RyR1 exhibits lower gain of CICR activity than RyR3 in the SR: evidence for selective stabilization of RyR1 channel

Takashi Murayama and Yasuo Ogawa

Department of Pharmacology, Juntendo University School of Medicine, Tokyo 113-8421, Japan

Submitted 16 September 2003 ; accepted in final form 10 February 2004


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We showed that frog {alpha}-ryanodine receptor ({alpha}-RyR) had a lower gain of Ca2+-induced Ca2+ release (CICR) activity than {beta}-RyR in sarcoplasmic reticulum (SR) vesicles, indicating selective "stabilization" of the former isoform (Murayama T and Ogawa Y. J Biol Chem 276: 2953–2960, 2001). To know whether this is also the case with mammalian RyR1, we determined [3H]ryanodine binding of RyR1 and RyR3 in bovine diaphragm SR vesicles. The value of [3H]ryanodine binding (B) was normalized by the number of maximal binding sites (Bmax), whereby the specific activity of each isoform was expressed. This B/Bmax expression demonstrated that ryanodine binding of individual channels for RyR1 was <15% that for RyR3. Responses to Ca2+, Mg2+, adenine nucleotides, and caffeine were not substantially different between in situ and purified isoforms. These results suggest that the gain of CICR activity of RyR1 is markedly lower than that of RyR3 in mammalian skeletal muscle, indicating selective stabilization of RyR1 as is true of frog {alpha}-RyR. The stabilization was partly eliminated by FK506 and partly by solubilization of the vesicles with CHAPS, each of which was additive to the other. In contrast, high salt, which greatly enhances [3H]ryanodine binding, caused only a minor effect on the stabilization of RyR1. None of the T-tubule components, coexisting RyR3, or calmodulin was the cause. The CHAPS-sensitive intra- and intermolecular interactions that are common between mammalian and frog skeletal muscles and the isoform-specific inhibition by FKBP12, which is characteristic of mammals, are likely to be the underlying mechanisms.

excitation-contraction coupling; ryanodine binding; ryanodine receptor


IN VERTEBRATE SKELETAL MUSCLE, depolarization of the T-tubule membranes triggers Ca2+ release from the sarcoplasmic reticulum (SR) in a process known as excitation-contraction (E-C) coupling (45). The Ca2+ release is mediated through the ryanodine receptor (RyR), a large homotetrameric channel complex (>2 MDa) in the SR membrane (16, 28, 36). In mammals, there are three genetically distinct isoforms of RyR (RyR1–RyR3), and the primary isoform in skeletal muscle is RyR1 (50). In many nonmammalian vertebrate skeletal muscles, in contrast, two isoforms of RyR, {alpha}- and {beta}-RyR, which are homologues of RyR1 and RyR3, respectively, exist in almost equal amounts (40). Recent studies have revealed that, in addition to RyR1, RyR3 is also expressed in some specific muscles in mammals, e.g., the diaphragm and soleus (11), although its amount is very low relative to that of RyR1 (1–5% at most in the diaphragm) (21, 33). RyR3 colocalizes with RyR1, forming clusters at the junctional triads (15, 42). It has recently been proposed (13) from electron microscopic observations that RyR3 is segregated from RyR1 into the parajunctional region immediately adjacent to the junctional region.

Ca2+ release through the RyR channel in skeletal muscle can be activated by two distinct modes: depolarization-induced Ca2+ release (DICR) and Ca2+-induced Ca2+ release (CICR). DICR is triggered by conformational change of the voltage sensor, the dihydropyridine receptor (DHPR), upon depolarization of the T tubule (43, 45). On this occasion, extracellular Ca2+ entry is not necessarily required. CICR is a ligand-gated mode in which Ca2+ itself activates the channel in a dose-dependent manner (12). Recent studies with transgenic mice lacking either RyR1 or RyR3 and a heterologous expression system with RyR-deficient "dyspedic" myotubes revealed that RyR1 can act as both DICR and CICR channels, whereas RyR3 can mediate only CICR but not DICR in mammalian skeletal muscle (14, 51, 52).

We (35) have recently determined the properties of Ca2+-dependent [3H]ryanodine binding, a biochemical measure of CICR activity, of the two isoforms coexisting in the native SR vesicles of frog skeletal muscle. The results demonstrated that [3H]ryanodine binding activity of {alpha}-RyR was as low as ~4% that of {beta}-RyR without alteration of sensitivity to CICR ligands such as Ca2+, adenine nucleotide, and caffeine. These results led to the conclusion that CICR of {alpha}-RyR was selectively "stabilized" in the native SR of frog skeletal muscle. This, in turn, means that CICR activity should be attributed largely to {beta}-RyR in the skeletal muscle (35). This could also be the case with mammalian skeletal muscle where the contribution of RyR3 to CICR activity could be assumed to be greater than that expected from its amount (3, 44, 53). The disproportionately greater contribution of RyR3 would be particularly remarkable in the skeletal muscle during development, because there were temporary changes in its content during growth (3, 54). The stabilized CICR activity of RyR1, the major isoform, also should be consistent with the finding that Ca2+ sparks are seldom observed in mammalian skeletal muscle, whereas they are frequently observed in frog skeletal muscle (47). To prove these possibilities, in this study we determined [3H]ryanodine binding of each of RyR1 and RyR3 in bovine diaphragm SR vesicles. Our results clearly demonstrated RyR1 with a lowered gain of CICR activity in mammalian skeletal muscle compared with RyR3, i.e., RyR1 selectively stabilized in the native SR vesicles. It was also shown that the stabilization of RyR1 was explained by isoform-specific inhibition both byFKBP12 and by some protein-protein or protein-lipid interactions that are sensitive to 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). The latter turned out to be common between frog and mammals.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Anti-RyR3 antibody against synthetic peptide KKRRRGQKVEKPE corresponding to amino acid sequence 4375–4387 of rabbit RyR3 (34) was purified by glutathione S-transferase fusion protein of amino acid sequence 4322–4410 of frog {beta}-RyR that had been immobilized on cyanogen bromide-activated Sepharose 4B (Amersham Biosciences). Antibodies against synthetic peptide HPASKQRSEGEKVR, which corresponds to a highly-conserved NH2-terminal amino acid sequence within RyRs (139–152 of human RyR1) (9), and YPSADFPGDDEEEE, corresponding to amino acid sequence 734–747 of frog DHPR {alpha}1S (23), were produced in rabbits and affinity-purified with peptide-coupled agarose beads. Monoclonal anti-calmodulin antibody (IM7) (22) was kindly provided by Prof. M. Yazawa (Hokkaido University, Sapporo, Japan). Monoclonal anti-RyR1 antibody (XA7) and polyclonal anti-FKBP12 antibody (PA1-026) were purchased from Upstate Biotechnology and Affinity Bioreagents, respectively. [3H]ryanodine (60–90 Ci/mmol) and calmodulin binding peptide were obtained from NEN Life Science Products and Calbiochem, respectively. Soybean phosphatide extract (lecithin, 95%) was obtained from Avanti Polar-Lipids. All other reagents were of analytic grade. Free Ca2+ and Mg2+ concentrations were calculated using association constants as follows (pH 6.8): 8.79 x 105 M–1, 1.82 x 103 M–1, and 5.37 x 103 M–1 for [Ca2+] of EGTA (18), for [Ca2+] of {beta},{gamma}-methyleneadenosine triphosphate (AMPPCP) (39), and for [Mg2+] of AMPPCP (39), respectively.

