Dantrolene Inhibition of Ryanodine Receptor Ca2+ Release Channels

MOLECULAR MECHANISM AND ISOFORM SELECTIVITY*

Fangyi ZhaoDagger , Pin Li§, S. R. Wayne Chen§, Charles F. LouisDagger , and Bradley R. FruenDagger ||

From the Dagger  Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455 and the § Department of Physiology and Biophysics, University of Calgary, Alberta, T2N 4N1, Canada

Received for publication, July 11, 2000, and in revised form, January 11, 2001




    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

As an inhibitor of Ca2+ release through ryanodine receptor (RYR) channels, the skeletal muscle relaxant dantrolene has proven to be both a valuable experimental probe of intracellular Ca2+ signaling and a lifesaving treatment for the pharmacogenetic disorder malignant hyperthermia. However, the molecular basis and specificity of the actions of dantrolene on RYR channels have remained in question. Here we utilize [3H]ryanodine binding to further investigate the actions of dantrolene on the three mammalian RYR isoforms. The inhibition of the pig skeletal muscle RYR1 by dantrolene (10 µM) was associated with a 3-fold increase in the Kd of [3H]ryanodine binding to sarcoplasmic reticulum (SR) vesicles such that dantrolene effectively reversed the 3-fold decrease in the Kd for [3H]ryanodine binding resulting from the malignant hyperthermia RYR1 Arg615 right-arrow Cys mutation. Dantrolene inhibition of the RYR1 was dependent on the presence of the adenine nucleotide and calmodulin and reflected a selective decrease in the apparent affinity of RYR1 activation sites for Ca2+ relative to Mg2+. In contrast to the RYR1 isoform, the cardiac RYR2 isoform was unaffected by dantrolene, both in native cardiac SR vesicles and when heterologously expressed in HEK-293 cells. By comparison, the RYR3 isoform expressed in HEK-293 cells was significantly inhibited by dantrolene, and the extent of RYR3 inhibition was similar to that displayed by the RYR1 in native SR vesicles. Our results thus indicate that both the RYR1 and the RYR3, but not the RYR2, may be targets for dantrolene inhibition in vivo.




    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Ryanodine receptors (RYRs)1 are intracellular Ca2+ channels specialized for the rapid and massive release of Ca2+, as is necessary for excitation-contraction (EC) coupling in striated muscle (1). Three different RYR isoforms have been identified in mammalian tissues: the RYR1, which is predominantly expressed in skeletal muscle; the RYR2, which is predominantly expressed in cardiac muscle; and the RYR3, which is expressed at comparatively low levels in a variety of tissues, including the brain. Because RYR channels play critical roles in the diverse physiologic and pathophysiologic cell processes that are controlled by Ca2+ release from intracellular stores (2), these channels represent potentially important pharmacologic targets for modulating cell regulation (3). However, the key functional properties that may distinguish the different RYR isoforms remain unclear (4, 5), and few if any drugs are known to act as isoform-specific modulators of these channels (3).

To date, the muscle relaxant dantrolene remains the only drug targeting RYR channels that is used clinically (3, 5). Early investigations indicated that dantrolene may act selectively on the physiologic mechanism responsible for activating Ca2+ release from the sarcoplasmic reticulum (SR) during skeletal muscle EC coupling (6-8). Accordingly, dantrolene (~10 µM) shifts the sensitivity of contractile activation to higher voltages and reduces the skeletal muscle twitch force by more than half (7, 8). Dantrolene inhibition of SR Ca2+ release in skeletal muscle has provided a lifesaving treatment for the pharmacogenetic disorder malignant hyperthermia (MH). Thus, the uncontrolled SR Ca2+ release, muscle contracture, and accelerated metabolism that threaten the MH-susceptible (MHS) patient exposed to volatile anesthetics during surgery are effectively suppressed upon treatment with dantrolene (9, 10). Dantrolene also reverses the increased sensitivity of MHS muscle to activation by caffeine (11), which constitutes the basis of in vitro diagnostic tests of this syndrome (12, 13). The efficacy of dantrolene in the treatment of MH is in large part a function of the selective action of this drug on SR Ca2+ release in skeletal muscle, while exerting no comparable negative inotropic effect on the beating heart (6, 10, 14). Notably, the absence of major effects of dantrolene on SR Ca2+ release in the heart is consistent with the possibility that dantrolene may act selectively on the RYR1 but not the RYR2 channel isoform (15). At the same time, it is also clear that the effects of dantrolene on Ca2+ release from intracellular stores are not strictly limited to skeletal muscle but extend to certain nonmuscle cells, including central neurons (16-19). The basis of the effects of dantrolene in nonmuscle cells remains unclear, however, because of uncertainty regarding the precise molecular mechanism of dantrolene inhibition and the selectivity of this mechanism for different Ca2+ release channel isoforms.

