Interaction of D-600 with the transmembrane domain of the
sarcoplasmic reticulum Ca2+-ATPase
Alicia
Ortega1,2,3,
V.
M.
Becker1,
R.
Alvarez1,
J. R.
Lepock2, and
H.
Gonzalez-Serratos3
1 Departamento de Bioquímica, Facultad de Medicina,
Universidad Nacional Autónoma de México, Mexico City 04510, México; 2 Departments of Biology and Physics, University
of Waterloo, Waterloo, Ontario, Canada N2L 3GI; and 3 Department
of Physiology, University of Maryland, Baltimore, Maryland
21201
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ABSTRACT |
Experiments were performed
to determine whether the organic Ca2+ channel blocker D-600
(gallopamil), which penetrates into muscle cells, affects sarcoplasmic
reticulum (SR) Ca2+ uptake by directly inhibiting the light
SR Ca2+-ATPase. We have previously shown that at 10 µM,
D-600 inhibits LSR ATP-dependent Ca2+ uptake by 50% but
has no effect on ATPase activity (21). These data suggest
that the SR Ca2+-ATPase might be a potential target for
D-600. The ATPase activity of the enzyme is associated with its
hydrophilic cytoplasmic domain, whereas Ca2+ binding and
translocation are associated with the transmembrane domain
(18). In the present experiments, we determined which of the two domains of the ATPase is affected by D-600. Thermal
inactivation experiments using the SR Ca2+-ATPase
demonstrated that D-600 decreased the thermal stability of
Ca2+ transport but had no effect on the stability of ATPase
activity. In addition, D-600 at a concentration of 160 µM did not
have any leaking effect of Ca2+ on the
Ca2+-loaded SR. Thermal denaturation profiles of SR
membranes revealed that D-600 interacts directly with the transmembrane
domain of the Ca2+-ATPase. No evidence for interaction with
the nucleotide domain was obtained. We conclude that the
Ca2+ blocker D-600 inhibits the SR Ca2+ pump
specifically by interacting with the transmembrane
Ca2+-binding domain of the Ca2+-ATPase.
calcium channel blocker; thermal denaturation profiles; transmembrane domain
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INTRODUCTION |
IN SKELETAL MUSCLE
CELLS both the magnitude and time course of the
contraction-relaxation cycle depend on the levels of the cytosolic-free
Ca2+ concentration, which is controlled by the transverse
tubules and sarcoplasmic reticulum (SR) membranes. It has been shown
that in skeletal muscle nearly all the voltage-dependent
Ca2+ channels are localized in the transverse tubular
membrane system (1, 2). The voltage-dependent
Ca2+ channels give rise to inward Ca2+ currents
(ICa) that can be inhibited by a diverse class
of organic compounds, conventionally grouped together as
Ca2+ antagonists or Ca2+ channel blockers. The
interaction of such drugs with voltage-dependent Ca2+
channels has been extensively studied (9-11). It has
been proposed that organic Ca2+ channel antagonists exert
their inhibitory effect by promoting a type of gating in which the
channels are unable to open. Colvin et al. (5) and Wang et
al. (24) have shown that relatively high concentrations
(0.1 and 3 mM) of verapamil, an organic Ca2+ blocker
from the fenylalkylamine series, may interact with the SR.
However, they did not determine the mechanism of interaction between
verapamil and the SR.
We have previously reported that another type of organic
Ca2+ channel blocker, diltiazem, produces up to an 80%
twitch potentiation in frog skeletal muscle cells (14). We
attributed this potentiation to a decrease in the rate of
Ca2+ uptake by the SR. This hypothesis was confirmed using
split muscle fibers in which we showed that diltiazem tends to maintain
a high level of cytosolic-free Ca2+ by exerting an
inhibitory effect on Ca2+ uptake by the SR
(12).
The Ca2+ antagonist D-600 is more frequently used than
other Ca2+ channel antagonists to study its effect on
skeletal muscle ICa, excitation-contraction
coupling, and the mechanisms by which they may produce these effects
(1, 2, 6, 8,
12-15, 19, 22). For this
reason and for our previous study (21), we chose D-600. It is interesting to note that D-600 has been used more despite
the fact that other organic Ca2+ channel blockers are more
efficient and widely used in clinical applications. We have recently
reported an inhibitory effect of D-600 on force development in skinned
skeletal muscle fibers and on Ca2+ uptake by isolated SR
(22). These observations, along with the fact that D-600
penetrates into cells (12), may explain the facilitation
of excitation-contraction coupling seen with D-600 by other researchers
(6).
