Differential Ca2+ sensitivity of skeletal and
cardiac muscle ryanodine receptors in the presence of calmodulin
Bradley R.
Fruen1,
Jennifer M.
Bardy1,
Todd M.
Byrem2,
Gale M.
Strasburg2, and
Charles F.
Louis1
1 Department of Biochemistry, Molecular Biology, and
Biophysics, University of Minnesota, Minneapolis, Minnesota 55455; and
2 Department of Food Science and Human Nutrition, Michigan
State University, East Lansing, Michigan 48824
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ABSTRACT |
Calmodulin (CaM) activates the skeletal muscle ryanodine receptor
Ca2+ release channel (RyR1) in the presence of nanomolar
Ca2+ concentrations. However, the role of CaM activation in
the mechanisms that control Ca2+ release from the
sarcoplasmic reticulum (SR) in skeletal muscle and in the heart remains
unclear. In media that contained 100 nM Ca2+, the rate of
45Ca2+ release from porcine skeletal muscle SR
vesicles was increased approximately threefold in the presence of CaM
(1 µM). In contrast, cardiac SR vesicle
45Ca2+ release was unaffected by CaM,
suggesting that CaM activated the skeletal RyR1 but not the cardiac
RyR2 channel isoform. The activation of RyR1 by CaM was associated with
an approximately sixfold increase in the Ca2+ sensitivity
of [3H]ryanodine binding to skeletal muscle SR, whereas
the Ca2+ sensitivity of cardiac SR
[3H]ryanodine binding was similar in the absence and
presence of CaM. Cross-linking experiments identified both RyR1 and
RyR2 as predominant CaM binding proteins in skeletal and cardiac SR,
respectively, and [35S]CaM binding determinations further
indicated comparable CaM binding to the two isoforms in the presence of
micromolar Ca2+. In nanomolar Ca2+, however,
the affinity and stoichiometry of RyR2 [35S]CaM binding
was reduced compared with that of RyR1. Together, our results indicate
that CaM activates RyR1 by increasing the Ca2+ sensitivity
of the channel, and further suggest differences in CaM's functional
interactions with the RyR1 and RyR2 isoforms that may potentially
contribute to differences in the Ca2+ dependence of channel
activation in skeletal and cardiac muscle.
sarcoplasmic reticulum; calcium release channel; excitation-contraction coupling
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INTRODUCTION |
IN BOTH SKELETAL AND
CARDIAC muscle, the increase in myoplasmic Ca2+ that
triggers contraction reflects the activation of ryanodine receptor
(RyR) channels in the sarcoplasmic reticulum (SR). The RyR isoforms
expressed in mammalian skeletal muscle (RyR1) and cardiac muscle (RyR2)
share 66% homology at the amino acid level and exhibit similar
responses to a number of physiological and pharmacological channel
effectors (33). Ca2+ itself is a principal
endogenous effector of RyR channels, and activating as well as
inhibitory Ca2+ binding sites within the primary sequence
of these channel proteins are suggested by studies describing the
Ca2+ dependence of channel activity in isolated
preparations (20). In situ, however, important actions of
Ca2+ may be dependent on Ca2+ binding not only
to RyR channels but also to RyR-associated proteins (19).
In this regard, calmodulin (CaM), the ubiquitous intracellular Ca2+ sensor, is now recognized as an integral component of
the intact RyR1 channel complex (35, 36).
CaM binds to RyR1 channels both in the absence and in the presence of
micromolar Ca2+ (24, 34, 37). In the presence
of micromolar Ca2+, CaM binding is associated with RyR1
inhibition, whereas at nanomolar Ca2+ concentrations, CaM
binding is associated with RyR1 activation (11, 12, 34).
Factors that may regulate CaM's interactions with RyR1 channels remain
largely undefined, however, and consequently, the magnitude of CaM's
effects on RyR1 activity has varied markedly in studies that utilize
different experimental conditions or preparations (8, 11, 26,
38). In addition, it remains unclear how the CaM-dependent
regulation of RyR1 channels may relate to channel regulation by
Ca2+ (i.e., Ca2+-induced Ca2+
release, or CICR) or by allosteric interactions with transverse tubule
voltage sensors (i.e., mechanical coupling). Importantly, detailed
studies of CaM's functional interactions with RyR channels have to
date focused on the RyR1 isoform, and in particular, no study has yet
determined whether, in the presence of nanomolar Ca2+, CaM
may also bind to and activate cardiac RyR2 channels. Thus the role of
CaM in the physiological mechanisms that initiate SR Ca2+
release in skeletal and cardiac muscle remains unclear.
In the present study, we examined the relationship between CaM- and
Ca2+-dependent mechanisms of RyR channel activation with
the use of SR vesicles prepared from porcine skeletal and cardiac
muscle. Our results indicate that CaM activation of RyR1 channels may reflect an increased sensitivity of these channels to activation by
Ca2+. In addition, our results demonstrate differences in
CaM's functional interactions with the RyR1 and RyR2 channel isoforms
that may potentially contribute to differences in the Ca2+
dependence of channel activation that characterize skeletal and cardiac
muscle excitation-contraction (E-C) coupling.
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EXPERIMENTAL PROCEDURES |
Materials.
Pigs were obtained from the University of Minnesota Experimental Farm.
