(Received for publication, March 6, 1995; and in revised form, May 18, 1995)
From the
Dantrolene, an intracellularly acting skeletal muscle relaxant,
inhibits Ca
Dantrolene
(1-[[5-(p-nitrophenyl)furfurylidine]amino]hydantoin
sodium) is a hydantoin derivative that acts as a postsynaptic muscle
relaxant (1) and is the only known effective treatment for
malignant hyperthermia (MH), ( The mechanism of ECC-induced Ca RyR opening can be modulated by a number of ligands and
cellular processes, including caffeine, ATP, ruthenium red, the
immunophilin FK506-binding protein (FKBP12), Ca We have
therefore begun a program to identify the molecular target of
dantrolene. In this paper we report the development of a
pharmacological assay demonstrating the existence of discrete
dantrolene binding sites in skeletal muscle SR membranes. Although
previous attempts have been made to identify a dantrolene receptor by
radioactive ligand binding assays (19) or by fluorescence
measurements (20) , the former assay suffered from poor
reproducibility (21) and both from the inability to distinguish
specific from nonspecific binding. Here we characterize a specific,
reproducible assay for [ Dantrolene sodium
Figure 1:
Dantrolene and its congeners.
Dantrolene and azumolene are physiologically active skeletal muscle
relaxants, whereas aminodantrolene is inactive. T denotes the
position of tritium in
[
Specific activity
was determined as follows. Unlabeled dantrolene was weighed in
triplicate and dissolved in DMF or ethanol to a 10 mM final
concentration. The samples were then diluted serially into ethyl
acetate, a solvent in which dantrolene acquires fluorescent
properties(20) , and excitation and emission maxima were
determined to be 383 and 540-580 nm, respectively, in an MPF-66
Perkin-Elmer fluorescence spectrophotometer (data not shown). DMF and
ethanol concentrations up to 10% do not quench dantrolene fluorescence
in ethyl acetate (data not shown). Fluorescence emission at 540 nm (
Figure 2:
Binding of
[
Figure 3:
Binding of
[
Figure 4:
Inhibition of
[
Figure 5:
Effect of pH on
[
Figure 6:
Ionic strength dependence of
[
Figure 7:
Effect
of Ca
Dantrolene, originally synthesized by Snyder et al.(1) , is one of a series of hydantoin derivatives found to
be active as muscle relaxants. Ellis and co-workers (39, 40) presented evidence that dantrolene inhibited
ECC in mammalian skeletal muscle, acting distal to the neuromuscular
junction, and Van Winkle (7) demonstrated in vitro evidence that the drug exerted its effects in skeletal muscle by
suppressing Ca There are only two
previous reports in the literature on the development of assays for
dantrolene binding to muscle membranes. Sengupta et al. (19) reported the binding of
[ The major
factors we found to be a hindrance during the development of a specific
[ The binding isotherms were
analyzed by nonlinear regression analysis rather than the conventional
Scatchard/Rosenthal plots to reduce the errors in calculation of the
binding parameters(45, 46) . Scatchard/Rosenthal plots
are methods for the linearization of nonlinear data and are valid only
when the signal-to-noise ratio is high(46) . Such was not the
case in our experiments. Indeed, a major impediment to the development
of the [ We found that the signal-to-noise ratio in the binding assay was
dependent on the solvent used to make stock solutions of
[ The pharmacological
specificity of [ The subcellular
distribution of the [ The optimum conditions for
[ Recent in vitro studies using SR membranes or
skinned fiber preparations may indicate that dantrolene has a biphasic
effect on mammalian skeletal muscle RyR, activation at nanomolar
concentrations and inactivation at micromolar concentrations, but do
not distinguish whether these are a direct or indirect effect of the
drug(53) . In conclusion, we have identified a putative
dantrolene receptor in skeletal muscle HSR by developing a specific,
reproducible [
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
release from the sarcoplasmic reticulum
during excitation-contraction coupling by an unknown mechanism. The
drug is used to treat malignant hyperthermia, a genetic sensitivity to
volatile anesthetics which results in the massive release of
intracellular Ca
from affected skeletal muscle. We
hypothesize that determination of the site of action of dantrolene will
lead to further understanding of the regulation of sarcoplasmic
reticulum calcium release. We report the identification of specific
dantrolene binding sites in porcine skeletal muscle sarcoplasmic
reticulum using a rapid filtration binding assay for
[
H]dantrolene. The binding isotherm in the heavy
sarcoplasmic reticulum fraction indicates a single binding site with a K
of 277 ± 25 nM and a B
of 13.1 ± 1.5 pmol/mg of protein.