Isolation of SR vesicles. SR vesicles were prepared from bovine diaphragm (32). The isolated vesicles were quickly frozen in liquid N2 and stored at –80°C until used. Membrane protein was measured by the biuret method using bovine serum albumin as a standard. In some experiments, SR vesicles were further treated with myosin light chain kinase-derived calmodulin binding peptide (Calbiochem) according to Balshaw et al. (1) to make them free from calmodulin. Western blot analysis with anti-calmodulin antibody IM7 (22) confirmed that treatment with 10 µM (but not 1 µM) of the peptide effectively removed calmodulin from the SR to <10% of its original content.

Western blot analysis. SR proteins were separated by SDS-polyacrylamide gel electrophoresis with 2–12% linear gradient gels. Gels were electrophoretically transferred onto polyvinylidene difluoride membranes. Western blotting was carried out with an enhanced chemiluminescence system (ECL; Amersham Biosciences) using peroxidase-conjugated secondary antibodies. Primary antibodies for RyR1, RyR3, DHPR, and FKBP12 were used at 1:20,000, 1:1,000, 1:5,000 and 1:1,000 dilutions, respectively.

Determination of [3H]ryanodine binding to RyR isoforms in native SR vesicles. Assays were essentially the same as the method described previously (35). Briefly, bovine diaphragm SR vesicles (200 µg of protein) were incubated with 8.5 nM [3H]ryanodine for 5 h at 25°C in 200 µl of a medium containing 0.17 M NaCl, 20 mM 3-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO)-NaOH, pH 6.8, 2 mM dithiothreitol, various Ca2+ concentrations buffered with 10 mM EGTA, and 1 mM AMPPCP, unless otherwise indicated. The vesicles were then supplemented with 20 µM nonradioactive ryanodine to terminate incorporation of the radioactive ligand, cooled to 4°C, and solubilized with 1% CHAPS and 0.5% lecithin. All subsequent steps were carried out at 4°C; this is critically important to prevent the bound [3H]ryanodine from dissociating from RyRs (see Ref. 35). RyR3 was specifically immunoprecipitated by overnight incubation with the anti-RyR3 antibody-agarose beads (33). The radioactivity responsible for RyR1 was determined from the supernatant by filtration through polyethyleneimine-treated Whatman GF/B filters. The precipitated agarose beads were washed three times with the ice-cold medium containing 0.5 M NaCl, 20 mM MOPSO/NaOH, pH 6.8, 1% CHAPS, 0.5% lecithin, and 2 mM dithiothreitol. The radioactivity responsible for RyR3 was recovered by incubating the beads with 0.1 M glycine-HCl (pH 1.5). Nonspecific radioactivity was determined in the presence of 20 µM unlabeled ryanodine at the onset of the binding reaction.

French press and sucrose density gradient ultracentrifugation. The SR vesicles were diluted with 0.15 M KCl and 20 mM MOPSO/NaOH, pH 6.8, at a concentration of 10 mg/ml. They were then passed through a French press (2 cycles at 6,000 lb./in.2) (7). The sample was placed on top of four layers of sucrose [0.8 M (12 ml), 1.1 M (10 ml), 1.3 M (10 ml), and 1.6 M (5 ml)], buffered at pH 6.8 with 20 mM MOPSO/NaOH, and centrifuged at 25,000 rpm in a Hitachi SRP28SA rotor overnight at 4°C. The 1.3 M-1.6 M sucrose interface that contains the terminal cisterna (TC) fraction was collected, diluted with the above medium, sedimented by ultracentrifugation, and resuspended in the medium containing 0.3 M sucrose.

Statistics. Quantitative data are given as means ± SE of the number of repeated experiments (n). For determination of the significance of the difference between mean values, Student's unpaired t-test was applied.


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Separation of RyR1 and RyR3 with bound [3H]ryanodine by immunoprecipitation with anti-RyR3. [3H]Ryanodine binding activity of RyR isoforms coexisting in bovine diaphragm SR was determined by a method recently developed in our laboratory (35) (see MATERIALS AND METHODS). The SR vesicles were incubated with [3H]ryanodine under various conditions to achieve ryanodine binding at the steady state. The reaction was then terminated by addition of nonradioactive ryanodine and by cooling to 4°C, and vesicles were solubilized with CHAPS at 4°C. Under low temperature with an excess amount of nonradioactive ryanodine, [3H]ryanodine that had been bound to RyR was hardly dissociated from it (35, 37). [3H]ryanodine binding in the 1 M NaCl medium before and after 16-h incubation in the presence of 20 µM nonradiolabeled ryanodine at 4°C was unchanged (1.9 ± 0.1 vs. 1.8 ± 0.2 pmol/mg, respectively; n = 3). Thus incubation under this condition provides an adequate period to separate the isoforms as described below.