45Ca2+ and [3H]ryanodine were
purchased from NEN Life Science Products (Boston, MA). Unlabeled
ryanodine and suramin were from Calbiochem (La Jolla, CA).
,[
]-Methyleneadenosine 5'-triphosphate [AMP-PCP (a
nonhydrolyzable ATP analog)] and porcine brain CaM were from Sigma
(St. Louis, MO).
Isolation of SR vesicles.
Skeletal muscle SR vesicles were isolated from pig longissimus dorsi
muscle as described (7). Briefly, vesicles obtained by
differential ultracentrifugation of a muscle homogenate were extracted
with 0.6 M KCl and subsequently fractionated on discontinuous sucrose
gradients. The terminal cisternae-derived (i.e., "heavy") SR
vesicles that band at the 36-40% interface were collected and stored frozen at
70°C. Cardiac muscle SR vesicles were isolated from porcine ventricular tissue (6). After homogenization
in 10 mM NaHCO3, membranes were extracted in 0.6 M KCl
and 20 mM Tris (pH 6.8) and then resuspended in 10% sucrose and stored
frozen at
70°C. All isolation buffers contained a mixture of
protease inhibitors.
45Ca2+ release.
SR vesicle 45Ca2+ release was assayed
essentially as described (21). Vesicles passively loaded
with 5 mM 45Ca2+ (± 1 µM CaM) were placed on
the side of a polystyrene tube that contained 120 mM potassium
propionate, 10 mM K-PIPES (pH 7.0), 8.6 mM EGTA, 3 mM
Na2AMP-PCP, 3 mM MgCl2 (free Mg2+
is ~0.4 mM), 2 mM Ca2+ acetate (free Ca2+ is
~100 nM), and ± 1 µM CaM. Ca2+ release was
initiated by rapid mixing and stopped at the indicated times by rapid
dilution into a release-inhibiting medium (120 mM potassium propionate,
10 mM K-PIPES (pH 7.0), 10 mM EGTA, 5 mM MgCl2, and 20 µM
ruthenium red) and then immediately collected on 0.45-µm Millipore
filters. The fraction of total loaded 45Ca2+
that was not released after 10-s incubations in a release medium that
promotes maximal RyR activation (450 mM KCl, 10 mM K-PIPES (pH 7.0), 10 mM Na2ATP, and 10 µM Ca2+) was considered
background and was subtracted from all determinations (<12% total
counts per minute for both skeletal and cardiac SR). Sample means were
compared with Student's t-test and were considered significantly different at P < 0.05. Estimates of the
time required for vesicles to release one-half of their
45Ca2+ contents (t1/2)
were based on fits to the equation R = Rmax×t/(t1/2 + t), where R is Ca2+ released,
Rmax is maximal Ca2+ release, and
t is time.
[3H]ryanodine binding.
SR vesicles were incubated for 90 min at 36°C in media that contained
120 mM potassium propionate, 10 mM K-PIPES (pH 7.0), 3 mM
Na2AMP-PCP, 100 nM [3H]ryanodine, and a
Ca2+ acetate-EGTA buffer set to give the desired
Ca2+ concentration. In experiments described in
RESULTS (see Fig. 2, C and D, and
Fig. 4), media also contained MgCl2 at the concentrations indicated in the figure legends. Free Ca2+ and
Mg2+ concentrations were calculated with the use of the
computer program Bound and Determined (S. P. J. Brookes,
Carleton University, Ottawa, Canada). Equilibrium
[3H]ryanodine binding was determined after collection of
SR vesicles on Whatman glass fiber filters. Nonspecific binding was
measured in the presence of 20 µM nonradioactive ryanodine. Data are
expressed as percentages of the maximal [3H]ryanodine
binding capacity of the SR vesicle preparation, as determined in media
containing 450 mM KCl, 10 mM Na2ATP, and 100 µM
Ca2+ (12.9 ± 1.3 pmol/mg SR protein for skeletal
muscle, n = 5; 3.7 ± 1.1 pmol/mg SR protein for
cardiac muscle, n = 4). Determinations of
half-maximally activating concentrations (Ka) of
CaM or Ca2+ were based on fits to the Hill equation
(SigmaPlot software; Chicago, IL).
[125I]CaM cross-linking.
Mammalian CaM Gln-143-Cys was site-specifically derivatized at Cys-143
with the photoactivatable cross-linking agent benzephenone-4-maleimide (32) followed by Bolton-Hunter iodination
(38). Iodinated, derivatized CaM (125I-Bz-CaM)
was dialyzed and concentrated in 1 mM HEPES (pH 7.4) with Centricon-10
concentrators (Millipore, Bedford, MA). SR vesicles (1 mg/ml) and
125I-Bz-CaM (50 nM) were preincubated in the dark for 30 min in ice-cold buffer containing 150 mM NaCl, 50 mM HEPES (pH 7.4),
and either 1 mM EGTA (free Ca2+
10 nM) or 100 µM
CaCl2 and then illuminated for 20 min on ice in an
ultraviolet cross-linker (
max = 365 nm; Hoefer, San
Francisco, CA). After electrophoresis of pelleted SR vesicles,
cross-linked proteins were identified on dried, Coomassie-stained gels
by storage phosphorimaging (Molecular Imager FX; BioRad, Hercules, CA),
and bands corresponding to the RyRs were quantified by densitometric analysis.
[35S]CaM binding.