Pharmacological specificity is characterized by inhibition of
[
H]dantrolene binding with unlabeled dantrolene,
or azumolene, a physiologically active congener, but not with
aminodantrolene, which is physiologically inactive. Drug binding is
maximal at pH 6.5-7.5, requires no Ca
or
Mg
, and is inhibited by salt concentrations above 100
mM. [
H]Dantrolene binding is greatest in
the sarcoplasmic reticulum, which contains the ryanodine receptor, the
primary calcium release channel. No binding is detected in the
fractions enriched for sarcolemma or transverse tubules. We suggest
that dantrolene inhibits calcium release from the sarcoplasmic
reticulum by either direct or indirect interaction with the ryanodine
receptor.
)a genetic disorder of
excitation-contraction coupling (ECC) in skeletal muscle (2) .
MH is triggered in susceptible individuals by volatile anesthetics and
depolarizing muscle relaxants (3) and is characterized by a
pattern of muscle physiology which resembles aberrant ECC. MH is
exemplified by a massive release of Ca
from the
sarcoplasmic reticulum (SR), which results in hypercontracture,
hypermetabolism, elevated temperatures, and death, if not treated with
dantrolene(3) . Porcine stress syndrome, a porcine model for MH
triggered by volatile anesthetics or stress, is also characterized by
an exaggerated increase in intracellular Ca
and is
linked to a genetic defect in the ryanodine receptor (RyR), the SR
calcium release channel(4, 5) . Dantrolene inhibits
the development or progression of MH by decreasing the levels of
myoplasmic Ca
in both human and porcine skeletal
muscle(2, 6, 7) . The evidence suggests that
dantrolene does so by inhibiting the release of SR Ca
(7) rather than its reuptake into the SR(8) .
release in
skeletal muscle has been reviewed
extensively(9, 10, 11, 12, 13) .
Briefly, ECC is initiated by a wave of depolarization across the
sarcolemma which is sensed by the transmembrane dihydropyridine
receptor (DHPR), an L-type calcium channel present in the transverse
tubules, specialized invaginations of the sarcolemma. The DHPR apposes
the RyR in the SR membrane forming the triad junction (14) and
is poised to sense depolarization and transmit a signal that results in
Ca
release via the RyR(9) . This signal
transduction involves intramembrane charge movement across the DHPR
rather than the calcium current seen in cardiac muscle(9) . The
exact relationship of this signaling event to the opening of the RyR is
not clear but may involve a direct interaction of the cytoplasmic loop
of the DHPR with the RyR(15) . The net result of this
interaction is the opening of the RyR, Ca
release
from the SR into the myoplasm, and the initiation of skeletal muscle
contraction.
, and
phosphorylation (12) . Furthermore, the cellular architecture
of the triad junction suggests that at least one of a number of
proteins known to span the junction, triadin, may modulate signal
transduction and/or RyR opening (16) . Most studies suggest
that dantrolene acts to inhibit Ca
release from the
SR at a site distal to the DHPR(7, 17) . Only one
study, in amphibian skeletal muscle, suggests that dantrolene may
affect charge movement across the DHPR(18) . Theoretically,
therefore, the pharmacological action of dantrolene to reduce
myoplasmic Ca
may occur as a result of the
drug's interaction with the DHPR, the RyR, or any of the
proteins, known or uncharacterized, of the triadic junction.
Identifying the molecular target of dantrolene will likely delineate
the component(s) that regulates SR Ca
release during
ECC in both normal and MH-susceptible skeletal muscle.
H]dantrolene binding to
pig skeletal muscle membranes. In addition, as dantrolene is such a
hydrophobic compound(22) , we also describe many of the
experimental obstacles overcome during development of the assay.
3½H
O, azumolene
sodium
2H
O, and aminodantrolene sodium were gracious
gifts of Proctor & Gamble, Norwich, NY.
[
H]Dantrolene was custom synthesized by ChemSyn
Science Laboratories, Lenexa, KS. [
H]Ryanodine
(61.5 Ci/mmol) and [
H]PN200-110 (78 Ci/mmol) were
purchased from DuPont NEN and Amersham Corp., respectively.
Phenylmethylsulfonyl fluoride, benzamidine, leupeptin, and pepstatin A
were purchased from Boehringer Mannheim. All HPLC solvents were
purchased from J. T. Baker and were HPLC grade. Cytoscint ES and
hyamine hydroxide were purchased from ICN, and glass fiber filters
(GF/C) were from Whatman. Other filters were gifts from Gelman and
Stratagene, as noted. Common laboratory chemicals were purchased from
Sigma.
