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RECEPTORS AND SIGNAL TRANSDUCTION

Mg²⁺ activates the ryanodine receptor type 2 (RyR2) at intermediate Ca²⁺ concentrations

¹Department of Pharmacology, Juntendo University School of Medicine, Tokyo; and ²Department of Biological Chemistry, Kyoto University Graduate School of Pharmaceutical Sciences, Kyoto, Japan

Submitted 18 May 2006 ; accepted in final form 6 September 2006

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
To clarify whether activity of the ryanodine receptor type 2 (RyR2) is reduced in the sarcoplasmic reticulum (SR) of cardiac muscle, as is the case with the ryanodine receptor type 1 (RyR1), Ca²⁺-dependent [³H]ryanodine binding, a biochemical measure of Ca²⁺-induced Ca²⁺ release (CICR), was determined using SR vesicle fractions isolated from rabbit and rat cardiac muscles. In the absence of an adenine nucleotide or caffeine, the rat SR showed a complicated Ca²⁺ dependence, instead of the well-documented biphasic dependence of the rabbit SR. In the rat SR, [³H]ryanodine binding initially increased as [Ca²⁺] increased, with a plateau in the range of 10–100 µM Ca²⁺, and thereafter further increased to an apparent peak around 1 mM Ca²⁺, followed by a decrease. In the presence of these modulators, this complicated dependence prevailed, irrespective of the source. Addition of 0.3–1 mM Mg²⁺ unexpectedly increased the binding two- to threefold and enhanced the affinity for [³H]ryanodine at 10–100 µM Ca²⁺, resulting in the well-known biphasic dependence. In other words, the partial suppression of RyR2 is relieved by Mg²⁺. Ca²⁺ could be a substitute for Mg²⁺. Mg²⁺ also amplifies the responses of RyR2 to inhibitory and stimulatory modulators. This stimulating effect of Mg²⁺ on RyR2 is entirely new, and is referred to as the third effect, in addition to the well-known dual inhibitory effects. This effect is critical to describe the role of RyR2 in excitation-contraction coupling of cardiac muscle, in view of the intracellular Mg²⁺ concentration.

[³H]ryanodine binding; CICR; stimulation by physiological Mg²⁺, excitation-contraction coupling in the heart

CICR plays a pivotal role in excitation-contraction coupling in cardiac muscle and is modulated by many endogenous and exogenous agents, including Ca²⁺, Mg²⁺, adenine nucleotides, SH-modifying agents, pH, calmodulin (CaM), and FK-506-binding protein (FKBP) 12/12.6, as well as many pharmacological reagents, including caffeine, procaine and halothane, and RyR domain peptides, which may affect interdomain interactions (8, 10, 18, 34, 37, 38). Ca²⁺ and Mg²⁺ are of primary importance among these factors. Ca²⁺ stimulates CICR at low concentrations (µM order), whereas it inhibits CICR at 0.1 mM or higher, showing biphasic Ca²⁺ dependence. This dependence is explained by the integration of the two independent effects on the activating (A-site) and inactivating (I-site) Ca²⁺ sites in the RyR molecules (13, 19, 21). In cardiac muscle, however, inhibition by a high Ca²⁺ concentration was not noticeable up to 0.1 mM, in contrast to the marked inhibition with RyR1 in skeletal muscle (5, 8, 17, 34, 37, 38). Therefore, the affinity for the divalent cation of the I-site of RyR2 was believed to be very low or negligible (5, 8, 17, 34, 37, 38). Mg²⁺ exerts an effect antagonistic to Ca²⁺ on the A-site and synergistic with Ca²⁺ on the I-site, inhibiting CICR (13, 19, 21). Therefore, Mg²⁺ shifts the pCa-CICR activity relationship to a higher Ca²⁺ concentration range (the first effect) and reduces the peak value (the second effect) (5, 6, 7, 17, 27, 35, 36, 37). An adenine nucleotide such as ATP or ,-methylene adenosine triphosphate (AMPPCP), a nonhydrolyzable ATP analog, stimulates CICR without a change in the Ca²⁺ dependence (21). The stimulation is as great as 10,000-fold (6, 27) in skeletal muscle, whereas it is not as marked (at most several-fold) in cardiac muscle (8, 18, 34, 37). For determination of [³H]ryanodine binding, a biochemical measure of CICR, in a medium simulating the cytoplasmic milieu, the addition of an adenine nucleotide is indispensable with skeletal muscle SR, whereas it is not always required with cardiac SR (5, 17, 27, 34, 35, 36). Recently, RyR1 in the SR was found to be suppressed selectively in Ca²⁺-dependent [³H]ryanodine binding (to about one-seventh), compared with RyR3 over the entire range of Ca²⁺ concentrations, without a change in its Ca²⁺ sensitivity, although purified RyR1 is very similar in activity to purified RyR3 (24, 30). A reduction in the affinity for the ligand, [³H]ryanodine, accompanied this suppression (24). The interdomain interaction between region 1 (the NH₂-terminal domain) and region 2 (the central domain) of RyR1 was found to be the main mechanism underlying the suppression (22). This interdomain interaction has also attracted the keen interest of investigators in view of its probable role in malignant hyperthermia (10).