In a previous report (15), we demonstrated that dantrolene acts directly on the RYR1 to reduce the extent of channel activation by calmodulin (CaM) and thereby decreases the Ca2+ sensitivity of channel activation. Here we further define the mechanism and isoform selectivity of dantrolene as an inhibitor of RYR channels.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

MHS pigs homozygous for the RYR1 Arg615 right-arrow Cys mutation were obtained from the University of Minnesota Experimental Farm; normal control animals were obtained from commercial suppliers. Skeletal muscle SR vesicles were prepared from longissimus dorsi muscles of MHS and normal pigs, and cardiac SR vesicles were prepared from porcine ventricular tissue as described previously (20, 21). All isolation buffers contained a mixture of protease inhibitors (100 nM aprotinin, 1 µM leupeptin, 1 µM pepstatin, 1 mM benzamidine, and 0.2 mM phenylmethylsulfonyl fluoride). [3H]Ryanodine was purchased from PerkinElmer Life Sciences, and nonradioactive ryanodine was purchased from Calbiochem. Dantrolene, porcine brain CaM, and AMPPCP (a nonhydrolyzable ATP analog) were from Sigma. Azumolene (a water-soluble analog of dantrolene) was manufactured by Procter & Gamble and provided by Dr. Esther Gallant (University of Minnesota). Dantrolene stock solutions (1 mM) were prepared fresh every 1-2 days in 50% methanol (0.5% methanol final concentration) and stored in the dark at room temperature.

The cloning and sequencing of the full-length cDNA encoding the mouse cardiac RYR2 (22) and the rabbit uterus RYR3 (23) have been described previously. RYR clones were transiently expressed in HEK-293 cells following transfection by Ca2+ phosphate precipitation. HEK-293 whole-cell lysates were prepared as described (24) in buffer containing 137 mM NaCl, 25 mM Tris/HEPES (pH 7.4), 1% CHAPS, and 0.5% phosphatidylcholine. [3H]Ryanodine binding to SR vesicles (2 mg/ml) or HEK-293 cell lysates (1.4-1.9 mg/ml) was determined following ~10-min preincubations in 37 °C media containing 120 mM potassium propionate, 10 mM PIPES (pH 7.0), 2 mM Na2AMPPCP, 1 µM CaM, and either 10 µM dantrolene or methanol vehicle. Following the addition of [3H]ryanodine (20 nM), samples were incubated for 90 min at 37 °C and then collected on Whatman glass fiber filters with three 3-ml washes with ice-cold 100 mM KCl. RYR expression levels in HEK-293 cells were quantitated via determinations of the maximal [3H]ryanodine binding capacity of cell lysates in media containing 600 mM KCl, 10 mM Na2ATP, and 100 µM Ca2+ (0.01 ± 0.009 pmol/mg for mock-transfected controls; 0.85 ± 0.06 pmol/mg for RYR2-expressing cells; 0.68 ± 0.5 pmol/mg for RYR3-expressing cells). Data were corrected for nonspecific binding determined in the presence of 100-fold excess nonradioactive ryanodine. Concentrations of ionized Ca2+ were obtained using calcium acetate-EGTA buffers (25).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Dantrolene Alters the Kd of [3H]Ryanodine Binding to the MHS and Normal RYR1-- Previous results from our laboratory demonstrated that dantrolene inhibition of skeletal muscle SR vesicle Ca2+ release was associated with reduced levels of [3H]ryanodine binding to the isolated RYR1 (15) and thus indicated that dantrolene may act directly at the RYR1 to inhibit channel activation. Other laboratories, however, have proposed that the effects of dantrolene on SR Ca2+ release may be mediated via its binding to non-RYR proteins (26, 27) and have questioned whether the magnitude of the actions of dantrolene on [3H]ryanodine binding in vitro may adequately explain the clinical effects of this drug. The current experiments therefore sought to further define the mechanism and selectivity of the actions of dantrolene on [3H]ryanodine binding and to determine whether this mechanism might reasonably account for the in vivo actions of dantrolene in reversing MH. The experiments shown in Fig. 1 compared the effects of dantrolene and the pig MHS mutation on the Kd and Bmax of [3H]ryanodine binding to skeletal muscle SR vesicles. The concentration of dantrolene in these experiments was 10 µM, a concentration that approximates therapeutic drug levels in vivo (28) and at which the inhibition of SR vesicle [3H]ryanodine binding by dantrolene was maximal (15). In the absence of dantrolene, the Kd for [3H]ryanodine binding to MHS SR vesicles was approximately one-third of that for normal SR vesicles (Table I), consistent with the magnitude of the effect of the MHS mutation on [3H]ryanodine binding affinity as documented previously (20). In comparison, the Kd for the binding of [3H]ryanodine to both MHS and normal SR vesicles was increased ~3-fold in the presence of dantrolene. Neither the MHS mutation nor dantrolene significantly altered the Bmax of SR vesicle [3H]ryanodine binding. Consequently, [3H]ryanodine binding to MHS SR vesicles in the presence of dantrolene was essentially equivalent to that of normal SR vesicle [3H]ryanodine binding in the absence of dantrolene over the range of the ryanodine concentrations examined (Fig. 1). These results thus demonstrate that dantrolene inhibition of the RYR1 was associated with a reduced affinity of the channel for [3H]ryanodine and further indicate that the magnitude of the effect of dantrolene on [3H]ryanodine binding was comparable with that of the MHS Arg615 right-arrow Cys mutation.