Mammalian CaM was metabolically radiolabeled according to the
procedures of Moore et al. (24). Briefly,
[35S]methionine was incorporated into CaM by bacterial
expression with the use of mCaM cDNA subcloned into the pET-28a
expression vector (Novagen, Madison, WI). Expressed
[35S]CaM was first purified by nickel affinity
chromatography, and then followed by phenyl sepharose chromatography.
The concentration of CaM was determined by spectroscopy with the use of
an extinction coefficient of 0.20 ml · mg
1 · cm
1 in the
presence of EGTA. SR vesicle [35S]CaM binding was
determined as described (24) in media that contained 300 mM NaCl, 50 mM MOPS (pH 7.4), 100 µg/ml BSA, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 1 mM EGTA,
and 1.2 mM CaCl2 (for high-Ca2+ media, as
indicated). After a 2-h incubation period, SR vesicles were collected
on Whatman GF/B filters and washed 5× with 3 ml of ice-cold binding
buffer. Nonspecific binding was determined in the presence of 5 µM
unlabeled CaM.
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RESULTS |
CaM regulation of 45Ca2+ release from SR
vesicles.
The effect of CaM on Ca2+ release from SR vesicles
passively loaded with 45Ca2+ was examined at
36°C in media approximating ionic conditions present in the myoplasm
of relaxed muscle. These media contained 120 mM potassium propionate,
10 mM K-PIPES (pH 7.0), 3 mM Na2AMP-PCP, and 3 mM
MgCl2 (free Mg2+ is ~0.4 mM). In these
initial experiments, ionized Ca2+ in the release media was
buffered to 100 nM with EGTA.
Figure 1A shows that CaM
significantly increased 45Ca2+ release
from porcine skeletal muscle SR vesicles in these media
(t1/2 for 45Ca2+ release
is ~30% of control in the presence of 1 µM CaM, Table 1). In contrast to CaM's effect on
45Ca2+ release from skeletal muscle SR,
45Ca2+ release from cardiac SR vesicles was
similar in the presence and absence of CaM (Fig. 1B).
Caffeine, however, significantly increased
45Ca2+ release from both cardiac and skeletal
muscle SR (Fig. 1, A and B). These results thus
indicate that CaM selectively activated 45Ca2+
release from skeletal muscle, but not cardiac, SR vesicles in media
that contained 100 nM Ca2+.

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Fig. 1.
Calmodulin (CaM) activation of sarcoplasmic reticulum (SR) vesicle
Ca2+ release in the presence of 100 nM Ca2+
(A and B) and 500 µM Ca2+
(C and D). SR vesicle
45Ca2+ release was determined as described in
EXPERIMENTAL PROCEDURES in media that contained 120 mM
potassium propionate, 10 mM PIPES (pH 7.0), 3 mM
Na2AMP-PCP, and 3 mM MgCl2. CaM (1 µM) or
caffeine (10 mM) were added to the media as indicated. Maximal
ryanodine receptor (RyR)-mediated 45Ca2+
release [determined in media that contained 450 mM KCl, 10 mM K-PIPES
(pH 7.0), 10 mM Na2ATP, and 10 µM Ca2+] was
33 ± 3.6 nmol/mg for skeletal SR and 20 ± 4.8 nmol/mg for
cardiac SR. Data are means ± SE from 3-4 independent
experiments (performed in duplicate and using different SR vesicle
preparations). * Significantly different from release in control media
(P < 0.05).
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For comparison, subsequent experiments examined SR
45Ca2+ release in media that contained 500 µM
Ca2+ (Fig. 1, C and D). In the
presence of 500 µM Ca2+, the rate of SR
45Ca2+ release was increased such that in the
control media, vesicles released more than one-half of their
45Ca2+ stores within 1 s. Nonetheless,
when CaM was included in the media, an approximate twofold increase in
t1/2 for 45Ca2+ release
was apparent for both skeletal and cardiac SR (Table 1). These results
are thus consistent with earlier reports that demonstrate similar
inhibitory effects of CaM on SR Ca2+ release from skeletal
(21) and cardiac (23) SR vesicles in media
containing micromolar Ca2+.
CaM effects on Ca2+ dependence of SR vesicle
[3H]ryanodine binding.
SR vesicle [3H]ryanodine binding was used to further
investigate the selective activation by CaM of the skeletal muscle RyR1 compared with the cardiac RyR2 isoform, and to further examine the
relationship between CaM- and Ca2+-dependent mechanisms of
RyR activation. Because ryanodine binds with high affinity to the open
state of RyR channels, changes in [3H]ryanodine binding
that occur in the presence of RyR effectors provide a useful index of
changes in RyR channel activity (3, 22). CaM's effects on
Ca2+ activation of [3H]ryanodine binding were
initially examined in media containing 3 mM Na2AMP-PCP and
3mM MgCl2 (i.e., as in Fig. 1). In these
Mg2+-containing media, the threshold for Ca2+
activation of [3H]ryanodine binding to both skeletal
muscle and cardiac SR was ~0.1 µM Ca2+, although the
maximal extent of activation by micromolar Ca2+ was
significantly less for skeletal than for cardiac muscle SR (24% vs.