It remains to be clarified whether suppression occurs with RyR2, whose CICR plays a central role in excitation-contraction coupling in cardiac muscle (3). This issue is also critical with respect to elucidation of the etiology of sudden death ascribed to the mutated RyR2, in which the mutation takes place in regions comparable to the sites associated with malignant hyperthermia (10). During the course of our investigation, we found that Mg²⁺ exerted a third effect of stimulating [³H]ryanodine binding in an intermediate range of Ca²⁺, roughly 10 to 100 µM, in addition to the antagonistic effect on the A-site (the first effect) and the synergistic effect on the I-site (the second effect) (13, 19, 21). In other words, Mg²⁺ relieves the partial suppression that becomes evident in the state of partial activation, usually in a limited range of Ca²⁺. The extent of this suppression was found to differ between rabbit and rat ventricular muscles.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Materials. Rabbit RyR2 cDNA was described previously (26). The expression vectors pcDNA5 FRT TO and pOG44, Flp-In T-REx-293 cells, Lipofectamine 2000 and the other reagents required for cell culture were purchased from Invitrogen (Carlsbad, CA). All other chemicals used were of reagent grade.

Cell culture and generation of an RyR2-inducible stable transfectant. Flp-In T-REx-293 cells were cultured in a high-glucose Dulbecco's modified Eagle's medium containing 10% fetal bovine serum at 37°C in a humidified CO₂ incubator. The day before transfection, 5 x 10⁵ cells were seeded onto a 12-well cell culture plate. The cells were cotransfected with two expression vectors, pOG44 and pcDNA5 FRT TO/RyR2, in a 3:1 ratio by lipofection. After 48 h, the cells were replated onto a 100-mm tissue culture dish and exposed to a selective medium containing 100 µg/ml hygromycin B. Four independent clones randomly selected among 14 surviving clones were cultured until a sufficient number of cells were grown. For expression, we usually used a 60–80% confluent cell layer, induced the protein with 1 µg/ml tetracycline, and harvested the cells after 18–20 h. Expression of RyR2 was detected in all clones by Western blot analysis.

Preparation of the SR vesicle fraction. All protocols in this study were approved by the Institutional Animal Care and Use Committee at the Juntendo University School of Medicine and were in accordance with the Guide for Care and Use of Laboratory Animals in the Institute for Laboratory Animal Research. The SR vesicle fraction was prepared as described previously, with some modifications (4). Briefly, animals (rats or rabbits) were anesthetized by intraperitoneal injection (50 mg/kg) of pentobarbital (50 mg/ml, Dainippon Seiyaku, Tokyo, Japan). Their hearts were quickly removed and perfused with 0.9% NaCl to remove the blood. In some cases, the rabbit back muscles were also used to prepare skeletal muscle SR vesicles. Both left and right ventricular muscles of animal hearts were chopped into pieces and homogenized in three volumes of distilled water containing 6.7 mM NaOH and 1 µg/ml protease inhibitor cocktail (P8340; Sigma, St. Louis, MO) in a blender-type homogenizer (AM-3; Nihonseiki Seisakusho, Tokyo, Japan), using three 15-s bursts at the maximum speed. The homogenate was centrifuged at a maximum force of 9,000 g for 20 min. The resulting supernatant was filtered with a Whatman no. 41 filter paper (Whatman, Brentford, UK), and the pellets were obtained by centrifugation at a maximum force of 27,000 g for 70 min. The pellets were resuspended in a buffer containing 50 mM KCl, 10 mM 3-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO)-KOH, pH 6.8 and 1 µg/ml protease inhibitor cocktail and repelleted. The microsomes were resuspended in the buffer supplemented with 0.18 M sucrose, quick-frozen in liquid nitrogen, and stored at –80°C until use.

Preparation of the crude endoplasmic reticulum (ER) fraction from HEK-293 cells expressing RyR2. After being washed once with PBS containing 0.1 mM CaCl₂, cells were detached from culture dishes by pipetting and collected by centrifugation at 1,400 rpm for 5 min in a TOMY (Tokyo) TS-7 rotor. Cell pellets were resuspended in PBS containing 0.1 mM CaCl₂, 0.5 mM diisopropyl fluorophosphate and 1/200 vol of protease inhibitor cocktail (P8340; Sigma) and homogenized by sonication. The homogenate was centrifuged at 1,400 rpm for 5 min (TS-7 rotor, TOMY), the supernatant was placed carefully onto a solution containing an equal volume of 0.5 M sucrose plus buffer A [50 mM KCl, 20 mM 3-(N-morpholino)propanesulfonic acid (MOPS)-KOH (pH 7.0) and 1/200 vol protease inhibitor cocktail], and recentrifuged at 25,000 rpm for 20 min in a 55T rotor in a Hitachi ultracentrifuge (Hitachi-Seisakusho, Ibaraki, Japan). The pellet was resuspended in 0.3 M sucrose plus buffer A, rapidly frozen in liquid nitrogen and then stored at –80°C until use. Typically, 10 mg of protein of the crude ER fraction was obtained from 18 culture dishes (100 mm in diameter).