Authentication of
[
[H]Dantrolene and Determination of
Specific Activity
H]Dantrolene (Fig. 1) was custom synthesized by ChemSyn using the four-step
reaction scheme of Snyder et al.(1) , with the first
step involving tritiation of o-bromoaniline by catalytic
halogen replacement. [
H]Dantrolene was received
as the crystalline salt (
3 H
O) and dissolved in
dimethylformamide (DMF). DMF was chosen as the solvent for two reasons:
1) dantrolene is soluble in DMF to a concentration of 10 mM (data not shown), and 2) it can be stored at -20 °C as a
solution, essential for radioactive compounds, since freezing tends to
concentrate solute locally, thus increasing the likelihood of
radiolysis(23) . Stock solutions of unlabeled dantrolene were
dissolved in DMF, ethanol, or water. Both labeled and unlabeled
dantrolene were chromatographed by HPLC on a Lichrosorb 100 RP-18
column (5 µm, Merck), 12.5
0.4 cm, using 25% acetonitrile
in 20 mM potassium phosphate buffer, pH 7.4, as solvent phase,
and peaks identified by absorbance at 385 nm(24) . In the case
of [
H]dantrolene, 0.2-ml fractions were
collected, and 50-µl aliquots were counted in 4 ml of Cytoscint ES
in an LKB 1209 RackBeta liquid scintillation counter. Both unlabeled
and [
H]dantrolene chromatographed as single peaks
with a retention time of 3.2 min (data not shown).
H]dantrolene.
, 383 nm) was measured as a function of
increasing dantrolene concentration and was found to be linear over the
range of 1-500 nM (INPLOT 4.0, GraphPad, r = 0.995, data not shown). A standard graph of fluorescence
intensity versus dantrolene concentration was then
constructed, and the concentration of
[
H]dantrolene was determined from the above graph
by measuring the fluorescence emission of the radioactive samples. To
determine the specific activity of [
H]dantrolene,
triplicate aliquots of known concentration were counted in Cytoscint ES
using an LKB RackBeta 1209 liquid scintillation counter (counting
efficiency 59%). The resultant specific activity of
[
H]dantrolene was found to be 8.92 Ci/mmol.
During the course of the experiments described below, specific activity
was routinely assessed and corrected for tritium exchange.
Determination of Dantrolene and Congener
Solubility
Solubility of dantrolene and congeners was
determined by filtration assay based on the finding that dantrolene and
congeners coprecipitate (see data below). Samples containing 1 ml of
assay buffer (see below) were mixed with 60,000 cpm
[H]dantrolene and increasing concentrations of
unlabeled dantrolene or congener in borosilicate glass tubes, vortexed,
and allowed to stand at 20 °C for 1 h. The final concentration of
DMF was 0.25%. Samples were filtered rapidly through Whatman GF/C
filters (average pore size 1.2 µm), as described for
[
H]dantrolene binding below, and washed with 1
5 ml of binding buffer at room temperature, and the amount of
radioactivity bound to the filters was determined. Increases in
filter-bound radioactivity in the presence of unlabeled drug are
presumably due to coprecipitation(25) .
Preparation of Pig Skeletal Muscle Membrane
Fractions
Pig (Yorkshire Landrace or homozygous
normal Yorkshire) fast twitch skeletal muscle were gifts of Dr. Donald
Wilkerson, Department of Surgery, UMDNJ-Robert Wood Johnson Medical
School, and Dr. Charles F. Louis, Department of Veterinary Biology,
University of Minnesota. Freshly dissected muscle was frozen
immediately in liquid nitrogen and maintained at -72 °C until
use. Membrane fractions corresponding to the sarcolemma, transverse
tubules, light (LSR) and heavy sarcoplasmic reticulum (HSR) were
prepared, with slight modification(26) , according to the
method of Meissner(27) . Briefly, 150-160 g of tissue was
homogenized in a Waring blender (2
30 s, high speed) with 750
ml of ice-cold buffer containing 5 mM Tris maleate, pH 6.8,
and 0.1 M NaCl. The homogenate was centrifuged at 2,600
g for 30 min at 4 °C. The supernatant was saved
and the pellet rehomogenized in the Waring blender with 150 ml of
buffer, 1
30 s at high speed, and centrifuged as above. The
supernatants were combined, filtered through six layers of cheesecloth,
and centrifuged at 10,000
g for 30 min at 4 °C.
The supernatant was discarded and the pellet resuspended in 30-40
ml of 0.6 M KCl, 5 mM Tris-Mes, pH 6.8, with two or
three strokes of a motor-driven, Teflon-glass homogenizer. These
salt-treated microsomes were sedimented at 120,000
g for 60 min. The supernatant was discarded and the pellets
resuspended in a total of 30 ml of 5 mM Tris-Mes buffer, pH
6.8, containing 0.4 M KCl, 10% sucrose (w/v), and 20
µM CaCl
. Ten ml of the resuspended pellet was
layered onto a discontinuous sucrose gradient (5 ml of 40%, 8 ml of
35%, 8 ml of 30%, and 5 ml of 20% sucrose containing 0.4 M
KCl, 20 µM CaCl
, in 5 mM Tris-Mes, pH
6.8) and centrifuged overnight at 130,000
g, with slow
acceleration and no braking at the end of the run. Membrane fractions
at the density interfaces were aspirated carefully, diluted slowly to
approximately 10% sucrose in buffer (0.1 M KCl, 5 mM
Tris-Mes, pH 6.8), and repelleted at 95,000
g for 40
min at 4 °C. The pellets were resuspended slowly in 1-2 ml of
5 mM Na-PIPES buffer, pH 6.8, containing 10% sucrose and 0.1 M KCl, aliquoted, frozen in liquid N
, and stored
at -70 °C until use. The following protease inhibitors were
present at all stages of membrane fractionation: 1 mM phenylmethylsulfonyl fluoride, 1 µM leupeptin, 1
µM pepstatin A, and 0.5 mg/ml benzamidine. Protein was
determined by the method of Bradford(28) . Membrane fractions
were characterized for their ability to bind both
[
H]ryanodine (29) and the dihydropyridine
antagonist [
H]PN200-110(30) .