82% activation, respectively; Fig. 2,
A and B). The addition of 1 µM CaM reduced the
Ka for Ca2+ activation of skeletal
SR [3H]ryanodine binding to approximately one-fourth of
control and decreased the cooperativity of Ca2+ activation
(Table 2). In contrast, the
Ca2+ dependence of cardiac SR [3H]ryanodine
binding was unaffected by CaM (P = 0.5).

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Fig. 2.
Effect of CaM on the Ca2+ dependence of SR vesicle
[3H]ryanodine binding. [3H]ryanodine
binding to normal skeletal muscle SR or cardiac SR was determined in
the absence ( ) or presence ( ) of 1 µM CaM.
Media contained 120 mM potassium propionate, 10 mM PIPES (pH 7.0), and
3 mM Na2AMP-PCP, either with (A and
B) or without (C and D) 3 mM
MgCl2. Data are expressed as percentages of the maximal
[3H]ryanodine binding capacity of the SR vesicle
preparations (12.0 ± 2.3 for skeletal SR; 3.9 ± 0.8 for
cardiac SR). Solid lines are based on fits to the Hill equation. Data
are means ± SE of 3-7 independent experiments (at least 3 different SR vesicle preparations).
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Table 2.
Effect of CaM on the Ca2+ dependence of
[3H]ryanodine binding to skeletal and cardiac muscle
SR vesicles
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CaM's effect on the Ca2+ dependence of
[3H]ryanodine binding was also examined in media
from which MgCl2 was omitted to optimize RyR activation by
Ca2+ and obviate differential effects of Mg2+
on the RyR1 and RyR2 channel isoforms (17). The omission
of Mg2+ increased the extent of Ca2+-activated
[3H]ryanodine binding to skeletal muscle SR but had
little effect on the threshold of activation (~100 nM
Ca2+ in control media; Fig. 2, C and
D). Consequently, in the absence of added CaM, the RyR1 and
RyR2 channel isoforms now displayed a similar Ca2+
dependence of activation (Ka for
Ca2+ is ~360 nM for both skeletal and cardiac muscle SR;
Table 2). This result is thus consistent with the similar affinities of Ca2+ activation sites on RyR1 and RyR2 channels (e.g.,
Refs. 6, 10, 17). In the presence of CaM, however, the Ca2+
dependence of [3H]ryanodine binding to skeletal and
cardiac muscle SR differed markedly (Fig. 2, C and
D). CaM reduced the threshold for Ca2+
activation of skeletal muscle SR [3H]ryanodine binding
~10-fold, decreasing the Ka for
Ca2+ from 360 nM to 60 nM. The Ca2+ sensitivity
of cardiac SR [3H]ryanodine binding, in contrast, was
again not significantly affected by CaM (Table 2; P = 0.2). These data therefore demonstrate that CaM activation was
associated with a pronounced shift in the Ca2+ dependence
of skeletal muscle SR [3H]ryanodine binding to lower
Ca2+ concentrations. Furthermore, in the presence of CaM,
the skeletal RyR1 and cardiac RyR2 isoforms displayed a marked
difference in their Ca2+ sensitivities that was not
apparent in the CaM-free media.
Modulation of CaM activation by CICR effectors.
To further investigate the relationship between CaM- and
Ca2+-dependent mechanisms of RyR1 activation, we examined
the modulation of CaM activation by effectors of CICR. In a previous
report, we documented that the extent of Ca2+ activation of
RyR1 is strictly dependent on the presence of adenine nucleotide when
media are composed primarily of organic anions (6).
Similarly, in a potassium propionate/PIPES medium containing 100 nM
Ca2+, the extent of CaM activation of RyR1 was also
dependent on adenine nucleotide. Thus in the absence of nucleotide, CaM
activated skeletal muscle SR [3H]ryanodine binding to
<5% of maximal activation, whereas including 3 mM
Na2AMP-PCP in the medium increased the CaM-dependent
activation by ~20-fold (Fig.
3A). In the presence of
Na2AMP-PCP, the CaM-dependent activation of skeletal muscle
SR [3H]ryanodine binding was monophasic and suggested
that CaM may act at a discrete high-affinity site on the RyR1. Caffeine
(5 mM) also increased the extent of CaM-activated
[3H]ryanodine binding to skeletal muscle SR (Fig.
3A). Determinations of apparent affinities and Hill
coefficients for CaM activation of [3H]ryanodine
binding suggested that AMP-PCP (Ka = 28 ± 3.1 nM; nH = 1.5 ± 0.2)
and caffeine (Ka = 38 ± 3.3 nM;
nH = 1.1 ± 0.1) increased CaM
activation of RyR1 independent of direct effects on CaM binding to
activation sites on the channel protein.

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Fig. 3.
Effect of Ca2+-induced Ca2+
release (CICR) activators on CaM-dependent activation of skeletal
muscle (A) and cardiac (B) SR
[3H]ryanodine binding in the presence of 100 nM
Ca2+. SR vesicle [3H]ryanodine binding was
determined as described in EXPERIMENTAL PROCEDURES. Control
media contained 120 mM potassium propionate, 10 mM PIPES (pH 7.0), and
100 nM Ca2+. Media were supplemented with either
Na2AMP-PCP (3 mM) or caffeine (5 mM in A, 10 mM
in B), as indicated. Data are means ± SE of 3-4
independent experiments.