[³H]ryanodine binding. [³H]Ryanodine binding was determined according to the method described by Murayama et al. (21). Briefly, the reaction was started at 25°C by the addition of [³H]ryanodine (specific activity 56 Ci/mmol; Perkin Elmer, Boston, MA) to a final concentration of 4.5 or 8.95 nM in an incubation mixture (total volume of 200 µl) containing 170 mM KCl, 20 mM MOPS-KOH (pH 7.2), CaCl₂ [sufficient to get a free Ca²⁺ concentration of less than 0.1 mM in the presence of 1 mM ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)], SR vesicles (final concentration, 0.5 mg protein/ml), and other ingredients if necessary. Ca²⁺ concentrations of 0.1 mM and higher were made by the addition of CaCl₂ alone, instead of the Ca²⁺-EGTA buffer. In experiments with Mg²⁺, MgCl₂ was added to the medium in a manner such that the SR vesicles were exposed to Mg²⁺ prior to Ca²⁺, and KCl was reduced to keep the ionic strength constant (21). The reaction was terminated after an 8-h incubation at 25°C by the addition of 3 ml ice-cold distilled water. Bound [³H]ryanodine was collected by filtration with a 5% polyethyleneimine-treated Whatman GF/B glass fiber filter under vacuum, followed by two more washes with 3 ml ice-cold water. The radioactivity of the bound [³H]ryanodine activity trapped on the filter was determined in a liquid scintillation counter (LS 6500, Beckman, Fullerton, CA). Specific [³H]ryanodine binding was obtained after correction for nonspecific binding in the presence of 10 µM unlabeled ryanodine. Free Ca²⁺ and Mg²⁺ concentrations were calculated using binding constants for divalent cations of EGTA (9) and AMPPCP (28).

SDS-PAGE and Western blot analysis. To detect RyR2, SDS-PAGE was carried out on a 2–12% polyacrylamide gradient slab gel using the discontinuous buffer system of Laemmli (12). Molecular mass markers used were myosin heavy chain (205 kDa), -galactosidase (116 kDa), phosphorylase b (97.4 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), and carbonic anhydrase (29 kDa). The electrophoresed proteins were transferred onto a PVDF membrane, and the membrane was probed with affinity-purified antibodies (1:1,000 dilutions) against NH₂-terminal or COOH-terminal RyR2 fragments (4). After washing, the membrane was incubated with secondary antibodies conjugated to horseradish peroxidase (1:3,000 dilutions, Bio-Rad, Hertfordshire, UK). Positive bands were detected by chemiluminescence (SuperSignal West Pico Substrate, Pierce, Rockford, IL) using Fujifilm RX-U (Tokyo). To detect CaM contaminating SR, SDS-PAGE and Western blot analysis were performed in a similar way and probed with a monoclonal antibody, IM7 (11), which was kindly provided by Professor Michio Yazawa, Hokkaido University.

Miscellaneous. Data are expressed as the means ± SE (number of determinations).

In the case of duplicate determinations, half the range of the variance was within the size of a symbol in most cases and was omitted from the plots for clarity. Values of P < 0.05 were considered significant by Student's t-test. Curve-fitting was made by using SigmaPlot software version 8.01 (Systat Software, Point Richmond, CA), where R² values were 0.99–0.90 in most cases.

The effects of Ca²⁺ and Mg²⁺ on [³H]ryanodine binding were analyzed according to the method of Murayama et al. (21). Briefly, the biphasic Ca²⁺ dependence can be explained by Ca²⁺ binding to the A-site and the I-site of RyR, respectively. Mg²⁺ serves as a competitive antagonist at the A-site and an agonist at the I-site. Thus, [³H]ryanodine binding, B, can be expressed by the following equation:

(1)

where B_p = the value B under the condition of f_A = 1 and f_I = 0; K_A,Ca, K_A,Mg,, K_I,Ca, and K_I,Mg are dissociation constants for Ca²⁺ and Mg²⁺ of the A- and I-sites of RyR, respectively, and n is the Hill coefficient for the corresponding site. Ca and Mg represent the concentrations of free divalent cations in the medium. In Fig. 1A, the parameters for Ca²⁺ of the A- and I-sites can be evaluated with a curve fit, using SigmaPlot software for nonlinear regression. In Fig. 1B, the parameters for Mg²⁺ of the A- and I-site can be evaluated in the presence of 3.2 µM and 0.1 or 1 mM Ca²⁺, respectively, in similar ways as described above. In Table 1, the dissociation constants for divalent cations at the A- and I-sites are summarized in the format of average ± SE (number of determinations). With rat cardiac SR, the apparent dissociation constants for Ca²⁺ are summarized in the presence of 0.3 mM Mg²⁺, where a biphasic Ca²⁺ dependence was obtained, as shown in Fig. 2.

