From the 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
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ABSTRACT |
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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 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.
MHS pigs homozygous for the RYR1 Arg615 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).
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 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
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.
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).
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.
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 (
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).
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 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 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
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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.
Effect of dantrolene on SR vesicle [3H]ryanodine binding
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.
Effect of dantrolene on RYR1 sensitivity to caffeine, Mg2+, and
Sr2+
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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.
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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.
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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.
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 (
) 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).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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
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.
-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.
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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
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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'-(,
-methylene)triphosphate;
PIPES, 1,4-piperazinediethanesulfonic acid;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Franzini-Armstrong, C.,
and Protasi, F.
(1997)
Physiol. Rev.
77,
699-729 |
2. | Berridge, M. J., Bootman, M. D., and Lipp, P. (1998) Nature 395, 645-648[CrossRef][Medline] [Order article via Infotrieve] |
3. |
Xu, L.,
Tripathy, A.,
Pasek, D. A.,
and Meissner, G.
(1998)
Ann. N. Y. Acad. Sci.
853,
130-148 |
4. |
Sutko, J. L.,
and Airey, J. A.
(1996)
Physiol. Rev.
76,
1027-1071 |
5. |
Zucchi, R.,
and Ronca-Testoni, S.
(1997)
Pharmacol. Rev.
49,
1-51 |
6. | Ellis, K. O., Butterfield, J. L., Wessels, F. L., and Carpenter, J. F. (1976) Arch. Int. Pharmacodyn. Ther. 224, 118-132[Medline] [Order article via Infotrieve] |
7. | Morgan, K. G., and Bryant, S. H. (1977) J. Pharmacol. Exp. Ther. 201, 138-147[Abstract] |
8. | Hainaut, K., and Desmedt, J. E. (1974) Nature 252, 728-729[Medline] [Order article via Infotrieve] |
9. | Loke, J., and MacLennan, D. H. (1998) Am. J. Med. 104, 470-486[CrossRef][Medline] [Order article via Infotrieve] |
10. | Ward, A., Chaffman, M. O., and Sorkin, E. M. (1986) Drugs 32, 130-168[Medline] [Order article via Infotrieve] |
11. | Britt, B. A., Scott, E., Frodis, W., Clements, M., and Endrenyi, L. (1984) Can. Anaesth. Soc. J. 31, 130-154[Medline] [Order article via Infotrieve] |
12. |
Tong, J.,
Oyamada, H.,
Demaurex, N.,
Grinstein, S.,
McCarthy, T. V.,
and MacLennan, D. H.
(1997)
J. Biol. Chem.
272,
26332-26339 |
13. | Jurkat-Rott, K., McCarthy, T., and Lehmann-Horn, F. (2000) Muscle Nerve 23, 4-17[CrossRef][Medline] [Order article via Infotrieve] |
14. | VanWinkle, W. B. (1976) Science 193, 1130-1131[Medline] [Order article via Infotrieve] |
15. |
Fruen, B. R.,
Mickelson, J. R.,
and Louis, C. F.
(1997)
J. Biol. Chem.
272,
26965-26971 |
16. | Mody, I., and MacDonald, J. F. (1995) Trends Pharmacol. Sci. 16, 356-359[CrossRef][Medline] [Order article via Infotrieve] |
17. | Wei, H., and Perry, D. C. (1996) J. Neurochem. 67, 2390-2398[Medline] [Order article via Infotrieve] |
18. |
Pelletier, M. R.,
Wadia, J. S.,
Mills, L. R.,
and Carlen, P. L.
(1999)
J. Neurophysiol.
81,
3054-3064 |
19. |
Mattson, M. P.,
Zhu, H., Yu, J.,
and Kindy, M. S.
(2000)
J. Neurosci.
20,
1358-1364 |
20. |
Mickelson, J. R.,
Gallant, E. M.,
Litterer, L. A.,
Johnson, K. M.,
Rempel, W. E.,
and Louis, C. F.
(1988)
J. Biol. Chem.
263,
9310-9315 |
21. |
Meissner, G.,
and Henderson, J. S.