[
[H]Dantrolene Binding
Assay
H]Dantrolene binding to pig
skeletal muscle membrane fractions was determined by a rapid filtration
assay. Membrane protein, at the appropriate concentration, was
incubated in buffer (10 mM Na-HEPES or Tris-HCl, pH 7.4), with
various concentrations of [
H]dantrolene, in the
presence or absence of either 30 µM unlabeled dantrolene
or 150 µM azumolene for 60 min at 37 °C in the dark,
in a final reaction volume of 250 or 500 µl, in triplicate.
[
H] Dantrolene binding to HSR at 37 °C
reached equilibrium by 60 min (data not shown). The final concentration
of DMF or ethanol in the assay mixture was less than 1%. A 50-µl
unfiltered aliquot/sample was placed in a vial containing 4 ml of
Cytoscint ES, and radioactivity was determined by liquid scintillation
counting to calculate directly the total ligand concentration/sample.
Samples were then filtered rapidly through Whatman GF/C filters using a
Hoefer model FH225V filtration apparatus (suction at 10 mm Hg for best
separation). Filters were washed rapidly (<2 s) with 1
5 ml
of ice-cold binding buffer and treated overnight with 200 µl of 1 M hyamine hydroxide/filter to hydrolyze the membranes and
proteins. Increasing the number of washes served only to decrease the
total radioactivity measured and did not increase the signal-to-noise
ratio (data not shown). Radioactivity was then determined by liquid
scintillation counting in 4 ml of Cytoscint ES. Total binding was that
achieved in the absence of unlabeled drug, and nonspecific binding,
that measured in the presence of 150 µM azumolene (Fig. 1), a more water-soluble, physiologically active congener
of dantrolene(31) . Since the maximum solubility of dantrolene
(20 °C, pH 7.4) was determined to be 30 µM (see
below), we used azumolene to determine the nonspecific binding of
[
H]dantrolene. Specific binding was determined by
subtracting nonspecific binding from total binding. The data for this
and the other binding assays described below were analyzed by nonlinear
regression analysis using INPLOT. Control assays were run in the
absence of skeletal muscle membranes to determine the degree of
[
H]dantrolene binding to the filters and to
ensure that there was no specific binding to filters, as has been
observed in other systems(32, 33) .
[
[H]Ryanodine Binding
Assay
H]Ryanodine binding to
skeletal muscle membrane fractions was determined according to the
method of Valdivia et al.(29) . Briefly, increasing
concentrations of [
H]ryanodine (1-50
nM) were incubated with membrane fractions in a buffer
containing 20 mM Tris-HCl, pH 8.5, 0.15 M NaCl, and
50 µM CaCl
, at 37 °C for 90 min. Samples
were filtered through Whatman GF/C filters using a Brandel cell
harvester and washed with ice-cold buffer (2
5 ml). The filters
were counted for radioactivity in Cytoscint ES. Nonspecific binding
corresponded to the binding measured in the presence of 10 µM ryanodine. Data were analyzed as above. The K
and B
for
[
H]ryanodine binding to HSR were 7.0 ±
0.05 nM and 9.0 ± 1.6 pmol/mg of protein, respectively.
[
[H]PN200-110 Binding
Assay
H]PN200-110 binding to
skeletal muscle membrane fractions was measured as described
previously(30) . Membranes were incubated at room temperature
(25 °C) in a solution containing 50 mM Tris-HCl, pH 7.4,
with different concentrations of [
H]PN200-110
(0.1-5 nM), in the dark. Membrane-bound ligand was
separated from the free ligand by using the Brandel cell harvester as
described for the [
H]ryanodine binding assay, and
the radioactivity on the filter was counted in Cytoscint ES. One
µM nitrendipine was used to determine the nonspecific
binding. Data were analyzed as above. The K
and B
values for
[
H]PN200-110 binding to the transverse tubules
were 0.8 ± 0.1 nM and 16 ± 2.4 pmol/mg of
protein, respectively.