View larger version (21K): [in this window] [in a new window]   Fig. 1. pCa-[3H]ryanodine-binding activity in rabbit and rat ventricular sarcoplasmic reticulum (SR) and effects of Mg2+. A and B, rabbit ventricular SR; C and D, rat ventricular SR. A and C show the pCa-[3H]ryanodine-binding relationship in the absence of Mg2+. B and D show the effects of Mg2+ on [3H]ryanodine binding in the presence of 3.2 µM (squares), 0.1 mM (triangles) and 1 mM (circles) Ca2+. The reaction medium contains 0.17 M KCl, 20 mM MOPS-KOH, pH 7.2, 0.5 mg SR protein/ml, 8.95 nM [3H]ryanodine and indicated concentrations of Ca2+ and Mg2+. Averages of duplicate determinations were plotted. Because half the range of the variance of determinations was within the size of a symbol in most points, it was omitted for clarity. This is also the case with the other figures, in which duplicate determinations were made in this manuscript. Curves in A and B were drawn by curve fitting to Eq. 1, but those in C and D were drawn arbitrarily by connecting data points smoothly. The curves in the following figures were drawn arbitrarily unless otherwise specified.

View larger version (12K): [in this window] [in a new window]   Fig. 2. Effect of Mg2+ on the pCa-[3H]ryanodine-binding relationship of rat ventricular SR. Open and closed circles represent results in the absence and presence of 0.3 mM Mg2+, respectively. Note that the complicated pCa-binding relationship in the absence of Mg2+ was converted into a biphasic Ca2+-dependence in the presence of 0.3 mM Mg2+, and the bound [3H]ryanodine was increased in the range of 10–300 µM Ca2+. The experimental conditions were similar to those in Fig. 1. Averages of duplicate determinations were plotted. The biphasic curve was drawn using a set of parameters described in the text.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In contrast, rat ventricular SR vesicles showed a peculiar Ca²⁺ dependence in [³H]ryanodine binding: it increased with increases in Ca²⁺ concentration up to around 10 µM, where a plateau was formed up to 100 µM. Thereafter, it increased further, and a higher peak was attained at about 1 mM Ca²⁺, followed by a decrease at higher Ca²⁺ concentrations (Fig. 1C). [³H]ryanodine binding in the presence of 3.2 µM and 1 mM Ca²⁺ was monotonically decreased by Mg²⁺, as shown in Fig. 1D. In the presence of 0.1 mM Ca²⁺, however, it surprisingly increased in the presence of low Mg²⁺ concentrations, and the decrease was not obvious until Mg²⁺ concentrations were 3 mM or higher. This Mg²⁺ dependence of rat SR is in marked contrast to rabbit SR, where the monotonic decrease was observed even in the presence of 0.1 mM Ca²⁺.

The third effect of Mg²⁺. As shown in Fig. 1D, [³H]ryanodine binding to rat ventricular SR in the presence of 0.1 mM Ca²⁺ was enhanced by 0.3 mM Mg²⁺. To get a deeper understanding of the finding, we determined the pCa-[³H]ryanodine-binding relationship in the presence of 0.3 mM Mg²⁺ (Fig. 2). The relationship became biphasic, which was familiar to us, giving an apparent K_A,Ca of 6.46 µM and K_I,Ca of 1.66 mM, as shown in Fig. 2. In the presence of Ca²⁺ concentrations less than 10 µM, Mg²⁺ shifted the curve rightward, whereas in the presence of Ca²⁺ concentrations higher than 1 mM, the shift was leftward. With further increases in Mg²⁺ concentrations, the stimulating curve was shifted more to the right along with the Ca²⁺ concentrations up to 0.1 mM, and the decreasing curve was shifted more to the left with decreased peak values, although the shift was minor in magnitude (data not shown). These effects were consistent with the predictions of a competitive antagonist effect for Ca²⁺ at the A-site and a synergistic effect with Ca²⁺ at the I-site. In the presence of 0.1 mM Mg²⁺, however, the pCa-binding relationship remained multiphasic, as was the case without Mg²⁺, although the binding was slightly increased.

Surprisingly, Fig. 2 shows that Mg²⁺ enhanced [³H]ryanodine binding in the plateau range of 10–100 µM Ca²⁺. This enhancing effect of Mg²⁺ is entirely new, and is referred to as the third effect of Mg²⁺ (in addition to its dual inhibitory effects).

It should be explained that rabbit RyR2 expressed in HEK-293 cells showed a multiphasic Ca²⁺ dependence in the presence of Ca²⁺ alone (Fig. 3). The presence of Mg²⁺ caused biphasic Ca²⁺ dependence with enhancement of [³H]ryanodine binding in the range of 10–300 µM Ca²⁺ (Fig. 3A). The dose-dependent effect of Mg²⁺ in the presence of 0.1 mM Ca²⁺ was upwardly convex, as is the case with rat cardiac SR: an increase with increases in Mg²⁺ followed by a decrease (Fig. 3B, compare with Fig. 1D). Although they presented similar results, Meissner and his colleagues (2, 40) made no comment on this matter. Rabbit RyR2, therefore, intrinsically has the potential to show multiphasic Ca²⁺ dependence.