Separation of the isoforms was achieved by immunoprecipitation with an anti-RyR3 antibody that specifically reacts with RyR3 among three mammalian RyR isoforms (34). As shown in Fig. 1A, immunoprecipitation with the antibody specifically and completely precipitated RyR3 into the beads from CHAPS-solubilized SR vesicles, with RyR1 remaining in the supernatant. This precipitation was prevented by addition of an epitope peptide (RyR3-peptide). With the use of vesicles that had been incubated with [3H]ryanodine, significant radioactivity was detected in the beads immunoprecipitated with the anti-RyR3 antibody, with the complementing radioactivity in the supernatant (Fig. 1B). In contrast, no radioactivity was detected in the beads with control IgG or in the presence of the RyR3-peptide, where RyR3 did not precipitate. These findings indicate that radioactivities of the supernatant and of the beads represent the [3H]ryanodine bound to RyR1 and RyR3, respectively. The antibody did not affect the [3H]ryanodine binding activity of the purified RyR3 (32), indicating no artificial modification of the RyR3 activity in the procedure. Thus this procedure determined the [3H]ryanodine binding activity of each of RyR1 and RyR3 in bovine diaphragm SR.



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Fig. 1. Immunoprecipitation with anti-RyR3 antibody separates RyR1 and RyR3 with their [3H]ryanodine binding activities. A: detection of ryanodine receptor (RyR) isoforms in the supernatant and precipitate after immunoprecipitation. Bovine diaphragm sarcoplasmic reticulum (SR) vesicles were solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and RyR3 was immunoprecipitated as described in MATERIALS AND METHODS. Proteins in the supernatant (Sup) and precipitated beads from 15 µg of SR were separated by SDS-PAGE, and each RyR isoform was detected by an isoform-specific antibody. Total SR proteins without immunoprecipitation (15 µg) are shown at left. Note that RyR1 was detected in the supernatant but not in the beads, whereas the reverse is true with RyR3. B: separation of [3H]ryanodine binding activity by immunoprecipitation. SR vesicles were incubated for 5 h at 25°C with 8.5 nM [3H]ryanodine in medium containing 1 M NaCl, 20 mM 3-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO)-NaOH, pH 6.8, 1 mM {beta},{gamma}-methyleneadenosine triphosphate (AMPPCP), and 100 µM free Ca2+. After termination of the reaction by addition of 20 µM nonradioactive ryanodine and cooling to 4°C, vesicles were solubilized with CHAPS and RyR3 was immunoprecipitated. The specific [3H]ryanodine binding in both the supernatant and precipitated beads was determined. Note that significant ryanodine binding in the beads was detected with anti-RyR3 antibody but not with control IgG. Binding in the beads was totally abolished by addition of 20 µM epitope peptide (+RyR3-peptide) that competitively inhibits interaction of the antibody with RyR3.

 
Determination of numbers of [3H]ryanodine binding sites for RyR1 and RyR3 in bovine diaphragm SR. Bovine diaphragm SR vesicles contain a major amount of RyR1 in addition to a minor amount of RyR3 (11, 21) (see Fig. 1A). To evaluate each activity in the SR, it is essential to know the number of active channels for each isoform. For this purpose, the maximal binding sites (Bmax) and the affinities for the ligand (Kd) were determined from dose-dependent [3H]ryanodine binding. For accurate determination of these values, we used high-salt medium containing 1 M NaCl in which the RyR activity is greatly enhanced (29, 38). The Scatchard plots revealed that the data could be fitted by straight lines for RyR1 and for RyR3 (Fig. 2, see also inset) indicating each single class of homogeneous binding site. The maximal binding sites (Bmax) for RyR1 and RyR3 were calculated to be 5.7 ± 0.5 and 0.23 ± 0.02 pmol/mg (n = 3), respectively. The number of active channels for RyR1 and RyR3 were thus estimated to be 96 and 4% of the total, respectively. The fraction of RyR3 is well consistent with a previous report on bovine diaphragm (<5%) (21) and higher than that of rabbit diaphragm (0.6%) (33). The Kd for [3H]ryanodine of RyR1 in the SR (21.6 ± 2.1 nM) was more than 11-fold greater than that of RyR3 in the SR (1.9 ± 0.3 nM). Kd values for ryanodine of the purified RyR1 and RyR3, on the other hand, were 1.6 and 2.3 nM under the corresponding conditions, respectively (33). This finding suggests that RyR1 has much lower affinity for ryanodine than RyR3 in the SR vesicles.



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Fig. 2. Scatchard plot analysis of [3H]ryanodine binding of the RyR isoforms in bovine diaphragm SR. [3H]ryanodine binding was carried out as described in Fig. 1 with 1.6–49 nM [3H]ryanodine. Data points are means of 3 similar experiments. Linear Scatchard plots indicate that each isoform had a single class of [3H]ryanodine binding site. Values for the maximal binding (Bmax) and affinity for the ligand (Kd) are 5.7 ± 0.5 pmol/mg protein and 21.6 ± 2.1 nM for RyR1 and 0.23 ± 0.02 pmol/mg protein and 1.9 ± 0.3 nM for RyR3, respectively (n = 3). From the Bmax values, the number of channels for RyR1 and RyR3 was estimated to be 96 and 4% of the total, respectively. The greater Kd value for RyR1 indicates its lowered channel activity under the conditions used.

 
Ca2+ dependence and magnitude of [3H]ryanodine binding of RyR1 and RyR3 in native SR vesicles. Figure 3A demonstrates the Ca2+-dependent [3H]ryanodine binding of RyR1 and RyR3 in bovine diaphragm SR as determined by the method described in Determination of [3H]ryanodine binding to RyR isoforms in native SR vesicles, but in the presence of 0.17 M NaCl instead of 1 M NaCl. Because activity of the RyR channels is low under such physiological salt conditions, 1 mM AMPPCP was supplemented to the medium. Both RyR1 and RyR3 showed biphasic Ca2+ dependence. The peak amplitude of the binding for RyR3 (0.057 pmol/mg SR protein at 100 µM Ca2+) was about one-fourth that for RyR1 (0.21 pmol/mg SR protein at 30 µM Ca2+). The Ca2+ sensitivity of RyR3 was significantly (P < 0.05) lower than that of RyR1 in both Ca2+ activation (EC50 = 12.6 ± 0.3 µM for RyR3 vs. 5.7 ± 0.2 µM for RyR1, n = 3) and Ca2+ inactivation (IC50 = 0.81 ± 0.16 mM for RyR3 vs. 0.27 ± 0.10 mM for RyR1, n = 3 ) (see also Fig. 3A, inset). The lower Ca2+ sensitivity of RyR3 than of RyR1 is also true with rabbit diaphragm (32, 33) and with mouse skinned fibers (53).