Solubility Limits of Dantrolene and
Congeners
Dantrolene is an extremely hydrophobic drug, and
therefore it precipitates easily out of aqueous solution. Hence, prior
to development of the binding assay, the solubility limits of
dantrolene and its congeners, azumolene and aminodantrolene, were
determined by coprecipitation, as described under ``Experimental
Procedures.'' The approximate solubility maxima at pH 7.4, 20
°C, were determined to be 30, 50, and 300 µM for
dantrolene, aminodantrolene, and azumolene, respectively. The above
solubility limit for dantrolene is in clear agreement with the value of
35.1 µM determined by Salata and Jalife(34) .
Solubility limits for these compounds at 37 °C are not
significantly different than those determined at 20 °C (data not
shown).Nonspecific Binding of
[
In developing
the rapid filtration assay for [H]Dantrolene
H]dantrolene
binding to pig skeletal muscle membranes, we immediately became aware
of the propensity of dantrolene to bind nonspecifically to many
laboratory materials. During attempts at developing a centrifugation
assay, we found that [
H]dantrolene binds
nonspecifically to the sides of polypropylene tubes (data not shown).
Moreover, polypropylene induces the precipitation of 30 µM azumolene in the presence of nanomolar amounts of
[
H]dantrolene (data not shown). Silanization of
polypropylene significantly reduced nonspecific binding (about 50%),
but the same process enhanced nonspecific binding to glass (data not
shown). Despite silanization, however, use of polypropylene tubes in
centrifugation assays for [
H]dantrolene binding
to skeletal muscle membranes resulted in unacceptable intersample
variation. These tubes, therefore, are unsuitable for use with
dantrolene or its congeners. Rapid filtration, rather than
centrifugation, was chosen as an assay method. Various filter media
were tested for their ability to bind
[
H]dantrolene nonspecifically, using the rapid
filtration method described above. Glass fiber filters (GF/C) were the
only ones not to bind significant amounts of
[
H]dantrolene (Table 1). Preincubating
these filters with unlabeled dantrolene, various detergents, or
chemicals commonly used to inhibit nonspecific binding in other systems
did not reduce nonspecific binding (Table 2). Hence, untreated
Whatman GF/C filters were used in the assay described below.
Specific Binding of
[
To
quantitate the interaction between [H]Dantrolene to HSR
H]dantrolene
and its binding sites in HSR at equilibrium, increasing concentrations
of [
H]dantrolene were incubated with HSR
membranes, in the presence or absence of azumolene, and radioactivity
was determined as described under ``Experimental
Procedures.'' Nonspecific binding, that measured in the presence
of 150 µM unlabeled azumolene, was subtracted from total
binding, that measured in the presence of
[
H]dantrolene alone (Fig. 2A), to
give a specific binding curve (Fig. 2, A and B). Using nonlinear regression analysis (INPLOT) to analyze
the calculated specific binding data, we determined that the resultant
curve is best described as a rectangular hyperbola, indicating a single
class of binding sites. The calculated K
and B
for
[
H]dantrolene binding to HSR under these
experimental conditions are 277 ± 25 nM and 13.1
± 1.5 pmol/mg of protein, respectively. These results were
obtained despite a signal-to-noise ratio between 8 and 15%. Recent
binding experiments carried out with
[
H]dantrolene dissolved in ethanol instead of DMF
yielded a signal-to-noise ratio of 20-30% without any significant
changes in the binding parameters (K
, 273
± 25 nM; B
, 12.9 ± 1.2
pmol/mg of protein; Fig. 3, A and B). Indeed,
the nonspecific, and, hence, the total
[
H]dantrolene bound, decreased by approximately
50% when the drug was dissolved in ethanol, as compared with that seen
when the drug was dissolved in DMF (see Fig. 2A and
3A). When control assays were carried out as described under
``Experimental Procedures'' but without membrane protein,
``specific'' binding of [
H]dantrolene
to glass fiber filters was not seen.
H]dantrolene to HSR membranes. Panel A,
total, nonspecific, and specific binding of
[
H]dantrolene to porcine HSR membranes as
determined by rapid filtration assay described under
``Experimental Procedures.'' Each point is the mean of
triplicate determinations ± S.E. from four different experiments
on membranes derived from three separate animals. Stock
[
H]dantrolene solutions were made in DMF. Panel B, specific binding, the difference between total and
nonspecific binding expanded from panel
A.
H]dantrolene to HSR membranes. Panel A,
conditions were identical to those in Fig. 2except that stock
[
H]dantrolene solutions were made in ethanol (n = 3). Panel B, specific binding curve
generated using data expanded from panel
A.