View larger version (12K): [in this window] [in a new window]   Fig. 3. [3H]Ryanodine binding to rabbit RyR2 expressed heterologously in HEK-293 cells. A: pCa-[3H]ryanodine-binding relationship. Open circles, in the absence of Mg2+; closed circles, in the presence of 1 mM Mg2+. The curve in the presence of Mg2+ was fit to Eq. 1 (Bp = 1.34 pmol/mg protein, apparent KA,Ca = 8.51 µM, nA,Ca = 1.85, apparent KI,Ca = 6.46 mM, nI,Ca = 1.40; R2 = 0.9993). B: Mg2+ dependence of [3H]ryanodine binding in the presence of 0.1 mM Ca2+. Experiments were carried out as in Figs. 1 and 2, except with 0.05 mg protein/ml ER fraction. Averages of duplicate determinations were plotted.

View larger version (14K): [in this window] [in a new window]   Fig. 4. Rabbit ventricular SR shows a rat-type Ca2+ dependence in the presence of AMP. Circles, in the absence of AMP; squares, with 3 mM AMP. Open and closed symbols represent determinations in the absence and presence of 0.3 mM Mg2+, respectively. The experimental conditions were similar to those in Fig. 1. Averages of duplicate determinations were plotted. The two biphasic curves were fitted to Eq. 1.

View larger version (14K): [in this window] [in a new window]   Fig. 5. Effect of caffeine on [3H]ryanodine binding to rabbit ventricular SR (A) and on its Mg2+ dependence in the presence of 0.1 mM Ca2+ (B). A: experiments were carried out as in Fig. 4, but using caffeine instead of AMP. Circles, without caffeine; triangles, with 10 mM caffeine. Open and closed triangles represent determinations in the absence and presence of 0.3 mM Mg2+, respectively. B: Mg2+ dependence also became a rat-like in the presence of caffeine. Open circles, without caffeine; open triangles, with 10 mM caffeine. See also Fig. 1B. Averages of duplicate determinations were plotted.

Effects of CICR modulators in the presence of 0.3 mM Mg²⁺. So far, the activity of RyR2 at 0.1 mM Ca²⁺ appeared as if it would freeze in the absence of Mg²⁺ and be relieved by 0.3 mM Mg²⁺. Figure 6 shows the dose-dependent effect of caffeine on [³H]ryanodine binding to rat ventricular SR in the presence of 0.1 mM Ca²⁺. In the absence of Mg²⁺, caffeine did not appreciably increase this binding. Addition of Mg²⁺ increased the binding up to twofold. Caffeine showed a dose-dependent enhancement in the presence of 0.3 mM Mg²⁺, and the maximum effect was attained at 3–10 mM of caffeine. A higher concentration of caffeine was somewhat inhibitory. The summary of this stimulatory effect of 10 mM caffeine at 0.1 mM Ca²⁺ with and without 0.3 mM Mg²⁺ is shown with the number of determinations in Fig. 7. Figure 7 also shows similar results with various other kinds of stimulators and inhibitors. They were examined at their optimum concentrations, which were found to be much lower with RyR2 compared with RyR1 of skeletal muscle. The amounts of bound [³H]ryanodine changed little in the absence of Mg²⁺, whereas they were greatly affected in the presence of Mg²⁺, as shown in Fig. 7 (see also Fig. 6). In other words, Mg²⁺ makes RyR2 more reactive to various agents. The enhancing effect of AMPPCP in the absence of Mg²⁺ was greater than that of caffeine, as is true with RyR1. In the presence of Mg²⁺, it was more remarkable. Whereas the stimulatory effect of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), which was used to remove the selective suppression of RyR1, was maximum at 2% on mammalian skeletal muscles (22), the maximum effect was obtained at 0.1%-0.2% with rat ventricular SR. CHAPS at 1% was rather inhibitory to rat or rabbit RyR2 (data not shown). It should be pointed out that the stimulatory effect of CHAPS was also dependent on the presence of Mg²⁺. FK-506, which deprives RyR of FKBP 12/12.6, is reported to increase the activity of RyR. FK-506, however, did not affect [³H]ryanodine binding in the absence or presence of Mg²⁺, excluding the possibility of the involvement of FKBP 12/12.6 in the third effect of Mg²⁺ (Fig. 7). FK-506 treatment did not affect the responses of RyR2 to caffeine or AMPPCP, either (data not shown).

View larger version (12K): [in this window] [in a new window]   Fig. 6. Mg2+ is necessary for increases by caffeine in [3H]ryanodine binding to rat ventricular SR. Closed triangles and open circles show determinations at 0.1 mM Ca2+ in the presence and absence of 0.3 mM Mg2+, respectively. The experimental conditions were similar to those in Fig. 1, except with 4.5 nM [3H]ryanodine. Note that the concentrations of caffeine are plotted on a logarithmic scale along the abscissa. Averages of duplicate determinations were plotted.