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Fig. 3. Ca2+-dependent [3H]ryanodine binding activity of separated RyR1 and RyR3. A: activity of total channels. Ca2+-dependent [3H]ryanodine binding of RyR1 and RyR3 was carried out as described in Fig. 1 in medium containing 0.17 M NaCl instead of 1 M NaCl. Data are means ± SE (n = 3). Inset: binding was normalized to the peak activity of each isoform. B: activity of single channels. Values in A were divided by the Bmax values for each isoform (see Fig. 2). RyR1 shows much lower activity than RyR3, suggesting that the former is stabilized in the Ca2+-induced Ca2+ release (CICR) activity in the SR.

 
Values of [3H]ryanodine binding on the ordinate in Fig. 3A (B values) refer to the activities of each isoform per unit weight of total protein in the SR as expressed in picomoles per milligram of protein. The B values calibrated with the respective Bmax values, i.e., B/Bmax values, refer to fractions of the maximum activity of each active isoform (Fig. 3B). The binding of RyR1 was thus much lower than that of RyR3 at every Ca2+ concentration: at the peak value, the binding of RyR1 (B/Bmax = 0.037 ± 0.003) was only 15% that of RyR3 (B/Bmax = 0.25 ± 0.02). Because the B/Bmax value was far less than 1 even at the peak under this condition, the binding was not saturated at the [3H]ryanodine concentration used (8.5 nM). Thus it would be reliable to compare the magnitude of activity of the two RyR isoforms. Consistent with a lower affinity for ryanodine of RyR1 in the SR (see Fig. 2), these results indicate that the RyR1 channels exhibit markedly lower CICR activity than RyR3 channels in native SR vesicles. The B/Bmax values of the purified RyR1 and RyR3 calculated from our previous article (32), in contrast, were 0.25 and 0.29, respectively, which are similar to that of RyR3 in the SR. Taken together, these findings strongly suggest that the gain of CICR activity of RyR1 is selectively set to a low level, i.e., "stabilized," in mammalian skeletal muscle SR.

The stabilized activity of RyR1 means that the functional contribution of RyR1 in CICR activity should be less than the magnitude expected from its abundance in the muscle. In other words, the contribution of RyR3 would be much higher. In fact, about one-fifth of the total binding was attributed to RyR3, although its amount was as small as 4% of the total RyR channels (Fig. 3A). These situations reasonably explain the previous unexpected results of the much greater contribution of RyR3 to Ca2+ release in skeletal muscles of RyR1- or RyR3-knockout mice (3, 53).

Similar responses of RyR1 and RyR3 to adenine nucleotides, Mg2+, and caffeine. Figure 4 demonstrates the dose-dependent enhancing effect of AMPPCP, a nonhydrolyzable ATP analog, on RyR1 and RyR3 in the presence of 100 µM Ca2+, a concentration at which Ca2+ activation is near the optimum. The [3H]ryanodine binding of the two isoforms similarly increased with increases in AMPPCP and reached a plateau around 1 mM. The apparent EC50 values of AMPPCP were estimated to be 0.3 mM for both isoforms. Thus the sensitivity of RyR3 to activation by adenine nucleotides is similar to that of RyR1.



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Fig. 4. Responses of RyR1 and RyR3 to AMPPCP. [3H]ryanodine binding to RyR1 ({circ}) and RyR3 ({triangleup}) were carried out as described in Fig. 3 at 100 µM Ca2+ in the presence of 0–3 mM AMPPCP. Values were normalized to Bmax for each isoform: 0.19 and 0.055 pmol/mg protein for RyR1 (at 1 mM) and RyR3 (at 3 mM), respectively. Data are means ± SE (n = 3). There was no difference in AMPPCP sensitivity between RyR1 and RyR3.

 
Figure 5 shows the effect of Mg2+ on the [3H]ryanodine binding activity. Mg2+ is known to inhibit RyR channels by two distinct mechanisms: it acts as a competitive antagonist at the Ca2+-activating site, which results in reduction of the Ca2+ sensitivity for activation, and also as an agonist at the Ca2+-inactivating site, which suppresses the activity (24, 30, 31). Mg2+ decreased the binding to the two isoforms in a similar dose-dependent manner in the presence of 30 µM Ca2+ (Fig. 5A). The apparent IC50 values for RyR1 and RyR3 were 0.15 ± 0.02 and 0.14 ± 0.02 mM (n = 3), respectively. Figure 5B shows the effect of Mg2+ on the Ca2+ dependence in the range of 0.1–30 µM Ca2+, which is of physiological relevance. Mg2+ (0.3 mM) decreased the binding to both the isoforms with a rightward shift of the Ca2+-dependent curve. These results suggest that effects of Mg2+ on the two RyR isoforms are likely to be similar.



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Fig. 5. Responses of RyR1 and RyR3 to Mg2+. A: effect of varied Mg2+ concentration on [3H]ryanodine binding to RyR1 ({circ}) and RyR3 ({triangleup}). Determinations were carried out as described in Fig. 3 at 30 µM Ca2+ with 0–3 mM Mg2+. Values were normalized to binding without Mg2+: 0.20 and 0.054 pmol/mg for RyR1 and RyR3, respectively. Data are means ± SE (n = 3). B: Ca2+ dependence of [3H]ryanodine binding to RyR1 ({circ}) and RyR3 ({triangleup}) in the presence of 0.3 mM Mg2+. Data are means ± SE (n = 3). Dotted and dashed lines indicate the Ca2+-dependent curve for RyR1 and RyR3, respectively, in the absence of Mg2+ (see Fig. 3A).

 
It is well known that caffeine stimulates CICR by increasing not only Ca2+ sensitivity but also the optimum activity (12, 36). Consistently, we showed that caffeine increased not only Ca2+ sensitivity but also the peak value at the optimum Ca2+ concentration (31). As shown in Fig. 6A, 10 mM caffeine enhanced [3H]ryanodine binding at 1 µM Ca2+, near the threshold for Ca2+ activation, and also at 100 µM Ca2+, near the optimum Ca2+ concentration; the extent of the enhancement was greater at 1 µM Ca2+ than at 100 µM Ca2+. There was no difference between RyR1 and RyR3 in the pattern of stimulation or in their magnitudes. The two RyR isoforms showed a similar dose-dependent activation curve by caffeine at both 1 and 100 µM Ca2+ (Fig. 6B). Thus RyR1 and RyR3 also may be similar in their responses to caffeine.