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Fig. 1.   Scatchard analysis of [3H]ryanodine binding to MHS and normal skeletal muscle SR vesicles in the presence or absence of dantrolene. The binding of [3H]ryanodine (10-500 nM) to skeletal muscle SR vesicles was determined in the presence (filled symbols) or absence (open symbols) of 10 µM dantrolene in media containing 120 mM potassium propionate, 10 mM PIPES (pH 7.0), 2 mM Na2AMPPCP, 1 µM CaM, and 100 nM Ca2+. Inset, fits of the data to a single rectangular hyperbola used for determinations of Kd and Bmax (Table I). Data are means ± S.E. from five independent experiments comparing four MHS (squares) and four normal (circles) skeletal muscle SR vesicle preparations.


                              
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Table I
Effect of dantrolene on SR vesicle [3H]ryanodine binding
[3H]Ryanodine binding was determined as described under "Experimental Procedures" in the absence or presence of 10 µM dantrolene. Media contained 120 mM potassium propionate, 10 mM PIPES (pH 7.0), 2 mM Na2AMPPCP, 1 µM CaM, and 100 nM Ca2+, except as otherwise indicated at left in the table. Kd and Bmax determinations are based on fits of the data presented in Figs. 1, 4B, and 5 to a single rectangular hyperbola.

Effect of Dantrolene on RYR1 Sensitivity to Caffeine, Mg2+, and Sr2+-- Previous results demonstrated that dantrolene decreased the sensitivity of the isolated RYR1 to activation by Ca2+ (15) and thereby suggested that a reduced affinity of Ca2+ binding to RYR1 activation sites may constitute the basis of dantrolene inhibition of Ca2+ release in skeletal muscle. To further examine this possibility, we determined the effect of dantrolene on the sensitivity of RYR1 to other effectors known to modulate RYR activation by Ca2+. Caffeine, for example, is known to activate the RYRI by increasing the Ca2+ sensitivity of the channel (29). Furthermore, a reduced threshold for caffeine activation is characteristic of MHS RYR1 mutations at diverse sites within the primary sequence of this channel protein (12). Accordingly, Fig. 2 indicates that the threshold for the caffeine activation of [3H]ryanodine binding to MHS SR was decreased relative to that of normal SR, and the EC50 for the caffeine activation of MHS SR was reduced by approximately one-half (Table II). Conversely, dantrolene (10 µM) shifted the caffeine threshold for the activation of [3H]ryanodine binding to higher caffeine concentrations and increased the EC50 for the caffeine activation of both MHS and normal SR vesicles by >= 2-fold. Dantrolene thus reduced the apparent caffeine sensitivity of the RYR1 and opposed the effect of the MHS mutation on the caffeine activation of the channel.



