were transfected with D1ER. FRET imaging was
performed as described in the legend to Figure 1. Single-cell FRET recordings from representative
HEK-293 cells expressing W3587A/L3591D/F3603A (70 cells) (A), W3587A (104 cells) (B)
and L3591D (81 cells) (C) are shown. The activation threshold (D) and termination threshold
(E) were determined using the equations shown in (A). The fractional Ca2 + release (F) was
calculated by subtracting the termination threshold from the activation threshold. The store
capacity (G) was calculated by subtracting Fmin from Fmax . Data shown are means +
− S.E.M.
(n = 5–7) (*P < 0.001 compared with the WT).
c The Authors Journal compilation c 2013 Biochemical Society
Stable inducible HEK-293 cell lines expressing RyR2 WT or the RyR2 F3603A mutant were
transfected with the FRET-based ER luminal Ca2 + -sensing protein D1ER 48 h before single-cell
FRET imaging. The expression of RyR2 WT and mutant F3603A was induced 24 h before
imaging. The cells were perfused with KRH buffer containing 0, 1 or 2 mM extracellular Ca2 + ,
or 2 mM extracellular Ca2 + plus 1 mM caffeine to induce SOICR. This was followed by the
addition of 20 mM caffeine to deplete the ER Ca2 + stores. Single-cell luminal Ca2 + dynamics
(FRET recordings) from representative HEK-293 cells expressing RyR2 WT (103 cells) (A) and
the F3603A mutant (112 cells) (B) are shown. The activation threshold (C) and termination
threshold (D) in the presence of 1 mM caffeine were determined using the equations shown
in (A and B). The fractional Ca2 + release (E) was calculated by subtracting the termination
threshold from the activation threshold with 1 mM caffeine. The store capacity (F) was calculated
by subtracting Fmin from Fmax . Data shown are means +
− S.E.M. (n = 4–5) (*P < 0.01 compared
with WT).
(the termination threshold) [13]. Importantly, this termination
threshold is altered in diseases and modulated by physiological
and pharmacological ligands [17–19]. These findings raise an
intriguing and important question of whether CaM also regulates
Ca2 + -release termination by altering the termination threshold.
CaM modulates the termination threshold
373
the present study, we found that RyR2-expressing HEK-293
cells transfected with WT CaM exhibited a higher termination
threshold than control HEK-293 cells (transfected with no DNA),
indicating that WT CaM facilitates the termination of RyR2mediated Ca2 + release. On the other hand, RyR2-expressing
HEK-293 cells transfected with the Ca2 + -binding-deficient CaM
mutant displayed a lower termination threshold than control
cells. The termination of Ca2 + release in control HEK-293 cells
is likely to be modulated by endogenously expressed CaM. It is
therefore possible that overexpression of WT CaM would enhance
the action of the endogenous CaM, leading to an increased
termination threshold, whereas overexpression of the Ca2 + binding-deficient CaM mutant would suppress the action of the
endogenous CaM, resulting in a reduced termination threshold.
Hence, these data indicate that Ca2 + binding to CaM is required
for the action of CaM in facilitating Ca2 + -release termination,
similar to the action of CaM in suppressing Ca2 + activation of
RyR2 reported previously [22,33]. Interestingly, we also found
that disabling Ca2 + binding to the N-lobe of CaM did not eliminate
its effect on the termination of Ca2 + release, whereas disabling
Ca2 + binding to the C-lobe of CaM was sufficient to abolish the
action of CaM in Ca2 + -release termination. Thus CaM-facilitated
termination of Ca2 + release requires Ca2 + binding to the C-lobe
of CaM.
Potential mechanisms of Ca2 + -release termination
Figure 6
SOICR
The RyR2 F3603A mutation markedly reduces the propensity for
Stable inducible HEK-293 cell lines expressing RyR2 WT or the RyR2 F3603A mutant were
loaded with 5 μM fura 2/AM in KRH buffer. The cells were then perfused continuously with KRH
buffer containing increasing levels of extracellular Ca2 + (0–2 mM) to induce SOICR. Fura-2
ratios of representative RyR2 WT (A) and F3603A (B) cells were recorded using epifluorescence
single-cell Ca2 + imaging. (C) The percentages of RyR2 WT (599 cells) and F3603A (464 cells)
cells that display Ca2 + oscillations at various extracellular Ca2 + concentrations. Data shown
are means +
− S.E.M. (n = 3) (*P < 0.05 compared with WT).