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Fig. 6. Responses of RyR1 and RyR3 to caffeine. A: [3H]ryanodine binding was carried out as described in Fig. 3 at 1 µM (pCa 6) and 100 µM (pCa 4) Ca2+ with (caffeine) or without (control) 10 mM caffeine. B: dose-dependent activation curve for caffeine. [3H]ryanodine binding to RyR1 (circles) and RyR3 (triangles) was carried out as in A with 0–10 mM caffeine at 1 µM (open symbols) and 100 µM Ca2+ (filled symbols). Values were normalized to Bmax for each condition: 0.33 and 0.51 pmol/mg protein at 1 and 100 µM Ca2+ for RyR1 and 0.11 and 0.12 pmol/mg protein at 1 and 100 µM Ca2+ for RyR3, respectively. Data are means ± SE (n = 3). There was no difference in responses to caffeine between RyR1 and RyR3.

 
FKBP12 stabilizes RyR1 but not RyR3 in bovine diaphragm SR. It is well established that FKBP12 or FKBP12.6 is associated with RyR1 or RyR2 and modulates its CICR activity (27). Pull-down assay with isoform-specific antibodies revealed that FKBP12 was coprecipitated with RyR1 (Fig. 7A). FK506 at 10 µM, which binds to dissociate FKBP12 from RyR, completely prevented coprecipitation of FKBP12. In contrast, no FKBP12 was coprecipitated with RyR3, as was true with RyR3 heterologously expressed in dyspedic myotubes (14) or with RyR3 of rabbit brain (6). [3H]ryanodine binding experiments showed that 10 µM FK506 increased the binding to RyR1 (0.13 ± 0.01 pmol/mg in control vs. 0.23 ± 0.03 pmol/mg with FK506, n = 4, P < 0.05) but not the binding to RyR3 (0.044 ± 0.002 pmol/mg in control vs. 0.045 ± 0.003 pmol/mg with FK506, n = 4) (Fig. 7B). No further increase in the binding to RyR1 was observed with 30 µM FK506, indicating that the effect of FK506 is the maximum at 10 µM (data not shown). These results suggest that FKBP12 is partly responsible for stabilization of RyR1 in the native SR vesicles. The in vitro binding experiments in which a FKBP12 protein affinity column was used demonstrated that RyR3 purified from rabbit diaphragm can interact with FKBP12 in a FK506-dependent manner (4, 32). It seems likely that RyR3 may have much less affinity for FKBP12 than does RyR1, as is the case with cardiac RyR2 (20). It is also possible that FKBP12 has no effect on the RyR3 function, even if it binds.



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Fig. 7. FKBP12 interacts with and stabilizes RyR1 but not RyR3. A: interaction of FKBP12 with RyR isoforms. RyR1 or RyR3 was immunoprecipitated using specific antibodies, and FKBP12 in the precipitated products was detected by Western blot analysis. FKBP12 was coprecipitated with RyR1 (IP-Ab, RyR1) but not with RyR3 (IP-Ab, RyR3). Addition of 10 µM FK506 completely prevented coprecipitation of FKBP12. B: effect of FK506 on [3H]ryanodine binding. [3H]ryanodine binding was carried out as described in Fig. 3 at 100 µM Ca2+ with (FK506) or without (control) 10 µM FK506. Data are means ± SE (n = 4). The binding of RyR1 was selectively destabilized by FK506 treatment. *P < 0.05 vs. control.

 
Partial reversal of stabilization of RyR1 by solubilization with CHAPS/phospholipids. In frog skeletal muscle, solubilization of the SR with CHAPS/phospholipids remarkably enhanced [3H]ryanodine binding to {alpha}-RyR in the SR vesicles; B/Bmax values with and without CHAPS/phospholipids were 0.22 and 0.007, respectively (see Fig. 6 of Ref. 35). Therefore, we examined the effects of CHAPS/phospholipids on bovine RyR isoforms. We used soybean lecithin, which is composed of 95% phosphatidylcholine, as phospholipids. Addition of 1% CHAPS with 0.5% lecithin increased more than fourfold the binding of RyR1 from 0.15 ± 0.01 pmol/mg in control to 0.66 ± 0.06 pmol/mg with CHAPS/phospholipids (n = 6) (Fig. 8A); in contrast, the binding of RyR3 was unchanged by the treatment (0.056 ± 0.008 pmol/mg in control vs. 0.062 ± 0.009 pmol/mg with CHAPS/phospholipids, n = 6). Thus CHAPS/phospholipids selectively increase the [3H]ryanodine binding to RyR1 in bovine diaphragm SR. A similar increase in the binding of RyR1 was also observed with 1% CHAPS without lecithin (data not shown). This finding may exclude the possibility of the direct activating action of lecithin.



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Fig. 8. CHAPS/phospholipids reverses the stabilization of RyR1. A: [3H]ryanodine binding was carried out as described in Fig. 3 at 100 µM Ca2+ with (CHAPS) or without (control) 1% CHAPS and 0.5% lecithin. Data are means ± SE (n = 4). The binding of RyR1 was selectively increased by CHAPS/phospholipids, indicating reversal of the stabilization by CHAPS. **P < 0.01 vs. control. B: summary of effects of FK506 and CHAPS on RyR1 and RyR3. Ryanodine binding activity in the absence (control, n = 11) and presence of 10 µM FK506 (FK506. n = 4), 1% CHAPS (CHAPS, n = 4), and both (FK506 + CHAPS, n = 3) is indicated as B/Bmax. The activity of RyR1 but not RyR3 was enhanced by these reagents. The additive effects of FK506 and CHAPS suggest that they may act through independent mechanisms. **P < 0.01.