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Fig. 2.   Effect of dantrolene on the activation of skeletal muscle SR vesicle [3H]ryanodine binding by caffeine. [3H]Ryanodine binding in the absence or presence of 10 µM dantrolene was determined in media containing 120 mM potassium propionate, 10 mM PIPES (pH 7.0), 2 mM AMPPCP, 1.7 mM MgCl2, 1 µM CaM, and 100 nM Ca2+. Inset, the data normalized to the maximal activation determined in the presence of 48 mM caffeine. Solid lines represent fits to the Hill equation. Data are means ± S.E. from four experiments comparing four MHS and three normal SR vesicle preparations.


                              
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Table II
Effect of dantrolene on RYR1 sensitivity to caffeine, Mg2+, and Sr2+
[3H]Ryanodine binding to skeletal muscle SR vesicles was determined as described under "Experimental Procedures" in the absence or presence of 10 µM dantrolene. Values for EC50, IC50, and Hill coefficients (nH) are based on fits of the data presented in Figs. 2 and 3 to the Hill equation.

Mg2+ is an endogenous inhibitor of RYR channels that may act by competing with Ca2+ at channel activation sites (30, 31). Consequently, the physiologic significance of any changes in RYR1 Ca2+ sensitivity in the presence of dantrolene will depend on whether dantrolene also effects corresponding changes in the Mg2+ sensitivity of the channel. To better understand how dantrolene may influence the selectivity of RYR1 activation sites for Ca2+ relative to Mg2+, we determined the Mg2+ sensitivity of [3H]ryanodine binding in media containing 100 nM Ca2+ (Fig. 3A). In the absence of dantrolene, [3H]ryanodine binding to MHS SR vesicles was slightly less sensitive to inhibition by MgCl2 than was binding to normal SR (the IC50 for MHS SR increased 23%, p = 0.03) (Table II). Dantrolene, however, did not significantly alter the IC50 for MgCl2 inhibition of [3H]ryanodine binding to either MHS or normal SR vesicles.



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Fig. 3.   Effect of dantrolene on RYR1 sensitivity to Mg2+ and Sr2+. [3H]Ryanodine binding in the absence or presence of 10 µM dantrolene was determined in media containing 120 mM potassium propionate, 10 mM PIPES (pH 7.0), 2 mM AMPPCP, and 1 µM CaM. The Mg2+ dependence of [3H]ryanodine binding (A) was determined in the presence of 100 nM Ca2+; the Sr2+ dependence of [3H]ryanodine binding (B) was determined in the presence of <10 nM Ca2+. Insets at right, normalized data to emphasize effects of the MHS mutation and dantrolene on RYR1 sensitivity to the divalent cations. Data are means ± S.E. from four experiments comparing four MHS and three normal skeletal muscle SR vesicle preparations.

Whereas Mg2+ competitively blocks RYR1 activation by Ca2+, Sr2+ ions may replace Ca2+ in activating RYR channels (31). Accordingly, the results in Fig. 3B indicate that SR vesicle [3H]ryanodine binding was activated by Sr2+ and that the EC50 for activation was significantly decreased for MHS SR vesicles (Table II). Furthermore, dantrolene significantly increased the EC50 for Sr2+. These results are thus in agreement with the effect of dantrolene on the Ca2+ sensitivity of the RYR1 (15) but are in contrast to the lack of any effect of dantrolene on RYR1 sensitivity to Mg2+ (Fig. 3B). Together, our results therefore support the possibility that dantrolene inhibits SR Ca2+ release in situ by reducing the sensitivity of the RYR1 to activation by Ca2+ and further indicate that this mechanism may operate independently of any effect of dantrolene on the Mg2+ sensitivity of the channel.