Inhibition of
[
We determined the ability of
dantrolene congeners to inhibit specific
[H]Dantrolene Binding to HSR by
Dantrolene Congeners
H]dantrolene binding to HSR to assess
pharmacological specificity. [
H]Dantrolene (200
nM) was incubated with 50 µg of HSR membrane protein in
the presence of increasing concentrations of dantrolene, azumolene, or
aminodantrolene (a physiologically inactive congener(35) ), in
binding buffer for 60 min at 37 °C, and radioactivity was
determined. The results, presented in Fig. 4, show that
dantrolene and azumolene have nearly identical inhibition
characteristics, whereas aminodantrolene, at concentrations up to its
solubility maximum, has no activity in this system. The apparent K
values for dantrolene and azumolene are
approximately 528 nM. This value was calculated using the
Cheng-Prusoff correction, in which K
is a
function of both the radiolabeled ligand concentration and the
concentration of inhibitor at which 50% of ligand binding to receptor
is inhibited(36) .
H]dantrolene binding to HSR membranes. HSR
membranes were incubated with [
H]dantrolene (200
nM) and increasing concentrations of dantrolene, azumolene, or
aminodantrolene as described under ``Results.'' Binding was
determined using rapid filtration, as described under
``Experimental Procedures.''
Comparison of Specific
[
To compare the
subcellular distribution of [H]Dantrolene,
[
H]PN200-110, and
[
H]Ryanodine Binding Sites in Subcellular
Skeletal Muscle Membrane Fractions
H]dantrolene and
[
H]ryanodine binding sites, we measured their
specific binding to skeletal muscle membrane fractions corresponding to
sarcolemma, transverse tubules, LSR, and HSR, prepared as described
under ``Experimental Procedures.'' Fifty µg of membrane
protein from each fraction was incubated, in triplicate, with
[
H]dantrolene (200 nM),
[
H]PN200-110 (0.8 nM), or
[
H]ryanodine (5 nM), in the presence or
absence of appropriate concentrations of the unlabeled counterpart of
each; specific binding was determined by the assay system described for
each drug under ``Experimental Procedures.'' The
concentration of each labeled ligand in the experiment approximated the
calculated K
for that ligand. The
results, shown in Table 3, indicate a parallel distribution of
[
H]ryanodine and
[
H]dantrolene binding sites in pig skeletal
muscle membrane fractions. The LSR fraction showed approximately half
the [
H]dantrolene and
[
H]ryanodine binding sites as compared with HSR.
No specific binding of either drug was seen in the sarcolemma or the
transverse tubule fraction, with the latter possessing the highest
concentration of [
H]PN200-110 binding sites.
Effects of pH, and Mono- and Divalent Cations, on
Specific [
The binding of [H]Dantrolene Binding to
HSR
H]ryanodine to
its receptor, the primary calcium release channel of the SR, is
affected by pH, divalent cations, and ionic
strength(11, 12, 37) . We therefore
investigated the effects of pH and increasing concentrations of NaCl,
KCl, Ca
, or Mg
on the specific
binding of [
H]dantrolene (200 nM) to
HSR, as described under ``Experimental Procedures.'' As shown
in Fig. 5, the optimum pH for
[
H]dantrolene binding is between 6.5 and 7.5,
with a rapid falloff in specific binding above pH 7.5. At pH 6.0,
dantrolene precipitates out of solution, and by pH 9.0 specific binding
is completely abolished. Both NaCl and KCl inhibit the binding of
[
H]dantrolene to HSR (Fig. 6), with
20-40% control binding evident at 150 mM. The specific
binding of [
H]dantrolene is not greatly inhibited
by Ca
or Mg
, as is shown in Fig. 7, although the patterns of the inhibition curves are
somewhat different. Calcium, at concentrations above 10
µM, inhibits specific binding to a maximum of
approximately 60% control values. On the other hand, the maximal
inhibitory effects of Mg
on
[
H]dantrolene binding also reach 60% control, but
at a concentration of 20 mM.
H]dantrolene binding. HSR membranes were
incubated with [
H]dantrolene (200 nM) in
the presence or absence of 150 µM azumolene at the
indicated pH and specific binding determined as described under
``Experimental Procedures.'' Buffers used were 20 mM Na-HEPES (pH 6.0-8.0) or 20 mM Tris-HCl (pH
7.5-9.5).
H]dantrolene binding to HSR. Specific
[
H]dantrolene binding to HSR was determined as
described under ``Experimental Procedures,'' in the presence
of increasing concentrations of NaCl or
KCl.
and Mg
on
[
H]dantrolene binding to HSR. Specific
[
H]dantrolene binding to HSR was determined in
the absence or presence of different concentrations of Ca
(panel A) or Mg
(panel B), as
described under ``Experimental
Procedures.''
release from the SR. Gronert et
al.(41) and Harrison (42) were the first to
report on the successful use of dantrolene in the treatment of porcine
MH. Soon after, dantrolene was shown to be effective in treating human
MH(43) , and the drug has been the mainstay of therapy since
that time. The effectiveness of dantrolene in treating MH has been
associated with the drug's ability to suppress the rise in
intramyoplasmic Ca
resulting from the triggering of
the syndrome(6, 44) . Although the molecular mechanism
of action of the drug is not yet known, its elucidation will help
define the regulation of Ca
release from skeletal
muscle SR. One experimental approach is to develop a pharmacological
binding assay that will ultimately lead to the identification of a
putative dantrolene receptor, via direct ligand-receptor interaction.