The present study was undertaken to determine whether the inhibitory
effect of D-600 on Ca2+ uptake by the SR is caused by a
direct interaction with the SR Ca2+-ATPase. The experiments
were performed in isolated light SR (LSR) vesicles from rabbit skeletal
muscle in which ATPase, Ca2+ uptake activities, and
Ca2+ leakage were measured. We also determined the effect
of D-600 on the denaturation profile of the Ca2+- ATPase
in LSR. This paper provides evidence of a direct interaction between
D-600 and the transmembrane domain of the SR Ca2+-ATPase.
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MATERIALS AND METHODS |
Preparation of LSR.
Microsomes, isolated from rabbit skeletal muscle, were loaded on a
discontinuous sucrose gradient composed of three layers, 25, 27.5, and
35% (wt/vol), containing 20 mM Tris-malate and 1 mM dithiothreitol
(DTT), pH 6.8. The microsomes were then centrifuged for 14 h at
23,000 rpm. The lighter bands were discarded, and the pellet was washed
and loaded on the top of a second discontinuous sucrose gradient of 28, 32, 35, and 45% (wt/vol). The fraction separated at the 32/35%
interface, which contains the highest specific Ca2+-ATPase
activity (21), was collected. This fraction is hereafter referred to as LSR. LSR has a protein content of ~90%
Ca2+-ATPase.
ATPase activity.
ATPase activity in LSR was determined by a colorimetric
technique, based on the reaction between Pi and malachite
green (15). Aliquots of 0.003 mg/ml LSR were incubated in
a solution containing (in mM): 0.1 CaSO4 or 1 EGTA, 5 MgSO4, 77 potassium methanosulfonate, 20 Tris-malate, and 1 NaATP, pH 6. The reaction was stopped after 30 min of reaction
with a solution containing 0.045% hydrochloride malachite green, 4.2%
ammonium molybdate in 4 N HCl, 0.8 ml Triton X-100 (10% for each 100 ml of solution), and 0.25 ml sodium citrate (34%), and the absorbance
was read at 660 nm.
Calcium uptake.
SR Ca2+ uptake was determined using the metallochromic
indicator Arsenazo III in a Cl
-free solution containing
(in mM) 0.1 CaSO4, 5 MgSO4, 77 potassium methanosulfonate, 20 Tris-malate, 1 NaATP, and 0.7 Arsenazo III, pH
6.8, as previously described (4). We used sulfates mainly because intracellular chlorine in mammalian and in amphibian
skeletal muscle is very low (7) and Cl
triggers Ca2+ release from SR (20).
Ca2+ transport was determined from the change in absorbance
at 660 nm. SR protein (0.05 mg/ml) was added to the reaction solution, and after a 30-s incubation, the reaction was started by adding 1 mM
ATP. Ca2+ uptake was measured in parallel with the above
reaction solution, but using 1 µCi of
45CaCl2. The Ca2+ remaining in the
LSR vesicles was determined by filtration, and the radioactivity was
measured using a scintillation counter.
Thermal inactivation.
LSR membranes (2 mg/ml) were incubated in 10% sucrose containing (in
mM) 1 DTT, 20 Tris-malate, and 1 CaSO4, pH 6.8, and were heated at one of several fixed temperatures in a water bath for 1, 3, 5, 7, or 10 min. Ca2+-ATPase activity and ATP-dependent
Ca2+ transport at room temperature (25°C) were determined
after 30 min of reaction and plotted as a function of incubation time
at temperatures between 37 and 53°C. Thermal inactivation and thermal denaturation were used to demonstrate the nature of the interaction between D-600 and the LSR Ca2+-ATPase. The values for
Ca2+ transport and ATPase activity were normalized with
respect to the first value obtained at each temperature after 1 min of
incubation. The rate of inactivation (i.e., conversion of active to
inactive enzyme) is indicated by the rate constant for inactivation,
k, which varies with temperature according to the Arrhenius
relation
where A is the Arrhenius constant defined as the
frequency factor, EA the activation energy,
R the gas constant, and T the absolute temperature.