 
Figure 8B summarizes the effects of FK506, CHAPS/phospholipids, and their combination on the [3H]ryanodine binding to RyR1 and RyR3 in bovine diaphragm SR. The activity is expressed as B/Bmax. In control (without FK506 or CHAPS/phospholipids) the activity of RyR1 (0.025 ± 0.001, n = 9) was only one-ninth that of RyR3 (0.22 ± 0.02, n = 9). The B/Bmax value of RyR1 was greater with CHAPS/phospholipids (0.12 ± 0.01, n = 4) than with FK506 (0.040 ± 0.004, n = 4). Their combination further increased the binding of RyR1 (0.18 ± 0.02, n = 3) to a level comparable to that of RyR3 (0.21 ± 0.01, n = 3). The extent of activation by FK506 (1.6 in control vs. 1.5 with CHAPS/phospholipids) and the extent by CHAPS/phospholipids (4.8 in control vs. 4.5 with FK506) were unaffected by the presence of the other reagent. In addition, the effect of FK506 was maximum at 10 µM, as described in FKBP12 stabilizes RyR1 but not RyR3 in bovine diaphragm SR. These results suggest that FK506 and CHAPS/phospholipids may act through mechanisms independent of each other. Again, the binding of RyR3 was unchanged by either reagent or by their combination. Under the conditions of the two combined reagents, the binding of RyR1 reached a level comparable to that of RyR3. These findings suggest that RyR1 may be stabilized partly by FKBP12 and partly by some protein-protein and/or protein-lipid interactions sensitive to CHAPS/phospholipids in native SR vesicles. Because oligomerization of RyR monomers is critical not only in functioning as the Ca2+ release channel but also in [3H]ryanodine binding, formation of a suitable configuration must be involved in the stabilization. CHAPS/phospholipids were used for solubilization and purification of RyR, and the purified RyR1 lacks FKBP12 (32). Thus greater activity of the purified RyR1 can be well explained by "destabilization" of the isoform by loss of FKBP12 and interactions sensitive to CHAPS/phospholipids.

Effect of high salt on stabilization of RyR1. High salt clearly stimulates the channel activity of RyRs (28, 36). To examine whether high salt destabilizes RyR1, we examined its effect on the [3H]ryanodine binding to RyR1 and RyR3 (Fig. 9). An increase in NaCl concentration from 0.17 to 1 M greatly enhanced the binding to RyR1 (from 0.14 ± 0.02 to 2.0 ± 0.2 pmol/mg, n = 3) and to RyR3 (from 0.051 ± 0.004 to 0.31 ± 0.03 pmol/mg, n = 3) (Fig. 9A). The extent of increase was greater in RyR1 (14-fold) than in RyR3 (6-fold). In 1 M NaCl medium, the binding of RyR1 was further increased by FK506 (3.6 ± 0.1 pmol/mg, n = 3) or CHAPS/phospholipids (4.7 ± 0.2 pmol/mg, n = 3), indicating that high salt and FK506 or CHAPS/phospholipids are additive to each other in activating RyR1 (Fig. 9B). By combining FK506 and CHAPS/phospholipids, the binding was further increased to 5.1 ± 0.3 pmol/mg (n = 3), which corresponds to Bmax. The combined effect of the two treatments was probably saturated in magnitude. In contrast, no further increase by these treatments was observed with RyR3, as was true in 0.17 M NaCl medium (see Figs. 7 and 8). It should be pointed out that [3H]ryanodine binding sites of RyR3 were almost saturated with the ligand in the 1 M NaCl medium. Taken together, these results suggest that stimulation by high salt may be different in the underlying mechanisms from that by FK506 or CHAPS/phospholipids.



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Fig. 9. Effect of high salt on the activity and stabilization of RyRs. A: effect of high salt on [3H]ryanodine binding to RyR1 and RyR3. Assays were carried out as described in Fig. 3 in 0.17 M (open bars) and 1 M (shaded bars) NaCl medium at 100 µM Ca2+. Data are means ± SE (n = 3). Note that high salt greatly increased binding to both RyR1 and RyR3. **P < 0.01 vs. 0.17 M NaCl. B: effect of FK506 and CHAPS/phospholipids on [3H]ryanodine binding under high-salt conditions. Assays were carried out as in A in 1 M NaCl medium in the absence (control) and presence of 10 µM FK506 (FK506) or 1% CHAPS/phospholipids (CHAPS). Data are means ± SE (n = 3). Binding of RyR1 was further stimulated by FK506 and CHAPS, suggesting that stabilization still occurred even under high-salt conditions. **P < 0.01.

 
T-tubule-SR interaction at triads, coexisting RyR3, and calmodulin are not involved in stabilization of RyR1. In skeletal muscle, the TC of SR interacts with the T tubule to form the triad structure. The SR vesicles we used contained a substantial amount of DHPR {alpha}1S, indicating the presence of T tubules (Fig. 10A, inset). Therefore, one might assume that some interaction with T-tubule components, e.g., DHPR, is involved in the stabilization of RyR1. To examine this possibility, we disrupted the triad structure by French press and separated TC through sucrose density gradient ultracentrifugation (see MATERIALS AND METHODS). Bmax values of [3H]ryanodine binding in the TC fraction were 33.7 and 1.6 pmol/mg for RyR1 and RyR3, respectively, indicating about a sixfold increase in the RyR content. Substantial removal of the T tubule was demonstrated by selective reduction in the DHPR {alpha}1S-subunit immunoreactivity (<10% of control) in reference to RyR1 and RyR3 (Fig. 10A, inset). Binding activity in the TC (B/Bmax = 0.049 ± 0.005 for RyR1 and 0.23 ± 0.01 for RyR3), however, was not significantly different from that in the SR (B/Bmax = 0.037 ± 0.003 for RyR1 and 0.25 ± 0.02 for RyR3) . Thus the T-tubule-SR interaction may not be involved in the stabilization of RyR1.



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Fig. 10. Association with T tubules or coexisting RyR3 is not involved in the stabilization. A: [3H]ryanodine binding of the terminal cisterna (TC) fraction that is depleted of T tubules. The TC fraction was isolated from SR vesicles as described in MATERIALS AND METHODS, and [3H]ryanodine binding to RyR1 and RyR3 was determined as described in Fig. 3 at 100 µM Ca2+. Data are means ± SE (n = 3). Values were normalized to each of the Bmax values of [3H]ryanodine binding: 5.7 and 33.7 pmol/mg for RyR1 and 0.23 and 1.6 pmol/mg for RyR3 in SR and TC, respectively. Inset: Western blot analysis for RyR1, RyR3, and dihydropyridine receptor (DHPR) {alpha}1S in the SR vesicles and the TC fraction. The {alpha}1S density upon immunoreaction was markedly reduced in the TC fraction, indicating depletion of T tubules. The ryanodine binding activity of RyR1 and RyR3, however, was similar in the 2 preparations. B: [3H]ryanodine binding of RyR1 in SR vesicles from bovine epicranial muscle lacking RyR3. Data are means ± SE (n = 3). Inset: Western blot analysis of RyR isoforms in diaphragm (Dia) and epicranial (Epi) muscles. RyR3 was not detected in epicranial muscle SR, whereas the expression level of RyR1 was similar to that in diaphragm. Ca2+-dependent [3H]ryanodine binding of the epicranial RyR1 ({triangleup}) was similar to that of the diaphragm RyR1 ({circ}).