View larger version (17K): [in this window] [in a new window]   Fig. 7. Stimulation by Mg2+ of known effects of modulators on rat ventricular SR. As shown in Fig. 6, the magnitudes of changes caused by a modulator were plotted in the absence of Mg2+ (open columns) and in the presence of 0.3 mM Mg2+ (hatched columns). Statistical evaluation was made between pairs for each modulator. With ,-methylene ATP (AMPPCP), 4 mM total MgCl2 and 4 mM total nucleotide were added, giving rise to about 0.8 mM free Mg2+. The controls without any reagent listed below in the presence of 4.5 nM [3H]ryanodine were 0.15 ± 0.006 (16) and 0.31 ± 0.006 (16) pmol/mg protein in the absence and presence of Mg2+, respectively. With 8.95 nM [3H]ryanodine, where the effect of FK-506 was examined, they were 0.31 ± 0.008 (4) and 0.56 ± 0.017 (4) pmol/mg protein, respectively. The numbers in parentheses represent the number of determinations, and the vertical bars indicate SE. The experimental conditions were similar to those in Fig. 6, and indicated reagents were added as follows: CAF (10 mM caffeine), AMPPCP (4 mM AMPPCP), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) (0.1% CHAPS), FK-506 (10 µM FK-506), PRO (1 mM procaine), and CaM (1 µM CaM). P < 0.01 and P < 0.001.

In conclusion, the presence of 0.3 mM Mg²⁺ greatly amplified the responses of RyR2, not only to stimulatory reagents, but also to inhibitory ones, with the exception of FK-506.

RyR2 is in a more activated state in the presence of Mg²⁺. In the standard assay medium containing 0.17 M KCl and 0.1 mM Ca²⁺, [³H]ryanodine binding to rat ventricular SR was determined in the presence and absence of 0.3 mM Mg²⁺ at varied concentrations of the ligand (Fig. 8). As shown in Fig. 8, the results can be explained by the following parameters: B_max = 2.09 pmol/mg protein, K_d = 33.3 nM in the absence of Mg²⁺, and B_max = 1.94 pmol/mg protein, K_d = 16.0 nM in the presence of 0.3 mM Mg²⁺. It should be explained that K_d would be 1–3 nM if determined in a medium containing 1 M KCl as reported (8, 18, 34, 37, 38). These results suggest that the single class of binding sites is homogeneous and that Mg²⁺ increased the affinity for the ligand without a change in the maximum number of these binding sites (B_max). The increased affinity of RyR2 for the ligand indicates that RyR2 is more activated in the presence of Mg²⁺. The results of determinations in the medium containing 0.17 M NaCl instead of KCl (Fig. 9) may help to deepen our understanding of the underlying mechanism of the third effect of Mg²⁺. As shown in Fig. 5A, rabbit ventricular SR showed a multiphasic Ca²⁺ dependence in the presence of 10 mM caffeine in the 0.17 M KCl medium. In the 0.17 M NaCl medium, however, the Ca²⁺ dependence was biphasic, with a broader plateau (1–1,000 µM Ca²⁺), even in the presence of 10 mM caffeine. The findings that Ca²⁺ dependence of [³H]ryanodine binding was changed by monovalent ions were already reported with RyR1 (20, 23) and with RyR2 (15). The results in Fig. 9 fit the following parameters: in the absence of caffeine or Mg²⁺ (open circles), B_p= 0.42 pmol/mg protein, K_A,Ca = 3.89 µM, n_A,Ca = 1.46, K_I,Ca = 1.91 mM, and n_I,Ca = 1.25, whereas in the presence of 10 mM caffeine without Mg²⁺ (open diamonds), B_p = 0.64 pmol/mg protein, K_A,Ca = 0.22 µM, n_A,Ca = 1.50, K_I,Ca = 6.31 mM, and n_I,Ca = 1.69. The effects of caffeine in 0.17 M NaCl medium would be described as follows: Ca²⁺ sensitization as great as 18-fold, increase in B_p, i.e., increase in CICR activity, and decrease in the affinity of the I-site for Ca²⁺ with the increase in the Hill coefficient. These effects of caffeine are consistent with the results already reported (1, 6, 18, 27, 29). The supplement of 0.3 mM Mg²⁺ (closed diamonds) further increased B_p (0.92 pmol/mg protein) and restored the affinity of the I-site for Ca²⁺(K_A,Ca = 0.40 µM, n_A,Ca = 1.63, K_I,Ca = 1.78 mM, n_I,Ca = 0.93). The increase in B_p was due to the increase in the affinity for the ligand, as shown in Fig. 8. These findings indicate that RyR2 may be susceptible to the allosteric change in the conformation and that coexistence of 0.3–1 mM Mg²⁺ is necessary for RyR2 to function properly.

View larger version (14K): [in this window] [in a new window]   Fig. 8. Effects of Mg2+ on dose-dependent [3H]ryanodine binding to rat ventricular SR at 0.1 mM Ca2+ (A) and their Scatchard plots (B). Open circles, in the absence of Mg2+ and closed triangles, in the presence of Mg2+. Determinations were made in medium containing 0.17 M KCl and varied amounts of [3H]ryanodine. The curves were drawn according to the usual binding curve using the following parameters, which were determined by Scatchard plots (B): Bmax = 2.09 pmol/mg protein, Kd = 33.3 nM (without Mg2+) and Bmax = 1.94 pmol/mg protein, Kd = 16.0 nM (with 0.3 mM Mg2+). The experimental conditions were similar to those in Fig. 2, except for the use of 0.1 mM Ca2+ and varied concentrations of [3H]ryanodine. Averages of duplicate determinations were plotted.