Dantrolene Inhibition Is Dependent on Adenine Nucleotide-- Ohta and co-workers (32) originally noted that the effect of dantrolene on skinned muscle fibers was more pronounced when the media contained ATP, and more recently, Palnitkar and co-workers (33) reported that adenine nucleotides enhanced the binding of [3H]dantrolene to SR vesicles. To better define the role of adenine nucleotides in the mechanism of RYR1 inhibition by dantrolene, we examined the effect of AMPPCP on the inhibition of skeletal muscle SR vesicle [3H]ryanodine binding by dantrolene (Fig. 4). Initial experiments compared the CaM-dependent activation of MHS SR vesicle [3H]ryanodine binding in the presence and absence of AMPPCP. The results in Fig. 4A show that SR vesicle [3H]ryanodine binding was dependent on both AMPPCP and CaM. Thus, in the presence of AMPPCP (2 mM), dantrolene decreased by one-third the extent of CaM-activated [3H]ryanodine binding to MHS SR vesicles. In contrast, dantrolene did not significantly inhibit [3H]ryanodine binding when AMPPCP was omitted from the binding media (Fig. 4A). This loss of dantrolene inhibition was confirmed in the determinations of Kd and Bmax values for [3H]ryanodine binding in the AMPPCP-free media (Fig. 4B, Table I). A comparison of the dose dependence of dantrolene inhibition at different [AMPPCP] also indicated that RYR1 inhibition was strictly dependent on the presence of adenine nucleotide (Fig. 4C); however, neither the extent nor the concentration dependence of dantrolene inhibition was altered when the concentration of the nucleotide was increased from 1 to 4 mM (IC50 for dantrolene = 129 ± 48 nM versus 112 ± 32 nM, respectively).



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Fig. 4.   Effect of AMPPCP on the inhibition of skeletal muscle SR vesicle [3H]ryanodine binding by dantrolene (10 µM). A, CaM activation of SR vesicle [3H]ryanodine binding determined in the absence (squares) or presence (circles) of 2 mM AMPPCP. Media also contained 120 mM potassium propionate, 10 mM PIPES (pH 7.0), 100 nM Ca2+, and 10 µM dantrolene, as indicated. B, ryanodine dependence of skeletal SR vesicle [3H]ryanodine binding in the presence (filled symbols) or absence (open symbols) of 10 µM dantrolene was determined as in Fig. 1 except that the media lacked AMPPCP. C, dose dependence of dantrolene inhibition of [3H]ryanodine binding at different concentrations of AMPPCP (1 µM CaM, 100 nM Ca2+ throughout). Data are means ± S.E. from three different MHS skeletal muscle SR vesicle preparations. Dan, dantrolene.

Effect of Dantrolene on the RYR2 and RYR3 Isoforms-- The possible effects of dantrolene on the Kd of [3H]ryanodine binding to cardiac SR vesicles were investigated in media that contained either 100 or 300 nM Ca2+ (Fig. 5). In 100 nM Ca2+-containing media, neither Kd nor Bmax determinations (Table I) were significantly affected by dantrolene (10 µM), whereas in the same media, dantrolene increased the Kd of [3H]ryanodine binding to skeletal muscle SR vesicles 3-fold (Fig. 1). When Ca2+ was increased to 300 nM, the affinity of [3H]ryanodine binding to cardiac SR vesicles was increased (~7-fold); however, dantrolene again had no effect on either Kd or Bmax determinations. These results thus demonstrate that, in comparison with the RYR1, the RYR2 isoform in cardiac SR vesicles was insensitive to clinical concentrations of dantrolene.



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Fig. 5.   Effect of dantrolene on cardiac SR vesicle [3H]ryanodine binding. The binding of [3H]ryanodine (10-500 nM) to cardiac SR vesicles in the presence or absence of 10 µM dantrolene was determined as in Fig. 1, in media containing either 100 or 300 nM Ca2+, as indicated. Inset, fits of the data to single rectangular hyperbola for determinations of Kd and Bmax (Table I). Data are means ± S.E. from four cardiac SR vesicle preparations. Dan, dantrolene.