The inactivation temperature (Ti) is defined as the
temperature resulting in half inactivation when the temperature is
increased at a rate of 1°C/min. For the thermal inactivation
experiments, we heated the sample at a temperature between 35 and
70°C. The value of Ti reflects the sensitivity of the
Ca2+-ATPase to thermal inactivation. Ti was
calculated using Eq. 1, which transforms the rate of
inactivation, determined by measurements of activity as a function of
time at a constant temperature, to inactivation, determined by
measurements of activity as a function of temperature when this is
increasing at a constant rate (18). Ti was
determined so that a direct comparison could be made with the
characteristics of denaturation, determined by differential scanning
calorimetry at a scan rate of 1°C/min (see below) and described by
Tm (the temperature of half denaturation).
Differential scanning calorimetry.
The thermal denaturation profile of LSR membranes was determined by
differential scanning calorimetry (DSC). A high-resolution Microcal MC2
DSC was used to obtain all scans. SR membranes (8-10 mg/ml) were
heated at a rate of 1°C/min from 10 to 100°C. The samples were then
cooled to 10°C and rescanned. For the DSC experiments, SR was
suspended in the absence or presence of 10 to 200 µM of D-600 in the
solution used for thermal inactivation. Denaturation was completely
irreversible after scanning up to 100°C. Intrinsic baseline curvature
was corrected by subtracting the rescan from the scan data, and a
correction was made for the shift in specific heat on denaturation
(Cp), as previously described (18).
DSC scans were deconvoluted assuming irreversible denaturation. This procedure requires that denaturation and inactivation can be
approximated by a two-state reaction of the form
that obeys pseudo first-order kinetics, and where the
temperature dependence of the rate constant k is given by
the Arrhenius relation. These assumptions have been shown to hold for
the Ca2+-ATPase of the SR (18). The fraction
of each component denatured or inactivated as a function of increasing
temperature at a constant rate (fD) is given as
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(1)
|
where Tc is the temperature at which
k = 1, t is time, and v is the
scan rate. The derivative of fD as a
function of temperature is proportional to the excess
Cp. The curves of excess
Cp as a function of temperature were
deconvoluted into individual components using a recursive minimization
routine (18). The Tm in the DSC of each
component is defined as the temperature at which the area under the
individual curve for each component is one-half of the total area.
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RESULTS |
The effect of 10 µM D-600 on the kinetics of ATP-dependent
Ca2+ uptake by LSR is illustrated in Fig.
1. The complete time course of
Ca2+ uptake is inhibited by 50% at 30 min. Such an
inhibition of Ca2+ uptake could reflect an interaction of
D-600 with either the Ca2+-ATPase nucleotide-binding
domain, with a subsequent inhibition of ATP hydrolysis, or the
Ca2+-ATPase transmembrane Ca2+-binding domain,
with subsequent inhibition of Ca2+ translocation. The
Ca2+-ATPase activity in LSR was measured to determine
whether D-600 affects the ATP hydrolytic activity. As shown in Fig.
2, D-600, at concentrations up to 160 µM, had no inhibitory effect on the ATPase activity. This result
suggested that the interaction of D-600 with the ATPase might be at a
site other than the nucleotide-binding site without affecting the ATP
hydrolytic activity.

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Fig. 1.
Effect of 10 µM of D-600 on ATP-dependent
Ca2+ uptake by light sarcoplasmic reticulum (LSR) as a
function of time ( ). ATP-dependent Ca2+
uptake is shown in the absence of D-600 ( ). Aliquots of
0.05 mg/ml of LSR were incubated in a medium containing (in mM): 5 MgSO4, 77 potassium methanosulfonate, 20 Tris-malate, 2 NaATP, and 0.1 CaSO4 in the presence of 1 µCi
45[Ca2+], pH 6.8. For further details see
MATERIALS AND METHODS. Values are means ± SE
(n = 5). SE bars in the presence of D-600 are within
the symbols.
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Fig. 2.
Ca2+-ATPase activity as a function of D-600
concentration. Aliquots of 0.003 mg/ml of LSR vesicles were incubated
during 30 min in a solution containing (in mM) 0.1 CaSO4, 5 MgSO4, 77 potassium methanosulfonate, 20 Tris-malate, and 2 NaATP, pH 6.8. Values are means ± SE (n = 5).
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D-600 potentiates the thermal inactivation of Ca2+
uptake, but not ATPase activity.