View larger version (15K): [in this window] [in a new window]   Fig. 9. NaCl instead of KCl affects the pCa-[3H]ryanodine-binding relationship. Rabbit ventricular SR vesicles were used for determinations in medium containing 0.17 M NaCl. Compare with Fig. 5A, where KCl was used as the main salt. Open circles, no extra additions; open diamonds, in the presence of 10 mM caffeine, but no Mg2+; closed diamonds, in the presence of 10 mM caffeine and 0.3 mM Mg2+. Averages of duplicate determinations were plotted. The curves were drawn using the sets of parameters described in the text.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Table 1 also summarizes the results of multiple similar determinations with rat ventricular SR in the presence of 0.3 mM Mg²⁺. Because Fig. 1, B and D, showed similar effects of Mg²⁺ on [³H]ryanodine binding in the presence of 3.2 µM and 1 mM Ca²⁺, the inhibitory effects of Mg²⁺ on rat ventricular SR may be similar to those for rabbit ventricular SR. The results shown in Table 1 indeed indicate that affinities of the A-site and the I-site for Ca²⁺ and Mg²⁺ would be similar between rabbit and rat ventricular SRs. Table 1 also indicates that the properties of frog skeletal muscles may differ from those of rabbit skeletal muscles.

The third, stimulatory effect of Mg²⁺ on RyR2. Rabbit RyR2 in the ventricular SR shows biphasic Ca²⁺ dependence in the presence of Ca²⁺ alone. The effect of Mg²⁺ on rabbit RyR2 is due to competitive antagonism to Ca²⁺ at the A-site and synergistic action with Ca²⁺ at the I-site (Fig. 1, A and B, and Table 1) (1, 13, 19, 21). Rat RyR2 in the ventricular SR, in contrast, showed a peculiar multiphasic Ca²⁺ dependence, with a plateau of an intermediate level of bound [³H]ryanodine in the range of 10–100 µM Ca²⁺ (Fig. 1, C and D). The presence of Mg²⁺ at 0.3 mM or higher converted the complexed Ca²⁺ dependence to a biphasic relationship, with enhanced [³H]ryanodine binding in the intermediate range of Ca²⁺ (Fig. 2). This biphasic Ca²⁺ dependence in the presence of Mg²⁺ can be explained by affinities for Ca²⁺ and Mg²⁺ of the A-site and I-site, which are similar to those of rabbit ventricular SR (Table 1). In the presence of an adenine nucleotide or caffeine, the multiphasic Ca²⁺ dependence with the intermediate level of the plateau was observed, irrespective of rabbit or rat RyR2. Mg²⁺ was found to exhibit the third effect on RyR2, i.e., enhanced binding in the intermediate Ca²⁺ concentrations in addition to the actions on the A- and I-sites (Figs. 2–5). The third, stimulatory effect of Mg²⁺ on RyR2 is dose-dependent, and its minimum concentration was about 0.3 mM. This prevents us from observing the third effect, if any, with RyR1 or RyR3, which has a K_I,Mg of around 0.2 mM (Table 1). Only RyR2, which has a large K_I,Mg of 2–3 mM, can be partly immune to the inhibitory effect of the I-site (Table 1). Furthermore, the third effect of Mg²⁺ is likely to be independent of the dual inhibitory effects and additive to them.

In the SR membrane of skeletal muscles, on the other hand, the activity of RyR1 is selectively suppressed to about one-seventh over the whole range of Ca²⁺ concentrations compared with RyR3, as evidenced by a reduced affinity for [³H]ryanodine to a comparable extent without a change in Ca²⁺ dependence (22, 24, 30). This means that the attenuating coefficient (1/7–1/8) for RyR1 is constant over the whole range of Ca²⁺ concentrations, without a change in the affinities for the divalent cations of the A- or I-site. The interdomain interaction between region 1 and region 2 in RyR1 is suggested to be involved primarily in the selective suppression (22). RyR2, in contrast to RyR1, appears to freeze to the state of an intermediate activity in the range of 10–100 µM Ca²⁺. In this sense, RyR2 is subjected to the partial suppression, which is removed by Mg²⁺. Because CHAPS stimulated RyR2 (Fig. 7), an interdomain interaction such as within RyR1 could be considered, although it is not identical.