To further investigate the isoform selectivity of dantrolene as an inhibitor of RYR channels, we also examined [3H]ryanodine binding to recombinant RYRs heterologously expressed in HEK-293 cells (22). HEK-293 cells provide a valuable system for the functional expression of the different RYR isoforms because any endogenous RYRs in these cells are expressed only at very low levels (34). Accordingly, Fig. 6A shows that the [3H]ryanodine binding activity of lysates prepared from mock-transfected HEK-293 cells was low (<= 5 fmol/mg protein) and was not significantly affected by dantrolene (10 µM). [3H]Ryanodine binding to RYR2-transfected HEK-293 cell lysates was increased ~20-fold relative to mock-transfected controls. Moreover, [3H]ryanodine binding to the recombinant RYR2 in these cells was unaffected by dantrolene (Fig. 6A), consistent with the results obtained using cardiac SR vesicles (Fig. 5). In comparison, [3H]ryanodine binding to lysates prepared from RYR3-expressing cells was significantly inhibited by dantrolene (p < 0.001). Furthermore, the magnitude of the effect of dantrolene on the recombinant RYR3 (60% of control [3H]ryanodine binding in the presence of dantrolene) was comparable with the effect of dantrolene on the RYR1 in native SR vesicles (Fig. 1 and data not shown). These results therefore demonstrate that the sensitivity of a cell to dantrolene may be determined by the RYR isoform(s) expressed and identify the RYR3 isoform as a target for dantrolene action.



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Fig. 6.   Effect of dantrolene on [3H]ryanodine binding to the RYR2 and RYR3 isoforms in heterologously expressed in HEK-293 cells. A, comparison of [3H]ryanodine binding to lysates prepared from HEK-293 cells mock-transfected with pcDNA vector with binding to lysates from cells transiently expressing either RYR2 or RYR3. [3H]Ryanodine binding was determined in the presence or absence of dantrolene (10 µM) as described under "Experimental Procedures." B, Ca2+ dependence of [3H]ryanodine binding to the expressed RYR3 in the presence () or absence (open circle ) of 10 µM dantrolene. Solid lines represent fits to the Hill equation. (Ca2+ EC50 values are given in the text.) C, dantrolene inhibition of RYR3; the effect of dantrolene analog, temperature, and adenine nucleotide. [3H]Ryanodine binding was determined as in panels A and B except that azumolene (10 µM) was substituted for dantrolene, or the temperature of the binding media was reduced to 20 °C, or the binding media lacked AMPPCP, as indicated. Data are means ± S.E. from three to nine independent experiments. Asterisks indicate significant differences from control binding in the absence of drug (p < 0.05, Student's t test).

Dantrolene inhibition of the recombinant RYR3 isoform was further characterized to investigate whether a similar mechanism may explain the effects of dantrolene on the RYR1 and RYR3 isoforms. The Ca2+ dependence of [3H]ryanodine binding to HEK-293 cell lysates containing heterologously expressed RYR3 (Fig. 6B) indicated that dantrolene inhibition of RYR3 was most pronounced at ~100 nM Ca2+. In the presence of dantrolene (10 µM), the EC50 for the Ca2+ activation of [3H]ryanodine binding was increased 2.3-fold (from 97.8 ± 37 nM to 229 ± 48 nM). Although this increase in the Ca2+ EC50 for RYR3 did not reach statistical significance (p = 0.07), the magnitude of the effect was comparable with that previously documented for dantrolene inhibition of the RYR1 (~2.5-fold increase in Ca2+ EC50 for RYR1 in the presence of 10 µM dantrolene (see Ref. 15)). Furthermore, the results shown in Fig. 6C indicated that the binding of [3H]ryanodine to RYR3 was also inhibited by azumolene (10 µM), a dantrolene analog known to inhibit the RYR1 isoform (33, 15). Finally, as was true for the RYR1 (15), RYR3 inhibition by dantrolene was also abolished when the temperature in the [3H]ryanodine binding media was reduced to 20 °C or when AMPPCP was omitted from the binding media (Fig. 6C). These results thus indicate that dantrolene inhibition of the RYR3 isoform exhibited properties similar to those previously demonstrated for the dantrolene inhibition of the RYR1 isoform in SR vesicles (15) (Fig. 4).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Despite the importance of dantrolene both in the treatment of MH and as a pharmacologic probe of Ca2+ release from intracellular stores, the molecular basis and specificity of the actions of dantrolene on RYR channels have remained uncertain. In this study, we have used [3H]ryanodine binding to further characterize the mechanism by which dantrolene may selectively inhibit the RYR1 but not the RYR2 channel isoform and to determine whether the RYR3 isoform may also be a target for dantrolene action.