To investigate in more detail whether D-600 interacts with the
ATP-binding domain or the Ca2+-binding domain, we used
thermal analysis techniques (i.e., thermal inactivation and
differential scanning calorimetry) that have been previously used to
distinguish between these domains in the Ca2+-ATPase of the
SR (18). To determine whether D-600 (10-320 µM) has
a direct effect on Ca2+-uptake activity, we performed
thermal inactivation experiments on LSR by measuring how much of the
activity at 25°C remained after LSR membranes were incubated for 1, 3, 5, 7, or 10 min at four different temperatures. Figure
3 illustrates the behavior of thermal
inactivation of Ca2+ uptake in the absence (A)
and presence (B-G) of D-600. The activity decays
exponentially with time, demonstrating pseudo first-order kinetics.
Figure 3 also shows that the rate of inactivation (slope) of
Ca2+ uptake is temperature dependent. At any temperature,
inactivation was faster in membranes incubated with D-600 than in those
incubated without the drug.

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Fig. 3.
Thermal inactivation of Ca2+ uptake (activity
vs. time). Aliquots of 0.05 mg/ml of LSR vesicles were incubated for
different times at 4 different temperatures (indicated within each
panel in °C) in the absence (A) or presence
(B-G) of the following micromolar concentrations of D-600:
(B) 10, (C) 20, (D) 40, (E)
80, (F) 160, and (G) 320. The incubation solution
contained (in mM) 0.1 CaSO4, 5 MgSO4, 77 potassium methanosulfonate, and 20 Tris-malate, pH 6.8. The reaction
was started by the addition of 1 mM NaATP.
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The rate constants of inactivation of Ca2+ uptake shown in
Fig. 3 were used to obtain Arrhenius plots (Fig.
4). It is clear from these plots that the
rate of thermal inactivation increases dramatically as the
concentration of D-600 is increased. Linear regression analysis was
used to obtain the activation energy (EA), corresponding to the slope of the curves, and the frequency factor (A), corresponding to the intercept.

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Fig. 4.
Arrhenius plots of the rate constants (obtained
from Fig. 3) for inactivation of LSR Ca2+ uptake. Plots are
shown for control membranes (C) and for membranes incubated with
increasing concentrations of D-600 (10-320 µM), as
indicated.
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Figure 5 shows the predicted plots
of the derivative of fractional inactivation as a function of
temperature (dfD/dT). These plots
correspond to the DSC profiles predicted for the protein domain, of
which denaturation is responsible for inactivation of Ca2+
uptake. The values of EA, A, and
Ti, where Ti corresponds to the temperature of
half-inactivation if temperature were to be increased at 1°C/min, are
given in Table 1. Ti was
determined from the fD, expressed as a function
of temperature (fD vs. T), and
calculated from the values of EA and A
obtained from the Arrhenius plots using Eq. 1. As shown
in Table 1, the Ti for Ca2+ uptake in the
absence of D-600 was 53.2 [0.8°C (n = 3)].
Inactivation proceeded with a high-activation energy in excess of 500 KJ/mol, suggesting that a protein conformational change such as partial unfolding is responsible for the inactivation. This table also shows
that 10 µM of D-600 decreased the Ti for inactivation of Ca2+ uptake by 4.9°C, from 53.2 to 48.3 [0.9°C
(n = 3)]. As the concentration of D-600 was increased
up to 160 µM, Ti progressively decreased, whereas
EA remained at a relatively constant value,
suggesting that the mechanism of inactivation remains unchanged at the
concentrations tested. When a concentration of 320 µM of D-600 was
used, a sharp decrease in EA and Ti
occurred. These results suggested that at very high concentrations of
D-600, the mechanism of inactivation of Ca2+ uptake
changes, probably due to an increase in the passive permeability of
Ca2+ from the SR.

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Fig. 5.
The derivative of fractional inactivation of
Ca2+ uptake as a function of temperature
(dfD/dT). The curves were calculated
from the EA (slope) and Arrhenius constant
A (intercept) obtained from Fig. 4; they correspond to LSR
in (A) 0, (B) 10, (C) 20, (D) 40, (E) 80, (F) 160, and
(G) 320 µM D-600.
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To rule out the possibility that the effect of D-600 on
Ca2+ uptake was due to Ca2+ leakage, we
measured passive Ca2+ permeability. Figure
6 shows the amount of Ca2+
remaining in LSR vesicles loaded in the absence of D-600 as a function
of time. Ca2+-loaded vesicles were diluted in a solution
containing 2 mM EGTA in the absence and presence of D-600. D-600 had no
effect on passive leakage at a concentration of 160 µM. However, 320 µM of D-600 produced a sharp decrease in intravesicular
Ca2+. Although Ca2+ leaks faster in the
presence of 320 µM, this does not appear to be the consequence of
membrane disruption because addition of the ionophore A-23178 produced
an immediate and full Ca2+ leakage.