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Subsequent experiments examined the possibility that AMP-PCP or
caffeine might also promote CaM activation of cardiac RyR2 channels in
100 nM Ca2+. In media containing either AMP-PCP (3 mM) or
caffeine (10 mM), CaM significantly increased cardiac SR vesicle
[3H]ryanodine binding (Fig. 3B;
P < 0.05 in the presence of 300 nM and 1 µM CaM).
However, the magnitude of the CaM-dependent activation of cardiac SR
[3H]ryanodine binding in these media was small compared
with the CaM-dependent activation of [3H]ryanodine
binding to skeletal muscle SR. Furthermore, compared with skeletal
muscle SR (Fig. 3A), the CaM-dependent activation of cardiac
SR [3H]ryanodine binding required higher concentrations
of CaM and did not plateau over the range of CaM concentrations
examined. Thus, in contrast to RyR1, CaM activation of RyR2 suggested a lower affinity or nonspecific interaction of CaM with the cardiac channel isoform.
The effect of CICR inhibitors on the CaM-dependent activation of RyR1
channels was examined in media containing 100 nM Ca2+ and 3 mM Na2AMP-PCP (Fig. 4). CaM
activation of skeletal SR [3H]ryanodine binding displayed
a marked sensitivity to inhibition by physiological concentrations of
Mg2+. The addition of 2.5 mM MgCl2 (free
Mg2+ is ~0.3 mM) reduced the CaM-dependent activation to
~25% of control, and 10 mM MgCl2 (free Mg2+
is ~3 mM) completely blocked the activation of
[3H]ryanodine binding by CaM. CaM activation of
[3H]ryanodine binding was similarly inhibited when
Ca2+ was buffered to lower concentrations with
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
(BAPTA; Fig. 4, free Ca2+ is ~3 nM). Both
Mg2+ and BAPTA reduced the maximal extent of CaM-activated
[3H]ryanodine binding without affecting the
Ka for CaM (24 ± 5.8 nM in 2.5 mM
MgCl2; 25 ± 7.8 nM in 1 mM BAPTA), consistent with noncompetitive inhibition of CaM activation. These results thus indicate that CaM activation of RyR1 was inhibited when activation of
the channel by Ca2+ was blocked and thereby further suggest
that CaM activation may reflect an increase in the sensitivity of the
RyR1 to Ca2+, rather than a Ca2+-independent
channel activation by CaM.

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Fig. 4.
Effect of CICR inhibitors on the CaM-dependent
stimulation of [3H]ryanodine binding to normal skeletal
muscle SR vesicles. [3H]ryanodine binding to SR vesicles
was determined as described in EXPERIMENTAL PROCEDURES in
media that contained 120 mM potassium propionate, 10 mM PIPES (pH 7.0),
100 nM Ca2+, and 3 mM Na2AMP-PCP (to optimize
the CaM-dependent stimulation of [3H]ryanodine binding).
MgCl2 or
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
(BAPTA) were included as indicated. Calculated ionized Mg2+
concentrations after addition of 2.5 mM and 10 mM MgCl2
were ~0.3 mM and ~3 mM, respectively. Calculated ionized
Ca2+ concentration in media containing 1 mM BAPTA was ~3
nM. Data are means ± SE of 3 or 4 independent experiments (at
least 3 different SR vesicle preparations, duplicate determinations).
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Competitive inhibition of CaM activation by suramin.
In a recent report, Klinger and colleagues (13)
demonstrated that the purinergic antagonist suramin reduced RyR1-CaM
binding and blocked RyR1 inhibition by Ca2+-CaM. Their
results thus indicated that suramin might directly compete with
Ca2+-CaM binding to its inhibitory site on the RyR1 channel
protein. Their study did not, however, determine whether suramin might also alter CaM's interactions with activation sites on the RyR1 in the
presence of submicromolar Ca2+. Accordingly, we examined
the effect of suramin on the CaM-dependent activation of skeletal
muscle SR [3H]ryanodine binding (Fig.
5). In the presence of 10 µM suramin, CaM-dependent activation of skeletal muscle SR
[3H]ryanodine binding was shifted ~10-fold to higher
CaM concentrations (Ka > 300 nM CaM).
Thus, in contrast to other RyR effectors (Figs. 3 and 4), these data
indicated that suramin may modulate CaM activation via a direct
competition with CaM binding site(s) on the RyR1. The increase of
suramin to 30 µM, however, not only fully blocked CaM-dependent
activation of [3H]ryanodine binding but also activated
[3H]ryanodine binding twofold, suggesting that at higher
concentrations, suramin may have multiple effects on RyR1 function.
Previously, activation of RyR1 and RyR2 channels by high concentrations
of suramin was associated with effects on both single channel
conductance and open probability of these channel proteins
(29).

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Fig. 5.
Competitive inhibition by suramin of CaM-activated SR
vesicle [3H]ryanodine binding.
[3H]ryanodine binding to skeletal muscle SR vesicles was
determined as in the absence and presence of suramin in media that
contained 120 mM potassium propionate, 10 mM PIPES (pH 7.0), and 3 mM
Na2AMP-PCP. Data are means ± SE of 2-4
independent experiments (triplicate determinations).
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Radiolabeled CaM binding to skeletal and cardiac muscle SR.