 
Ca2+ sparks in cultured myotubes from skeletal muscle (48) or in freshly isolated vascular smooth muscle cells (25) of RyR3–/– mice were more frequent than in those of wild-type mice. Therefore, it is suggested that coexisting RyR3 might suppress RyR1 or RyR2 channel activity. To test this possibility, we examined [3H]ryanodine binding of RyR1 in bovine epicranial muscle that does not express RyR3 (Fig. 10B). Western blot analysis did not detect RyR3 in SR vesicles prepared from the epicranial muscle, whereas the expression level of RyR1 was comparable to that in the diaphragm (Fig. 10B, inset). However, Ca2+-dependent [3H]ryanodine binding of RyR1 was similarly stabilized to a low level. Consistently, the Kd value for [3H]ryanodine of RyR1 in epicranial muscle was 23.6 ± 3.4 nM (n = 3), which was similar to that of RyR1 in the diaphragm (21.6 ± 2.1 nM). These findings indicate that coexisting RyR3 is not involved in the stabilization of RyR1 in skeletal muscle.

We also examined whether calmodulin coexisting in SR vesicle preparation is involved in the stabilization. SR vesicles were treated with myosin light chain kinase-derived calmodulin-binding peptide, which is reported to bind to calmodulin with a high affinity and thus remove it from the vesicles (1). Western blot analysis with anti-calmodulin monoclonal antibody IM7 (22) revealed that this treatment with an excess amount of the calmodulin binding peptide (10 µM) effectively removed calmodulin from SR (<10% of original content), but the [3H]ryanodine binding of RyR1 was not significantly changed (0.20 ± 0.02 pmol/mg in control vs. 0.24 ± 0.02 pmol/mg in treated SR, n = 3). This excludes the possible involvement of calmodulin in the stabilization of RyR1.


    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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In the present study, we investigated [3H]ryanodine binding to RyR1 and RyR3 in native bovine diaphragm SR that were separated into each activity of the two isoforms by immunoprecipitation with RyR3-specific antibody. The determined activity for each isoform was expressed by the term B/Bmax, which refers to the extent of activation of the single active channels. The results presented here thus demonstrate properties of CICR activity of single RyR1 and RyR3 channels in mammalian skeletal muscle, where they are embedded in the intact SR membrane under the influence of interacting molecules. The B/Bmax expression of [3H]ryanodine binding clearly demonstrated that RyR1 exhibited much lower ryanodine binding activity than RyR3 (Fig. 3B). Consistently, RyR1 showed a considerably lower affinity for ryanodine than RyR3 (Fig. 2). In marked contrast, RyR1 and RyR3 purified from rabbit skeletal muscle showed similar B/Bmax values (32) and affinities for ryanodine (33), which were close to the values of RyR3 in the SR of the present study. Taken together, these findings suggest that RyR1 channel has a lower gain of CICR activity than RyR3 in mammalian skeletal muscle, i.e., RyR1 selectively stabilized in the native SR vesicles.

We (35) previously showed that {alpha}-RyR is selectively stabilized in the CICR activity in frog skeletal muscle. The common characteristics of the stabilization between mammalian and frog muscles are as follows: 1) stabilized isoforms (RyR1 and {alpha}-RyR) show greatly lowered affinity for ryanodine; 2) modulation by Ca2+, Mg2+, adenine nucleotides, or caffeine remains unaffected even in the stabilized state; and 3) stabilization is reversed by CHAPS/phospholipids. Thus the stabilized CICR activity of RyR1 homologues may be well conserved among vertebrate skeletal muscles. Some interesting differences also were obvious between them. First, the extent of stabilization was greater in frog than in bovine muscle; B/Bmax values of {alpha}-RyR and RyR1 were 0.009 and 0.037, respectively. In other words, the magnitude of the stabilization with {alpha}-RyR (~4%) was greater than that with RyR1 (~15%), because {beta}-RyR and RyR3 showed similar B/Bmax values of 0.20–0.25. Second, whereas the stabilization of RyR1 was caused by both FKBP12 and the CHAPS/phospholipids-sensitive mechanism, the latter is likely to be fully responsible for the stabilization of {alpha}-RyR because of lack of the effect of FK506 (35). Thus {alpha}-RyR might not be regulated by FKBP12 in frog skeletal muscle. It seems interesting that the partial amino acid sequence of FKBP12 coprecipitated with {alpha}-RyR was more similar to that of mammalian FKBP12.6 than to FKBP12.0 (35).

It was reported that RyR3 is sensitive to ligands for CICR, e.g., Ca2+, Mg2+, adenine nucleotide, and caffeine (8, 21, 32, 33). The present results demonstrated that the responses of RyR3 to Mg2+, adenine nucleotide, and caffeine are similar to those of RyR1 (Figs. 46). RyR3 was slightly lower than RyR1 in Ca2+ sensitivity to not only activating but also inactivating Ca2+ (Fig. 3A), consistent with the previous findings with the purified isoforms (32, 33) and with skinned muscle fibers from dyspedic mice (53). The most notable difference is that RyR3 was not stabilized at all in the SR vesicles, in marked contrast to RyR1. The B/Bmax value for RyR3 was sevenfold greater than that for RyR1 (Fig. 3B). Thus RyR3 may form Ca2+ release channels characteristic of the unstabilized activity compared with the RyR1 channels. This means that RyR3 could contribute to the CICR activity much more than that expected from its amount in the muscle. This can reasonably explain the previous results of an unexpectedly much greater contribution of RyR3 to Ca2+ release in skeletal muscles of RyR-knockout mice (3, 53). It is also possible that the unstabilized activity of RyR3 might appear to enhance the sensitivity of CICR to Ca2+ or ligands such as caffeine. In fact, the B/Bmax values of single RyR3 were higher than those of the single RyR1 at all Ca2+ concentrations (Fig. 3B). These situations might explain the previous reports of greater sensitivity of RyR3 to caffeine in Ca2+ sparks with myotubes from RyR-knock out mice (10) or Ca2+ transients in 1B5 cells expressing RyR (14).