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Fig. 6.
Effect of D-600 on Ca2+ efflux from LSR in 1 mM EGTA as a function of time. Aliquots of 0.05 mg/ml of LSR were
loaded with 100 µM CaCl2 and 1 mM ATP at room temperature
during 30 min (MATERIALS AND METHODS). Symbols represent
uptake in the absence of D-600 ( ), in the presence of
160 µM D-600 ( ), in the presence of 320 µM D-600
( ), and in the presence of 1 mM ionophore A-23187
( ). The line represents the best fit curve.
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To provide further evidence of a direct interaction between D-600 and
the transmembrane domain of the Ca2+-ATPase, which explains
the inhibition of Ca2+ uptake, DSC was used to determine
the actual site of interaction.
Differential scanning calorimetry.
Lepock et al. (18) have shown that the DSC profile
obtained from highly purified LSR membranes has two peaks (endotherms) corresponding to the denaturation of the cytosolic nucleotide-binding and the transmembrane Ca2+-binding domains of the
Ca2+-ATPase. Based on these data, we also used LSR
membranes to obtain a set of ATPase DSC denaturation profiles at
different D-600 concentrations. Figure 7
shows the denaturation profile of LSR in which the Tm values were determined by deconvolution of the two endotherms as
previously described (18). Ca2+-ATPase
denatures in two major endotherms. The main component, A,
which corresponds to the denaturation of the cytosolic
nucleotide-binding domain, had a Tm of 50°C. This
component was not affected by D-600. The second component,
B, which corresponds to the denaturation of the
transmembrane Ca2+-binding domain, had a Tm of
60 [1.01°C (n = 3)]. The Tm of
component B was shifted to lower temperatures in the
presence of D-600 (10-250 µM). A concentration of 100 µM
shifted the Tm of component B up to the point at
which it overlapped with the Tm of component A. However, this concentration of D-600 did not affect the thermal stability of the nucleotide-binding domain.

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Fig. 7.
Differential scanning calorimetry profiles [excess
specific heat on denaturation (Cp) vs.
temperature] of LSR. LSR membranes (8-10 mg/ml) were scanned with
temperature increasing at a rate of 1°C/min in a solution containing
10% sucrose, 1 mM dithiothreitol, and 20 mM TES, either without D-600
(solid line) or with 10 µM (dashed line), 20 µM (dot-dashed line),
or 80 µM (dotted line) D-600. Peaks A and B are
labeled (see text for additional description).
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The transition temperatures at various concentrations of D-600
calculated from the denaturation profile of the Ca2+-ATPase
component B are given in Table
2. These results show that the
transmembrane domain is the ATPase component affected by
D-600. The effect of D-600 on component B is dose dependent at the same concentrations in which Ca2+ uptake is
inhibited.
In addition to the endotherms described for components A and
B, an endotherm at ~95°C was detected in some of the
scans. This is likely due to the denaturation of an additional protein
component. An exotherm (valley) in the region of 70-75°C
occurred at high D-600 concentrations. The underlying cause of this
exotherm is unknown.
To show that the effect of D-600 on the LSR function, specifically on
the Ca2+ uptake, is related to a conformational change in
the transmembrane domain of the ATPase, we plotted the changes in
Tm of component B and the changes in
Ti for Ca2+ uptake, both as a function of D-600
concentration (Fig. 8). Qualitatively, the curves for Tm and Ti are similar; however,
Ti values are higher than Tm values. These
thermal analyses results demonstrate that D-600 interacts with the
transmembrane domain of the SR Ca2+-ATPase, causing a
conformational change that may be responsible for sensitizing the
thermal inactivation of Ca2+ uptake in LSR.

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Fig. 8.
Decrease in temperature of half denaturation
(Tm) for peak B (see Fig. 7) as a function of
D-600 concentration ( ), obtained from the
differential scanning calorimetry profiles shown in Fig. 7. Decrease in
inactivation temperature (Ti) for the inactivation of
Ca2+ uptake in isolated SR ( ), obtained
from the curves in Fig. 3. The ordinate shows change (decrease) from
the value obtained in the absence of D-600.