To investigate potential differences in CaM's physical interactions
with the skeletal RyR1 and cardiac RyR2 isoforms, initial experiments
utilized photoactivatable, iodinated CaM, site-specifically derivatized
at Cys-143 with benzophenone-4-maleimide (32). SR vesicles
were preincubated in media that contained 50 nM 125I-Bz-CaM
and either ~10 nM Ca2+ or 100 µM Ca2+ and
then irradiated with UV light to initiate cross-linking. The
autoradiogram in Fig. 6A
identifies SR proteins cross-linked with 125I-Bz-CaM as
resolved by SDS-PAGE. The Coomassie stain of this gel (Fig.
6B) confirms that approximately equivalent amounts of ~565
kDa proteins, corresponding to the skeletal RyR1 and cardiac RyR2
isoforms, were present in the different lanes. For both skeletal and
cardiac SR preparations, these ~565 kDa RyR proteins were the
predominant 125I-Bz-CaM cross-linked species, and for both
skeletal and cardiac SR, cross-linking was apparent whether media
contained nanomolar or micromolar Ca2+. Nevertheless, these
results suggested potential differences in the Ca2+
dependence of 125I-Bz-CaM cross-linking to RyR1 and RyR2.
Thus we found that the cross-linking of 125I-Bz-CaM to RyR1
in nanomolar Ca2+ was consistently increased relative to
that in micromolar Ca2+ (Fig. 6A). Conversely,
the cross-linking of 125I-Bz-CaM to RyR2 in nanomolar
Ca2+ was reduced relative to that in micromolar
Ca2+ (Fig. 6A).

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Fig. 6.
Cross-linking of a photoactivatable derivative of
[125I]CaM to skeletal and cardiac muscle SR vesicles. SR
vesicles from 2 different skeletal SR preparations (left 4 lanes) and 2 different cardiac SR preparations (right 4 lanes) were covalently labeled with 125I-Bz-CaM (50 nM) as
described in EXPERIMENTAL PROCEDURES. Media contained
either ~10 nM Ca2+ or 100 µM Ca2+, as
indicated. A: autoradiogram of
[125I]CaM-labeled SR proteins separated on 5-12%
linear gradient PAGE. Arbitrary optical density units of bands that
correspond to the RyRs were as follows: lane 1, 2,132;
lane 2, 1,074; lane 3, 3,396; lane 4,
1,587; lane 5, 2,600; lane 6, 5,460; lane
7, 3,745; and lane 8, 7,561. B: the
Coomassie blue-stained gel is shown to document that the different
lanes in A contained comparable amounts of RyR protein.
Arrows indicate locations of the two RyR isoforms, free
125I-Bz-CaM, and the positions of molecular weight markers.
Experiment shown is representative of 5 independent experiments.
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It is possible that Ca2+-dependent conformational changes
in the 125I-Bz-CaM molecule influenced the efficiency of
cross-linking independent of actual effects on RyR CaM binding. In
addition, recent evidence has suggested that CaM binding determinations
may be affected by Bolton-Hunter iodination of CaM (24).
Experiments that utilize expressed CaM metabolically labeled with
35S ([35S]CaM), however, have indicated
high-affinity binding to RyR1 in both the presence and absence of
micromolar Ca2+, with a binding stoichiometry of ~1 CaM
per channel subunit (24). We therefore utilized
[35S]CaM to further characterize CaM binding to cardiac
SR. Figure 7 shows cardiac SR
[35S]CaM binding in media in which Ca2+ was
buffered to either ~10 nM or ~200 µM. Scatchard analysis (Fig. 7,
inset) indicates that, in the presence of micromolar Ca2+, [35S]CaM binding to cardiac SR was
consistent with a single population of high-affinity sites
(Kd = 15.8 ± 1.8 nM,
Bmax = 25.3 ± 2.3 pmol/mg). In
comparison, in nanomolar Ca2+, [35S]CaM
binding to cardiac SR was reduced to one-fifth
(Bmax = 4.9 ± 0.9 pmol/mg), and the
apparent affinity of binding was also significantly decreased relative
to that in micromolar Ca2+ (Kd = 83.6 ± 11.4 nM; P < 0.5).

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|
Fig. 7.
[35S]CaM binding to cardiac SR vesicles.
[35S]CaM binding to SR vesicles was determined as
described in EXPERIMENTAL PROCEDURES in media that
contained 300 mM NaCl, 50 mM MOPS (pH 7.4), 100 µg/ml BSA, 0.1%
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and 1 mM
EGTA (free Ca2+ is ~10 nM, ). As
indicated ( ), media were also supplemented with 1.2 mM
CaCl2 (free Ca2+ is ~200 µM). Binding
parameters based on 3 independent SR vesicle preparations were
Bmax = 4.9 ± 0.9 pmol/mg in nanomolar
Ca2+ vs. 25.3 ± 2.5 pmol/mg in micromolar
Ca2+; Kd = 83.6 ± 11 nM
in nanomolar Ca2+ vs. 15.8 ± 1.8 nM in micromolar
Ca2+.
|
|
Table 3 directly compares the binding of
[35S]CaM to cardiac and skeletal SR. Parallel
determinations of [3H]ryanodine receptor density allowed
for estimates of the number of CaM binding sites per RyR tetramer. In
agreement with earlier findings (24), the extent of
[35S]CaM binding to skeletal muscle SR was similar in the
presence of nanomolar and micromolar Ca2+, and binding was
consistent with approximately four [35S]CaM binding sites
per [3H]ryanodine binding site in our skeletal SR
preparations. Likewise, in media that contained micromolar
Ca2+, cardiac SR exhibited approximately five
[35S]CaM binding sites per [3H]ryanodine
binding site (Table 3). In contrast to skeletal SR, however, cardiac SR
[35S]CaM binding was reduced to ~1 mol of
[35S]CaM per [3H]ryanodine binding site in
media containing nanomolar Ca2+. These results thus suggest
that the selective activation by CaM of Ca2+ release from
skeletal compared with cardiac SR at nanomolar Ca2+ (Fig.