To address this question, we directly monitored the ER luminal
Ca2 + dynamics in RyR2-expressing HEK-293 cells transfected
with CaM WT or mutants. We demonstrate, for the first time,
that CaM increases the termination threshold and thus facilitates
the termination of Ca2 + release in a manner dependent on Ca2 +
binding to CaM. We also show that mutations in the CaM-binding
domain of RyR2 that either eliminate or retain the interaction
between CaM and RyR2, all affect the termination threshold.
These data indicate that the CaM-binding domain of RyR2 is an
important determinant of Ca2 + -release termination.
Ca2 + binding to the C-lobe of CaM is required for CaM-dependent
facilitation of Ca2 + -release termination
It has been demonstrated that CaM inhibits Ca2 + activation
of single RyR2 channels and [3 H]ryanodine binding to RyR2
[20,22]. Although both apoCaM and Ca2 + –CaM are able to bind
to RyR2, the inhibitory effect of CaM on RyR2 requires Ca2 +
binding to CaM, as a Ca2 + -binding-deficient CaM mutant, in
which all four EF-hand Ca2 + -binding sites were disabled, was
still able to interact with RyR2, but had no effect on RyR2
channel activity [22]. This indicates that it is the Ca2 + -bound
form of CaM that exerts the inhibitory effect on RyR2. In
Although it is clear that CaM facilitates Ca2 + -release termination,
the exact molecular mechanism by which CaM modulates the
termination threshold for Ca2 + release is currently unknown.
During Ca2 + release, the cytosolic Ca2 + level is elevated, whereas
SR luminal Ca2 + is depleted. Elevated cytosolic Ca2 + would
activate CaM by forming the Ca2 + –CaM complex, which has
been shown to inhibit the Ca2 + -dependent activation of single
RyR2 channels [22]. It is then possible that this Ca2 + -bound
CaM would facilitate the termination of Ca2 + release by inhibiting
RyR2. Consistent with this possibility, we found that the effect of
CaM on Ca2 + -release termination requires Ca2 + binding to the
C-lobe of CaM. Thus CaM may act as a cytosolic Ca2 + sensor
mediating, in part, the cytosolic Ca2 + -dependent inactivation
of RyR2 and thus the termination of Ca2 + release. It is also
possible that elevated cytosolic Ca2 + may exert a direct effect on
RyR2 gating and thus Ca2 + release via its low-affinity inhibitory
Ca2 + -binding sites [34]. In addition, SR luminal Ca2 + depletion
during Ca2 + release is believed to play an important role in
Ca2 + -release termination [5,7–13]. However, how SR luminal
Ca2 + modulates RyR2 gating and Ca2 + release is not completely
understood. It has been proposed that reduced SR luminal Ca2 +
deactivates RyR2 via the SR Ca2 + -binding protein calsequestrin
(CASQ2), leading to Ca2 + -release termination [15]. However,
it is important to note that Ca2 + -release termination is still
present in CASQ2-deficient cardiomyocytes [35], indicating that
CASQ2-mediated termination of Ca2 + release is not the only
mechanism of Ca2 + -release termination. It is also important to
know that HEK-293 cells lack muscle-specific proteins such as
CASQ2, triadin and junctin. Hence it will be of interest and
importance to determine whether these proteins modulate the
threshold for Ca2 + -release termination. It is also possible that
reduced SR luminal Ca2 + could desensitize RyR2 via its luminal
Ca2 + -regulatory sites [34]. Alternatively, long-range allosteric
conformational changes in RyR2 induced by the reduced SR
luminal Ca2 + content as a consequence of Ca2 + release may
contribute to the CaM-dependent facilitation of release termination in addition to elevated cytosolic Ca2 + . Therefore it is likely
c The Authors Journal compilation c 2013 Biochemical Society
374
X. Tian and others
that multiple mechanisms are involved in the termination of Ca2 +
release. Further detailed and systematic studies will be required
to fully understand the mechanism of Ca2 + -release termination.
Role of the CaM-binding domain of RyR2 in Ca2 + -release
activation and termination
The CaM-binding site in RyR2 has been localized to residues
3583–3603. Deletion of this sequence or a triple mutation
(W3587A/L3591D/F3603A) within this region abolishes both
CaM binding to RyR2 and the inhibitory effect of CaM on single
RyR2 channels [21]. Interestingly, single mutations, W3587A,
L3591D and F3603A, partially affect CaM binding, but eliminate
the inhibitory action of CaM. It was suggested that the CaMbinding domain plays a role not only in binding with CaM, but also
in transducing the binding of CaM to a functional effect on RyR2
via interactions with other domains of RyR2 [21]. Consistent
with this view, we found that deletion of the CaM-binding site
and the triple and single mutations, regardless of whether they
eliminate CaM binding or not, all abolished the action of CaM
in facilitating Ca2 + -release termination. Furthermore, we showed
that the point mutation F3603A also affected the activation of
spontaneous Ca2 + release by increasing the activation threshold,
thereby reducing the propensity for SOICR, in addition to its
impact on the termination threshold. Therefore the CaM-binding
domain of RyR2 is an important determinant of both the activation
and termination of Ca2 + release, and the function of this domain
is likely to be modulated by CaM in a Ca2 + -dependent manner.