As a first step toward realization of this goal, we have developed an
assay demonstrating the binding of [
H]dantrolene
to a specific receptor(s) in skeletal muscle.
C]dantrolene (specific activity not reported)
to pig skeletal and cardiac muscle SR using equilibrium dialysis.
However, the receptor concentration (binding sites) used in their assay
(6 nM) is higher than the K
of
the high affinity site (5 nM), whereas ideally the
concentration should have been 0.1
K
to minimize ligand depletion(45) . Moreover, being
lipophilic, dantrolene binds nonspecifically to both biological
membranes as well as the membranes used for equilibrium dialysis
experiments (see ``Results''), and the authors make no
mention of the correction for, or even the extent of, nonspecific
binding. Indeed, White et al.(21) attempted to
reproduce the results of these experiments without success. Dehpour et al. (20) demonstrated binding of dantrolene to
rabbit skeletal muscle SR membranes using fluorescence techniques.
Their methods, however, were unable to distinguish specific from
nonspecific binding. Hence, to demonstrate specific binding of
dantrolene to a receptor, we have custom synthesized
[
H]dantrolene and developed a reproducible
binding assay that controls for nonspecific binding.
H]dantrolene binding assay were the poor
solubility of the ligand and its capacity to bind nonspecifically to
laboratory materials. Despite these obstacles, we have developed a
binding assay that, under the present experimental conditions,
indicates the presence of a single class of binding sites with
calculated K
and B
values of approximately 275 nM and 13 pmol/mg of
protein, respectively. In ligand binding assays, proper assessment of
nonspecific binding should be done at concentrations of unlabeled
congener at least 100 times, preferably 1,000 times, the estimated K
of the ligand(45) . Under our
assay conditions, dantrolene precipitates at concentrations
30
µM. Hence, we could not measure nonspecific binding by
maintaining 100-fold excess of unlabeled dantrolene at
[
H]dantrolene concentrations above 300 nM without inducing precipitation(25) . The specific binding
curves shown in Fig. 2and Fig. 3were generated after
determination of nonspecific binding in the presence of 150 µM azumolene rather than dantrolene. However, extrapolation of the
nonspecific binding curve generated in the presence of 25 µM dantrolene by linear regression analysis (INPLOT, r = 0.996) to values above 300 nM [
H]dantrolene and subtraction of those
calculated values from the directly determined total binding curve
yielded a [
H]dantrolene specific binding curve
virtually identical to that generated in the presence of azumolene
(data not shown). This demonstrates the validity of our assay using
azumolene to measure nonspecific binding.
H]dantrolene binding assay was the high
degree of nonspecific binding to skeletal muscle membranes. The
signal-to-noise ratio in the experiments from which Fig. 2B was generated varied between 8 and 15%, whereas, classically, this
value should be more than 50% and preferably
75%(46) .
Careful attention to experimental detail, however, has allowed us to
obtain highly reproducible results, even when results are compared
between different muscle preparations. The binding data shown in Fig. 2are the individual mean binding values ± S.E. from
four different experiments, carried out with HSR membranes obtained
from three different animals. The points lie on a single isotherm,
demonstrating both the reproducibility and the mathematical
defensibility of the results, despite the low signal-to-noise ratio.
H]dantrolene. This is evident in the higher
nonspecific binding of [
H]dantrolene seen when
ligand was dissolved in DMF rather than ethanol. Dantrolene, therefore,
may form a complex with DMF which allows for greater nonspecific
adsorption and/or dissolution into hydrophobic membrane components.
Alternatively, DMF may have a direct effect on the membrane itself,
resulting in an increase in the solubility of the drug in the
membrane's hydrophobic environment.
H]dantrolene binding was assessed
using inhibition assays with unlabeled dantrolene or known
physiologically active (azumolene) or inactive (aminodantrolene)
congeners(35, 47) . Both unlabeled dantrolene and
azumolene were equipotent as inhibitors of
[
H]dantrolene binding, paralleling their reported
equivalence in physiological studies(31, 48) .
However, the calculated K
value for
dantrolene is different from the calculated K
value despite the Cheng-Prusoff correction(36) ,
which is likely due to the limitations of the assay: namely, the low
specific activity of the radioactive ligand, the poor solubility of the
drug, and the relatively low signal-to-noise
ratio(22, 23) . Indeed, the concentration of the
labeled compound in competition binding assays should ideally be 0.1
K
(45) . However, because
of the low specific radioactivity of the synthesized
[
H]dantrolene, it is impossible to detect a
specific signal at the prescribed ligand concentration. Hence, we chose
a concentration of [
H]dantrolene (200
nM) which would give specific counts between 2,000 and 4,000
cpm/assay to obtain reproducible data. Experiments carried out at lower
[
H]dantrolene concentrations (25-50
nM) and higher membrane protein concentrations gave
inconsistent results because of increases in both nonspecific binding
and ligand depletion (data not shown). Despite these limitations, the
results described above indicate that the binding of
[
H]dantrolene to its putative receptor in
skeletal muscle SR is indeed specific and that the binding activity
parallels reported physiological activity.