Purified RyR2 showed monophasic Ca²⁺ dependence without inhibition by high Ca²⁺, independent of animal sources. Similar monophasic Ca²⁺ dependence was observed in 1 M KCl-containing medium with cardiac SR vesicles (8, 18, 34, 37, 38). These results might strongly suggest the involvement of some accessory proteins in multiphasic Ca²⁺ dependence. In these cases, however, the activation by Ca²⁺ was very steep, nearly of an all-or-none type. Because the Hill coefficient for the activating Ca²⁺ was near 2 with RyR2 in the SR, the allosteric conformation may be changed after purification. Yamaguchi et al. (40) reported that inhibition by CaM can be detected by lipid bilayer experiments only under a charged voltage difference, but not by [³H]ryanodine binding. The effect of CaM may be dependent on the conformation of RyR molecules, as reported by Rodney et al. (32). Addition of exogenous CaM to crude SR intriguingly reduced [³H]ryanodine binding to one-third in the presence of 0.3 mM Mg²⁺, but caused no inhibition in the absence of Mg²⁺ (Fig. 7). It could be hypothesized that added Mg²⁺ would bind to CaM to remove the inhibitory effect. The stimulatory effect of Mg²⁺ on RyR2, however, was observed in a limited range of Ca²⁺ concentrations, whereas the inhibitory effect of CaM on RyR2 was homogeneous over the whole range of Ca²⁺ concentrations (2, 18, 40). CaM (17), therefore, is excluded from the list of candidates for the responsible cofactor. Treatment of the SR with FK-506 did not change [³H]ryanodine binding in the presence or absence of Mg²⁺(Fig. 7). Washing of rat SR with 0.17 M KCl medium containing 1 mM EGTA, which is effective to deprive sorcin of RyR2 (16), did not change the multiphasic Ca²⁺ dependence (data not shown). These findings suggest that either FKBP (38) or sorcin (16) would play a minor role in the third effect of Mg²⁺. The possibility of other unidentified proteins cannot be excluded, and further investigations are required to clarify the underlying mechanism of the third effect of Mg²⁺.

The third effect of Mg²⁺ was not detectable at 0.1 mM, but was clearly observed at 0.3 mM or more. Correspondingly, the second increase of [³H]ryanodine binding in multiphasic Ca²⁺ dependence in the absence of Mg²⁺, was observed at 0.3 mM Ca²⁺ and higher, with the apparent peak around 1 mM Ca²⁺ (Figs. 1C and 2–5). These findings strongly suggest that Ca²⁺ and Mg²⁺ are of equal importance in the third effect.

The regulation will be dependent on the extent of activation of RyR2 molecules, but not on the Ca²⁺ concentration itself, because the plateau of [³H]ryanodine binding started at a lower Ca²⁺ concentration in the presence of caffeine with the unchanged plateau level. The results in Figs. 4 and 5A show that the intermediate plateau with rabbit ventricular SR was gradually increased as Ca²⁺ increased up to 100 µM in the absence of Mg²⁺, whereas it was not appreciably changed with rat ventricular SR. This gradual increase in the plateau level, with the increase in Ca²⁺, was also observed with rabbit RyR2 expressed in HEK-293 cells (Fig. 3). These results may suggest that rabbit RyR2 is more vulnerable to the third effect than rat RyR2 or that rabbit RyR2 may be looser in its regulation than rat RyR2. More comparative studies are required, because it is well known that species specificity is great even among mammalian hearts.

Physiological relevance of the current findings. It has so far been claimed that no inhibition of CICR at high Ca²⁺ concentrations is characteristic of RyR2 (3, 5, 8, 17, 34, 37). We showed clearly the effectiveness of the I-site, although RyR2 shows a lower affinity than RyR1 and RyR3 (Table 1). One explanation for this discrepancy is the difference in experimental conditions: a high salt-medium, 1 M KCl, was used instead of 0.17 M KCl, and/or purified RyR2 was used for the experiments (8, 14, 15, 18, 34, 37). Another explanation is the limited range of Ca²⁺ concentrations of up to pCa 4 (14, 15). Another intriguing determination was made by Xu et al. (39) in which 5 mM Mg-AMPPCP was used. In this case, the free Mg²⁺ concentration inevitably amounts to about 0.8 mM, which certainly would exert the third effect.

Mg²⁺ makes RyR2 sensitive to well-known modulators (Fig. 7); therefore, the presence of Mg²⁺ is inevitable with experiments on RyR2. In the absence of Mg²⁺, the partial suppression of RyR2 may be obvious. In the presence of Mg²⁺, RyR2 is very similar among mammalian hearts by removal of the suppression. The physiological intracellular concentration of Mg²⁺ in cardiac muscle is assumed to be 0.5–1 mM (3, 20, 25), which is sufficient for the stimulatory effect of Mg²⁺ on RyR2. Mg²⁺, on the other hand, will move the pCa-activity curve about 0.5–0.7 pCa unit to a higher Ca²⁺ concentration range. The several-fold enhancement of the activity will compensate for this handicap in the presence of Mg²⁺ and ATP. The presence of Mg²⁺ at the physiological intracellular concentration is essential for RyR2 to play a key role in excitation-contraction coupling of cardiac muscle.

	ACKNOWLEDGMENTS

 
We thank Drs. N. Kurebayashi, T. Murayama, and T. Kashiyama of Juntendo University for suggestions and encouragement.

Present address of O. Sato: Dept. of Physiology, University of Massachusetts Medical School, 55 Lake Ave. North, Worcester, MA 01655.

	FOOTNOTES

 

Address for reprint requests and other correspondence: A. Chugun, Dept. of Pharmacology, Juntendo Univ. School of Medicine, 2–1-1 Hongo, Bunkyo-ku, Tokyo 113–8421, Japan (e-mail: chugun{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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