Role of CaM–RyR2 interactions in the pathogenesis of
cardiomyopathies and CPVT (catecholaminergic polymorphic
ventricular tachycardia)
Naturally occurring RyR2 mutations have been linked not only
to cardiac arrhythmias and sudden death, but also to cardiomyopathies [36]. We have previously shown that RyR2 mutations
linked to CPVT reduce the activation threshold for SOICR [27–
29,37]. Recently, we have demonstrated that RyR2 mutations
associated with both cardiac arrhythmias and cardiomyopathies reduce both the activation and termination thresholds
for SOICR, and suggested that abnormal termination of Ca2 +
release can lead to cardiomyopathies [16]. Considering the
finding that CaM modulates the termination threshold for Ca2 +
release, an altered CaM–RyR2 interaction may be involved
in cardiomyopathies. Consistent with this view, knock-in
mice harbouring a triple mutation (W3587A/L3591D/F3603A)
in RyR2 that abolishes CaM binding and the action of CaM in
Ca2 + -release termination developed severe cardiomyopathy [23].
A relatively modest cardiomyopathy was also observed in knockin mice expressing the RyR2 L3591D mutation that affects Ca2 + release termination [38].
An altered CaM–RyR2 interaction may also be involved
in the pathogenesis of CPVT. A CPVT-associated RyR2
mutation, R2474S, has been shown to affect interdomain
interactions, which in turn alter CaM–RyR2 interactions upon
protein kinase A-dependent phosphorylation of RyR2, leading
to enhanced spontaneous Ca2 + release [39]. A defective
CaM–RyR2 interaction may result from mutations in CaM.
Recently, genome-wide linkage analysis and DNA sequencing
have linked the calmodulin gene (CAML1) to a dominantly
inherited form of CPVT-like cardiac arrhythmia, and identified
several disease-associated CaM mutations [40]. It is unknown,
however, how these CaM mutations cause CPVT-like arrhythmias.
Further investigations will be required to assess whether
c The Authors Journal compilation c 2013 Biochemical Society
these disease-associated CaM mutations affect the termination
and/or activation threshold for RyR2-mediated Ca2 + release.
A potential explanation, on the basis of the results of the
present study, is that disease-linked CaM mutations may reduce
the termination threshold for RyR2-mediated Ca2 + release. A
reduced termination threshold would increase the fractional Ca2 +
release and thus the amplitude of spontaneous Ca2 + release
under conditions of SR Ca2 + overload, which would in turn
enhance the propensity for spontaneous Ca2 + -release-induced
DADs (delayed afterdepolarizations) and triggered activities.
Therefore an abnormal CaM–RyR2 interaction may be, in part,
attributable to the pathogenesis of cardiomyopathies and CPVT.
In summary, the present study demonstrates for the first time
that CaM modulates the termination threshold for RyR2-mediated
Ca2 + release, and that the CaM-binding domain of RyR2 is an
important determinant of Ca2 + -release termination and activation.
A defective CaM–RyR2 interaction may lead to cardiomyopathies
and cardiac arrhythmias.
AUTHOR CONTRIBUTION
Xixi Tian, Yijun Tang and S.R. Wayne Chen designed the research. Xixi Tian, Yijun Tang,
Yingjie Liu and Ruiwu Wang performed the research; Xixi Tian, Yijun Tang and Yingjie Liu
analysed the data. Xixi Tian and S.R. Wayne Chen wrote the paper.
FUNDING
This work was supported by the Canadian Institutes of Health Research and the Heart
and Stroke Foundation of Alberta, NWT and Nunavut (to S.R.W.C.) X.T. is a recipient of
the Alberta Innovates-Health Solutions (AIHS) Studentship Award. S.R.W.C. is an AIHS
Scientist.
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Received 17 June 2013/8 August 2013; accepted 30 August 2013
Published as BJ Immediate Publication 30 August 2013, doi:10.1042/BJ20130805
c The Authors Journal compilation c 2013 Biochemical Society