The selective stabilization of RyR1 is entirely due to the state of RyR1 at which the isoform shows a reduced affinity for [3H]ryanodine. It is important to note that the stabilization cannot be brought about by changes in the sensitivity to CICR ligands such as Ca2+, Mg2+, adenine nucleotide, or caffeine (Figs. 36). Thus stabilization may be independent of the responses to CICR ligands. These findings suggest that the activity of RyR1 channels would always be stabilized in muscle, irrespective of the changes in the environment or state of muscle (resting or contracting). This, in turn, implies that abnormality in the stabilization of RyR1 would cause substantial changes in Ca2+ homeostasis that might lead to critical disorder in the muscle as discussed below.

We found that the stabilization of RyR1 is attributed partly to FKBP12 (Fig. 7) and partly to some mechanism sensitive to CHAPS/phospholipids (Fig. 8). The fact that the effects of FK506 and CHAPS/phospholipids are additive suggests that the mechanisms are independent of each other. We found that FKBP12 interacts with RyR1 but weakly with RyR3 in skeletal muscle. The lack of FKBP12 interaction of RyR3 is consistent with recent reports (6, 14). Interestingly, stabilization still occurred under high salt conditions where the RyR activity is known to be greatly enhanced (28, 36) (Fig. 9). Thus destabilization is different in a mechanism than stimulation by high salt. The fact that high salt enhanced the binding not only to RyR1 but also to RyR3 may support this idea.

The stabilization of RyR1 was partially reversed by CHAPS/phospholipids (Fig. 8A). It was also demonstrated that CHAPS/phospholipids alter Ca2+ sensitivity of RyR1 (41). These results suggest that some protein-protein or protein-lipid interactions sensitive to CHAPS are critically involved in the stabilization and function of RyR1. It is also possible that lecithin added with CHAPS might directly increase the activity of RyR1, because a recent report (5) demonstrated that the RyR channel activity was affected by the composition of phospholipids. Lecithin used here, however, cannot be the cause of destabilization of RyR1 because destabilization by CHAPS was also found in the absence of lecithin. The stabilization was still observed with the TC-rich fraction, indicating that it occurs in the SR vesicle itself but not through interaction with T tubules (Fig. 10B). Thus proteins or lipids of TC membranes are plausible candidates for the stabilization. Neither coexisting RyR3 (Fig. 10A) nor calmodulin was the cause. Several molecules have been reported to inhibit the RyR channel activity, such as triadin (17), calsequestrin (2), and sphingolipids (46). It is also likely that the stabilization of RyR1 may be caused by interactions within or between four monomers of RyR1 tetramers. This is supported by findings that the "purified" RyR1 channels display lower open probability than RyR3 channels in planar lipid bilayer experiments (8, 32). Thus stabilization may be the inherent nature of the RyR1 channels in vertebrate skeletal muscles. Recently, Yamamoto et al. (55) demonstrated that a peptide corresponding to amino acid sequence 2442–2477 of rabbit RyR1 (DP4) increased the Ca2+ release activity of RyR1. They suggested that the peptide probably acts by competing for the NH2-terminal domain that normally interacts with the central domain of RyR1 to stabilize the channel activity (19). It is quite possible that such intermolecular interactions are sensitive to CHAPS. Further characterizations are necessary to clarify the mechanisms of stabilization.

In physiological Ca2+ release, RyR1 channels are activated by conformational changes of DHPR upon depolarization of the T tubule (45). In the triad junction, tetrad (four assembled DHPRs) in the T tubule is tightly apposed alternately to every other foot (RyR tetramer) in the SR (16). This indicates that half of the RyR1 channels are coupled to DHPR and the other half are not. It is therefore postulated that the "uncoupled" RyR1 channels might act as the CICR channels amplifying Ca2+ that was released by DICR via the "coupled" channels (45). Our results indicate that the CICR activity of RyR1 may be greatly stabilized in the native SR vesicles. In addition, Mg2+ in the cytoplasm also strongly inhibits RyR in skeletal muscle (28, 36) (see Fig. 5). Thus the CICR activity of RyR1 may be very low in mammalian skeletal muscle. This seems unfavorable for the suggestion that RyR1 could amplify Ca2+ by the CICR mechanism. The contribution of CICR via the RyR1 channels to the physiological Ca2+ release might be minor, if any; rather, RyR3 would be favorable as an amplifier of Ca2+ because it is not stabilized at all in skeletal muscle. In this sense, it seems reasonable that Ca2+ sparks are easily observed in frog skeletal muscle, where an almost equal amount of {beta}-RyR is expressed, whereas no or very few sparks are detected in adult mammalian muscles, which primarily express RyR1 (47).

Our present results also provide an interesting idea about etiology for malignant hyperthermia (MH) (26). MH is linked with a missense mutation of a single amino acid residue of RyR1 that should enhance the CICR activity. The mutated sites are largely classified into three regions, but the mutations are broadly distributed within these regions (19, 26). Hypotheses proposed to date are that these mutations should alter the sensitivity of mutated RyR1 to Ca2+/Mg2+ or to the Ca2+-releasing agents (19, 26). It would be quite interesting if the MH mutations cause destabilization of RyR1, resulting in an enhanced CICR activity as seen with CHAPS. This is consistent with the idea proposed by Yamamoto et al. (55). In pigs, it was reported that solubilization with CHAPS/phospholipids or the presence of high salt eliminated the differences in the [3H]ryanodine binding between MH and normal SR vesicles (49). Further investigations are required to clarify the mechanism and the physiological and/or pathological significance of this selective stabilization of CICR in RyR1.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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This work was supported in part by a Grant-in-Aid for Scientific Research and by a High Technology Research Center grant from the Ministry of Education, Culture, Sports, Science and Technology, Japan.


    ACKNOWLEDGMENTS
 
We thank Prof. Michio Yazawa for kindly providing the monoclonal anti-calmodulin antibody and for valuable advice and suggestions in detection and removal of contaminating calmodulin.


    FOOTNOTES
 

Address for reprint requests and other correspondence: T. Murayama, Dept. of Pharmacology, Juntendo Univ. School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan (E-mail: takashim{at}med.juntendo.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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