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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 |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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 |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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 |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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 · cm1 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 |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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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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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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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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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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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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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 |
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We thank Drs. Z. Grabarek and S. L. Hamilton for providing the mammalian CaM clone, and Dr. E. Balog for helpful discussions.
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FOOTNOTES |
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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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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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) 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).