(1987)
J. Biol. Chem.
262,
3065-3073 |
22. |
Zhao, M.,
Li, P.,
Li, X.,
Zhang, L.,
Winkfein, R. J.,
and Chen, S. R.
(1999)
J. Biol. Chem.
274,
25971-25974 |
23. |
Chen, S. R. W.,
Li, X.,
Ebisawa, K.,
and Zhang, L.
(1997)
J. Biol. Chem.
272,
24234-24246 |
24. |
Du, G. G.,
Imredy, J. P.,
and MacLennan, D. H.
(1998)
J. Biol. Chem.
273,
33259-33266 |
25. | Brooks, S. P., and Storey, K. B. (1992) Anal. Biochem. 201, 119-126[Medline] [Order article via Infotrieve] |
26. |
Parness, J.,
and Palnitkar, S. S.
(1995)
J. Biol. Chem.
270,
18465-18472 |
27. | Pessah, I. N., Lynch, C. I. I. I, and Gronert, G. A. (1996) Anesthesiology 84, 1275-1279[Medline] [Order article via Infotrieve] |
28. | Flewellen, E. H., Nelson, T. E., Jones, W. P., Arens, J. F., and Wagner, D. L. (1983) Anesthesiology 59, 275-280[Medline] [Order article via Infotrieve] |
29. | Herrmann-Frank, A., Luttgau, H. C., and Stephenson, D. G. (1999) J. Muscle Res. Cell Motil. 20, 223-237[CrossRef][Medline] [Order article via Infotrieve] |
30. | Laver, D. R., Baynes, T. M., and Dulhunty, A. F. (1997) J. Membr. Biol. 56, 213-229 |
31. |
Liu, W.,
Pasek, D. A.,
and Meissner, G.
(1998)
Am. J. Physiol.
274,
C120-C128 |
32. | Ohta, T., Ito, S., and Ohga, A. (1990) Eur. J. Pharmacol. 178, 11-19[CrossRef][Medline] [Order article via Infotrieve] |
33. | Palnitkar, S. S., Bin, B., Jimenez, L. S., Morimoto, H., Williams, P. G., Paul-Pletzer, K., and Parness, J. (1999) J. Med. Chem. 42, 1872-1880[CrossRef][Medline] [Order article via Infotrieve] |
34. | Tong, J., Guang, G., Chen, S. R. W., and MacLennan, D. H. (1999) Biochem. J. 343, 39-44[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Shomer, N. H.,
Mickelson, J. R.,
and Louis, C. F.
(1994)
Am. J. Physiol.
267,
C1253-C1261 |
36. |
Lacampagne, A.,
Klein, M. G.,
and Schneider, M. F.
(1998)
J. Gen. Physiol.
111,
207-224 |
37. |
Lamb, G. D.,
and Stephenson, D. G.
(1992)
News Physiol. Sci.
7,
270-274 |
38. |
Murayama, T.,
Kurebayashi, N.,
and Ogawa, Y.
(2000)
Biophys. J.
78,
1810-1824 |
39. | Fruen, B. R., Bardy, J. M., Byrem, T. M., Strasburg, G. M., and Louis, C. F. (2000) Am. J. Physiol. 279, C724-C733 |
40. | Wedel, D. J., Quinlan, J. G., and Iaizzo, P. A. (1995) Mayo Clin. Proc. 70, 241-246[Medline] [Order article via Infotrieve] |
41. | Furuichi, T., Furutama, D., Hakamata, Y., Nakia, J., Takeshima, H., and Mikoshiba, K. (1994) J. Neurosci. 14, 4794-4805[Abstract] |
42. | Giannini, G., Conti, A., Mammarella, S., Scrobogna, M., and Sorrentino, V. (1995) J. Cell Biol. 128, 893-904[Abstract] |
43. |
Ledbetter, M. W.,
Preiner, J. K.,
Louis, C. F.,
and Mickelson, J. R.
(1994)
J. Biol. Chem.
269,
31544-31551 |