H]dantrolene binding sites
was assessed using partially purified membrane fractions corresponding
to the four major subcellular membrane regions in skeletal muscle
involved in ECC. We found that the [
H]dantrolene
binding sites were most abundant in HSR followed by LSR, whereas they
were absent from the transverse tubule and sarcolemma fractions. The
distribution of [
H]dantrolene binding among these
subcellular fractions parallels that of
[
H]ryanodine binding. This leads us to suggest
that the binding sites for the two ligands either have opposite actions
on the same receptor, i.e. the RyR, or that the receptors for
the two ligands are separate molecules that interact indirectly. The
fact that no binding of [
H]dantrolene is detected
in the transverse tubule fraction makes it extremely unlikely that
dantrolene exerts its effects by inhibiting the action of the DHPR
during ECC directly. This diminishes the significance of earlier
results showing that azumolene, at very high concentrations, inhibits
the binding of the dihydropyridine antagonist
[
H]PN200-110 to transverse tubule
membranes(49) .
H]dantrolene binding were determined by varying
assay solution conditions. These studies allowed us to compare the
binding conditions with those reported for
[
H]ryanodine binding to the RyR. Maximal
[
H]dantrolene binding to its receptor occurs in
the pH range 6.5-7.5, whereas maximal
[
H]ryanodine binding occurs around pH
8.5(50) , a value at which [
H]dantrolene
binding is profoundly inhibited (Fig. 5). Further,
[
H]dantrolene binding to the SR requires no
Ca
and is not affected even when the buffer in our
binding assay is supplemented with 1 mM EGTA (data not shown).
On the other hand, the lack of Ca
in the binding
buffer results in 99% inhibition of [
H]ryanodine
binding to the RyR(50, 51) . High concentrations of
Ca
in the range that maximally stimulates
[
H]ryanodine binding (50, 51) only partially inhibit
[
H]dantrolene binding to HSR (Fig. 7A). The latter is both physiologically and
pharmacologically fortuitous, for were dantrolene binding inhibited by
Ca
, dantrolene could not be effective in reversing
MH. Myoplasmic Ca
concentrations during an episode
have been demonstrated to reach sustained concentrations of at least
7-10 µM(44, 52) . High ionic
strength is essential for [
H]ryanodine binding to
the RyR(38, 51) , whereas the same inhibits
[
H]dantrolene binding (Fig. 6). Yet, the
extents of both [
H]dantrolene and
[
H]ryanodine binding are similarly inhibited by
Mg
to 60% of control values (see (51) and Fig. 7B). These results indicate that the in vitro requirements for dantrolene and ryanodine binding to their
receptor sites are distinct. They do not distinguish whether the
proposed modulation of the RyR by dantrolene is by a direct or indirect
mechanism.
H]dantrolene binding assay. Because
of the low signal-to-noise ratio evident in our binding assay, we had
set the following criteria to convince ourselves of the validity of our
conclusions. First, the assay must be reproducible, the results for
total and nonspecific binding must be significantly different from each
other, and the specific binding curve must fit a mathematically
defensible model for receptor-ligand interactions. Second, the assay
must demonstrate pharmacological specificity, i.e. active
congeners of dantrolene should inhibit the binding of
[
H]dantrolene, whereas inactive congeners should
not. Third, specificity should be evident in the subcellular
distribution of binding. The evidence presented herein fulfills these
criteria and suggests that the dantrolene binding site modulates the
opening of the RyR, directly or indirectly. Future studies will be
directed at distinguishing whether the binding site for
[
H]dantrolene is on the RyR or on a separate,
regulatory molecule.
We acknowledge gratefully the many discussions,
suggestions, and assistance of Drs. H. M. Geller, D. J. Wolff, and M.
P. McCarthy which contributed directly to this work and Drs. P. J.
Munson and H. L. Weiner for discussions relating to data analysis and
binding studies for hydrophobic compounds. We are beholden to Drs. Tom
Schwan (retired) and T. J. Moorehead of Proctor & Gamble
Pharmaceuticals for many helpful discussions on the chemistry and
pharmacology of dantrolene and its congeners. We sincerely appreciate
the continued support of Dr. Sanford L. Klein and colleagues of the
Department of Anesthesia, UMDNJ-RWJMS, and we are indebted to Drs. S.
B. Horwitz and N. Ron Morris for continued encouragement.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.