Dantrolene Opposes Increased RYR1 Activity Resulting from the MHS Arg615 right-arrow Cys Mutation-- Although dantrolene is only a partial inhibitor of the isolated RYR1, the current results clearly demonstrate that the magnitude of the effect of dantrolene on the functional activity of this channel is comparable with the effect of the MHS RYR1 Arg615 right-arrow Cys mutation. Thus, dantrolene effectively opposed the ~3-fold increase in the affinity of SR vesicle [3H]ryanodine binding resulting from the MHS mutation (Fig. 1). Dantrolene similarly opposed the ~2-fold increase in RYR1 caffeine sensitivity resulting from the MHS mutation (Fig. 2). Increased RYR1 caffeine sensitivity is diagnostic for human MHS mutations at diverse sites in the primary sequence of the channel protein (12), and this result is therefore consistent with the general efficacy of dantrolene in the treatment of this genetically heterogeneous disorder. The basis of the increased caffeine sensitivity of MHS channels is uncertain (29) but is unlikely to reflect the actual effects of the various mutations on the affinity of caffeine binding to the RYR1 (35). Rather, increased caffeine sensitivity may indicate that MHS mutations, like caffeine, act by reducing the threshold for RYR1 activation by Ca2+ (29). In this view, the observed 2-3-fold increase in the caffeine EC50 in the presence of dantrolene is consistent with the 2-3-fold shifts in the sensitivity of MHS and normal channels to both Ca2+ (15) and Sr2+ (Fig. 3B) in the presence of dantrolene.

Dantrolene Alters Selectivity of RYR1 Activation Sites for Ca2+ Relative to Mg2+-- Physiologic Mg2+ concentrations (~1 mM) may fully block RYR1 activation by Ca2+ (30, 36), and this suggests that dynamic changes in the affinity of this channel for either Ca2+ or Mg2+ may be a requisite step for channel activation in situ (29, 36). According to the model of Lamb and Stephenson (37), for example, RYR1 activation during EC coupling is dependent on a decrease in the Mg2+ affinity of the channel, which is mediated via the coupling of RYRs to transverse tubule voltage sensors. It is also possible, however, that the Ca2+ affinity of RYR1 activation sites might be modulated independently of major changes in the sensitivity of the channel to [Mg2+]i. In light of the effects of dantrolene both on EC coupling in intact muscle (7, 8) and on the Ca2+ sensitivity of the isolated RYR1, we examined the potential role of altered Mg2+ sensitivity in the mechanism of dantrolene inhibition. Our results (Fig. 3) indicate that dantrolene inhibition of SR vesicle [3H]ryanodine binding was not associated with an altered IC50 for Mg2+ (Table II). Because this inhibition reflects the competitive binding of Mg2+ to RYR1 Ca2+ activation sites (30, 31), our results indicate that the effect of dantrolene on the apparent Ca2+ affinity of RYR1 activation sites (15) is not associated with corresponding changes in the affinity of these same sites for Mg2+. Similarly, Murayama and co-workers (38) recently concluded that the effect of caffeine on the Ca2+ affinity of RYR activation sites was also not associated with changes in the affinity of Mg2+ binding to these sites. Thus, in intact muscle, both dantrolene and caffeine may modulate the Ca2+ sensitivity of the RYR1 via mechanisms that operate independently of any changes in the affinity of the channel for Mg2+.

Isoform-specific Action of Dantrolene on RYR Channels-- Accumulating evidence now supports a model in which the effects of dantrolene on SR Ca2+ release in skeletal muscle may be explained by the direct binding of dantrolene to the RYR1 channel protein without invoking putative non-RYR dantrolene receptors (26). Accordingly, purified, solubilized preparations of the RYR1 channel protein retain sensitivity to dantrolene (15), and cross-linking experiments have identified the RYR1 as the major SR protein labeled with a photoaffinity dantrolene analog (33). Nonetheless, the explanation for the dantrolene insensitivity of EC coupling in cardiac muscle has remained uncertain. For example, cardiac insensitivity to dantrolene might potentially be explained by a difference in the dantrolene binding properties of the RYR2 isoform itself, by some cardiac-specific modification of the RYR2 protein, or by other differences in the molecular machinery that controls SR Ca2+ release in cardiac as compared with skeletal muscle. We therefore investigated the possible effects of dantrolene on the RYR2 isoform heterologously expressed in a nonmuscle cell (22). Our results (Fig. 6A) show that the RYR2 expressed in HEK-293 cells remained insensitive to dantrolene. In comparison, the RYR3 isoform expressed in the same cell type was significantly inhibited by dantrolene. These results indicate that the RYR2 itself is intrinsically insensitive to dantrolene and thus suggest that this isoform may lack a high affinity dantrolene site that is present in both the RYR1 and the RYR3 isoforms. We conclude that the absence of major effects of dantrolene on SR Ca2+ release in the heart is likely a simple function of the predominant expression of the RYR2 channel isoform in cardiac muscle.