1, A and B) may be associated with increased CaM
binding to the RyR1 compared with the RyR2 isoform in nanomolar
Ca2+.
 |
DISCUSSION |
CaM activates RyR1 channels in the presence of nanomolar
Ca2+ concentrations, and this might suggest that the
CaM-dependent activation of RyR1 may operate independently of
Ca2+-dependent channel activation (i.e., CICR). On the
contrary, our results indicate that CaM activation of RyR1 likely
operates by increasing the sensitivity of the
Ca2+-dependent activation mechanism of this channel
protein. Furthermore, the magnitude of CaM's effect at
Ca2+ concentrations present in resting muscle (~100 nM)
is consistent with a major role for CaM in controlling the sensitivity
of CICR in vivo. Finally, our results indicate that CaM's effect on
the Ca2+ sensitivity of channel activation may be far
greater for the RyR1 than for the RyR2 isoform. We therefore suggest
that differential interactions of CaM with RyR1 and RyR2 channels may
potentially contribute to differences in the Ca2+
dependence of SR Ca2+ release in skeletal and cardiac muscle.
RyR1 activation by Ca2+ and CaM are regulated by common
effectors.
In media containing 100 nM Ca2+ and adenine nucleotide, CaM
activation of [3H]ryanodine binding to skeletal muscle SR
vesicles was monophasic (Ka is ~30 nM) and
Hill coefficients for activation indicated only weak cooperativity
(nH is ~1.5). Thus despite early evidence that
CaM may bind to as many as four sites per subunit of the RyR1 tetramer
(9, 34, 37), the observed activation is also consistent
with more recent data suggesting that there is CaM action at a single
high-affinity site within the RyR1 primary sequence (Ref. 24, Table 3).
Moreover, that suramin competitively blocks not only CaM inhibition of
RyR1 at micromolar Ca2+ (Ref. 13, data not shown) but also
CaM activation of RyR1 at nanomolar Ca2+ (Fig. 5) further
supports the possibility that CaM activation and CaM inhibition reflect
action at the same site on the RyR1 channel protein (24,
34).
The extent of CaM activation of RyR1 was dependent on the presence of
adenine nucleotide. In the absence of AMP-PCP, CaM activated normal
skeletal muscle SR vesicle [3H]ryanodine binding to <5%
of maximal, whereas in the presence of 3 mM AMP-PCP, activation of
[3H]ryanodine binding by CaM approached 50% of maximal
activation (Fig. 3A). Conversely, CaM activation was
inhibited by physiological concentrations of Mg2+ (Fig. 4).
Caffeine increased the extent of CaM-dependent activation of
[3H]ryanodine binding to skeletal muscle SR (Fig.
3A), whereas BAPTA (free Ca2+ is ~3 nM)
noncompetitively inhibited CaM activation (Fig. 4). Together these
results demonstrate that CaM activation of RyR1 is modulated by
effectors of CICR and further support the findings of Ikemoto and
co-workers (11, 12) in indicating that CaM activation may
reflect an increased sensitivity of a CICR activation mechanism. A
similar role for CaM in sensitizing RyR channels to activation by CICR
was previously proposed to account for CaM activation of RyR channels
in sea urchin egg microsomes (18). Notably, CICR in
skeletal muscle has generally been considered to depend on an initial
increase in myoplasmic Ca2+ above resting concentrations
(28, 31). However, these results raise the possibility
that CaM may provide a means by which CICR may operate even at resting
Ca2+.
Distinct functional interactions of CaM with RyR1 and RyR2.
D-myo-inositol 1,4,5-trisphosphate receptors
[Ins(1,4,5)P3Rs] are intracellular
Ca2+ release channels that exhibit important structural and
functional similarities with RyR channels (19), and
differences in CaM's interactions with type 1 and type 3 Ins(1,4,5)P3Rs are postulated to
contribute to differences in the Ca2+-dependent regulation
of the two Ins(1,4,5)P3R isoforms
in situ (2, 27). Likewise, our results demonstrate that
CaM may differentially effect the Ca2+-dependent activation
of skeletal RyR1 and cardiac RyR2 isoforms. In the presence of CaM, the
threshold for activation of skeletal muscle SR
[3H]ryanodine binding was shifted to ~10-fold lower
Ca2+ concentrations (Fig. 2C), and the
apparent Ka for Ca2+ was decreased
to near or below resting Ca2+ concentrations (Table 2). In
comparison, the Ca2+ dependence of cardiac SR
[3H]ryanodine binding was only minimally affected by CaM,
and in all media the Ka for Ca2+
activation of [3H]ryanodine binding to cardiac SR
remained above resting Ca2+ concentrations (Fig. 2, Table
2). Although cardiac SR [3H]ryanodine binding was
significantly increased by CaM when Mg2+-free media were
supplemented with either caffeine or Na2AMP-PCP (Fig.