The insensitivity of the cardiac RYR2 to dantrolene is associated with other notable differences in the regulation of this channel isoform. Thus, in comparison with both the RYR1 and the RYR3 isoforms, the RYR2 isoform is less responsive to activation by adenine nucleotide (21, 24) and CaM (39, 23). Recently, we reported that CaM, together with adenine nucleotide, activates the RYR1 by increasing the Ca2+ sensitivity of the channel (39). Conversely, dantrolene inhibits the RYR1 by reducing Ca2+ sensitivity via a mechanism that is dependent on both adenine nucleotide and CaM. Thus, we postulate that the selective action of dantrolene on the RYR1 and RYR3 may in effect oppose the nucleotide- and CaM-dependent activation of these channel isoforms.

Dantrolene Effects in Nonmuscle Cells-- The identification of the RYR3 as a target for dantrolene suggests that this more broadly expressed channel isoform may potentially underlie the effects of dantrolene in various nonmuscle tissues and cell types. In this regard, the effects of dantrolene on Ca2+ signaling in central neurons are of particular interest. For example, dantrolene has been shown to inhibit the elevations of neuronal Ca2+ evoked by N-methyl-D-aspartate, glutamate, or potassium depolarization (16). Moreover, dantrolene may protect central neurons from disruptions in Ca2+ homeostasis resulting from ischemic injury (16, 17), epileptic seizure (18), or exposure to amyloid beta -peptide (19). Central nervous system effects of dantrolene are also suggested by reports that subjects treated with the drug may experience dizziness, blurred vision, and fatigue (40). However, resolving the molecular targets that underlie the effects of dantrolene on central neurons is difficult because all three RYR isoforms are expressed in the brain and multiple isoforms may be present within a single cell type (41-43). Yet, notably, the predominant RYR in the brain as a whole is RYR2 (41, 42), whereas our results indicate that RYR1 and RYR3, but not RYR2, may be targets for dantrolene. In light of our results, it may now be of interest to define the specific RYR isoforms that may be responsible for the various effects of dantrolene on Ca2+ signaling in different neuronal cells. Regardless, it is clear that understanding the molecular basis of the effects of dantrolene on intracellular Ca2+ release channels may have implications that extend beyond skeletal muscle and MH to diverse cell types and disease states.


    ACKNOWLEDGEMENTS

We thank Jennifer Bardy and Rachel Bloomquist for excellent technical assistance and Ed Balog for helpful discussions.


    FOOTNOTES

* This work was supported by grants from the American Heart Association (to B. R. F.), Grant GM-31382 from the National Institutes of Health (to C. F. L.), and grants from the Medical Research Council and Heart and Stroke Foundation (to S. R. W. C.).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.

Senior Scholar of the Alberta Heritage Foundation for Medical Research.

|| To whom correspondence should be addressed: 6-155 Jackson Hall, 321 Church St. S.E., Minneapolis, MN 55455. Tel.: 612-625-3292; Fax: 612-625-2163; E-mail: fruen001@tc.umn.edu.

Published, JBC Papers in Press, February 5, 2001, DOI 10.1074/jbc.M006104200


    ABBREVIATIONS

The abbreviations used are: RYR, ryanodine receptor; SR, sarcoplasmic reticulum; MH, malignant hyperthermia; MHS, MH-susceptible; EC, excitation-contraction; CaM, calmodulin; AMPPCP, adenosine 5'-(beta ,gamma -methylene)triphosphate; PIPES, 1,4-piperazinediethanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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