3B), this activation was smaller in magnitude and required higher CaM concentrations than did CaM activation of skeletal muscle SR
[3H]ryanodine binding under the same conditions.
Moreover, CaM failed to activate 45Ca2+ release
from cardiac SR vesicles (Fig. 1B, Table 1), suggesting that
CaM effects on cardiac SR [3H]ryanodine binding may
reflect lower affinity or possibly nonspecific interactions with RyR2
at nanomolar Ca2+. Consistent with this possibility, the
binding of [35S]CaM to cardiac SR vesicles was reduced at
nanomolar Ca2+ (Fig. 7, Table 3), indicating that
differential activation of RyR1 and RyR2 by CaM may be associated with
important differences in CaM binding to the two channel isoforms at
nanomolar Ca2+. These results, therefore, suggest that the
RyR2 isoform may lack a CaM activation site that is present in RyR1.
Alternatively, an RyR2 site may be occluded, for example, by covalent
modification of the channel (24, 25) or by endogenous CaM
that has remained tightly bound. At micromolar Ca2+,
however, skeletal and cardiac SR displayed similar
[35S]CaM binding stoichiometries (Table 3), a result that
is consistent with earlier reports that document similar inhibitory
actions of Ca2+-CaM on RyR1 (21) and RyR2
(Ref. 30; see also Fig. 1, C and D).
Nevertheless, it remains to be determined whether similar mechanisms
may underlie Ca2+-CaM inhibition of the RyR1 and RyR2
isoforms, and whether these mechanisms may involve, for example, the
modulation of Ca2+-dependent channel inhibition.
Potential role of CaM activation in skeletal muscle E-C coupling.
The primary, voltage-dependent mechanism responsible for activating SR
Ca2+ release during skeletal muscle E-C coupling almost
certainly involves a direct mechanical interaction between transverse
tubule voltage sensors and RyR1 channels that results in RyR1
activation at resting Ca2+ concentrations (31,
33). Less certain is the degree to which this mechanism may be
dependent on endogenous effectors of CICR, including Ca2+,
Mg2+, ATP, and CaM (1). In muscle fiber
preparations, a major fraction of the SR Ca2+ released
during an action potential may be attributed to CICR (28,
31). Yet, paradoxically, CICR from isolated skeletal muscle SR
may be largely suppressed under ionic conditions that exist in vivo
(e.g., Refs. 5 and 7). This paradox might be resolved if the
sensitivity of CICR in skeletal muscle was in part controlled by the
transverse tubule voltage sensors. According to the model proposed by
Lamb and Stephenson (15, 16), SR Ca2+ release
is blocked by physiological Mg2+, and during E-C coupling,
voltage sensors activate Ca2+ release by promoting the
dissociation of Mg2+ from low-affinity sites on RyR1
channels. More recently, Lacampagne and co-workers (14)
demonstrated that lowering myoplasmic Mg2+ in fiber
preparations increased the frequency of spontaneous Ca2+
release events (i.e., "sparks") without altering the properties of
the individual release events. These effects were attributed to
decreased Mg2+ block of high-affinity Ca2+
activation sites on RyR channels and a resultant shift in the threshold
for CICR nearer to resting Ca2+ (14). In this
regard, our results indicate that lowering the Mg2+
concentration increased the maximal extent of
Ca2+-activated [3H]ryanodine binding to
skeletal muscle SR but had comparatively little effect on the threshold
for Ca2+ activation, except when CaM was also present (Fig.
2). Thus we suggest that if the mechanism that activates SR
Ca2+ release in skeletal muscle is dependent on a major
shift in the Ca2+ threshold of CICR, then this mechanism
might also be dependent on CaM-RyR1 interactions. In this view, our
results that indicate a greater effect of CaM on the Ca2+
sensitivity of the RyR1 compared with the RyR2 isoform would be
consistent with the activation of Ca2+ release from the
cardiac SR being more strictly dependent on an initial increase in
myoplasmic Ca2+ above resting levels (31, 33).
In conclusion, our results indicate that CaM activates RyR1 by
increasing the Ca2+ sensitivity of the channel. Although
the significance of CICR in skeletal muscle remains in question, these
results provide additional evidence that CaM may play a critical role
in controlling the activation of RyR1 channels at resting
Ca2+ concentrations. Finally, apparent differences in
CaM's interactions with the RyR2 isoform at submicromolar
Ca2+ underscore the importance of characterizing the CaM
binding domains of the different RyR isoforms (24, 25, 36)
and of further defining the potentially complex roles of CaM-RyR
interactions in both skeletal and cardiac muscle E-C coupling.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Z. Grabarek and S. L. Hamilton for providing the
mammalian CaM clone, and Dr. E. Balog for helpful discussions.
 |
FOOTNOTES |
This work was supported by grants from the National Institutes of
Health (to C. F. Louis), the Muscular Dystrophy Association (to
G. M. Strasburg), and the American Heart Association (to B. R. Fruen).
Address for reprint requests and other correspondence: B. R. Fruen, 6-155 Jackson Hall, 321 Church St. SE, Minneapolis, MN 55455 (E-mail: fruen001{at}tc.umn.edu).
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. §1734 solely to indicate this fact.
Received 20 September 1999; accepted in final form 4 April 2000.
 |
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