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DISCUSSION |
Our results show that D-600 1) inhibits
Ca2+ uptake in isolated LSR membranes in a dose-dependent
manner, 2) does not affect the hydrolytic activity of the
Ca2+-ATPase at doses in which Ca2+ uptake is
inhibited, 3) does not affect LSR passive permeability of
Ca2+ at concentrations up to 160 µM, 4)
decreases the thermal stability of the ATP-dependent
Ca2+-uptake activity in a dose-dependent manner, and
5) decreases the thermal stability of the transmembrane
domain of the Ca2+-ATPase in a dose-dependent manner.
We conclude that the inhibition of Ca2+ uptake by D-600 is
not the consequence of Ca2+ leakage because membrane
permeability is not affected by the drug (Fig. 6). These results also
lead us to conclude that the organic Ca2+ channel blocker
D-600 interacts directly with the Ca2+-ATPase at a
concentration in which muscle fiber contractility is also affected
(21). Assessing from the DSC results and the ATPase
hydrolytic activity, D-600 does not affect the nucleotide-binding domain of the ATPase. Instead, the impaired thermal stability of
Ca2+ uptake and the impaired Ca2+-binding
domain indicate that D-600 interacts with the ATPase transmembrane
domain. These results confirm and extend our previous finding that
D-600 affects SR Ca2+ loading in skinned muscle fibers
(21). The failure of D-600 to inhibit caffeine-induced
Ca2+ release from isolated junctional sarcoplasmic
reticulum strongly supports the premise that the SR
Ca2+- ATPase, rather than the SR Ca2+
channel, is affected by this drug (21).
Lepock et al. (18) showed that in the absence of
Ca2+, the LSR Ca2+-ATPase denatures as a single
component with a Tm of 48-49°C, and in the presence
of Ca2+, a second component appears that denatures at a
Tm of 60°C. These transitions in temperature represent
the denaturation of the nucleotide-binding domain of the
Ca2+-ATPase (detectable by DSC and with FITC in the absence
of Ca2+) and denaturation of the Ca2+-binding
domain (detectable by DSC and by tryptophan fluorescence in the
presence of Ca2+), respectively (18). Previous
studies by Lepock et al. (18) and Cheng and Lepock
(3) have shown that a conformational change in a region of
the protein closely associated with the Ca2+-binding sites
in the transmembrane domain causes uncoupling of Ca2+
transport from ATP hydrolysis. This may be due to the
unfolding of a conformationally flexible site involved in
Ca2+ translocation, but not in ATP hydrolysis before the
complete unfolding of the Ca2+-ATPase, as has been
suggested to occur for a number of other enzymes (23).
These data support our conclusion that D-600 specifically affects the
transmembrane domain of the Ca2+-ATPase containing the
Ca2+-binding sites.
The experiments presented here further support our previous results
obtained in skeletal muscle fibers (21, 22)
that demonstrate that D-600 has a twitch-potentiating effect. This
twitch potentiation (22), observed despite the inhibitory
effect of D-600 on contractures at low temperatures (8),
shows that early steps in the excitation-contraction coupling chain of
events remain functional in the presence of D-600. We had previously
suggested that D-600 affects muscle contraction by exerting an
inhibitory effect on the SR Ca2+-ATPase (22).
A potential mechanism is that D-600 penetrates into the myoplasm in
intact fibers and interferes with the excitation-contraction coupling
mechanism by inhibiting the SR Ca2+-ATPase. As a result,
myoplasmic Ca2+ is not completely sequestered into the SR
after each twitch. Consequently, cytosolic Ca2+ increases
slightly after each activation and twitches are thus potentiated
(19, 22).
Colvin et al. (5) reported that high concentrations (1 mM)
of Ca2+ channel blockers (dihydropyridines) stimulated the
Ca2+-ATPase activity in isolated SR from cardiac and
skeletal muscle, but no such effect was observed at lower
concentrations. Wang et al. (24) also observed that
verapamil, felodipine, and diltiazem at a concentration of 40 µM
produced a 40-60% activation of the SR Ca2+-ATPase.
As we show in the present studies, 320 µM of the organic Ca2+ channel blocker D-600 causes LSR Ca2+
leakage (Fig. 6). Thus the data of Colvin et al. (5) and
Wang et al. (24) could not be interpreted as the result of
an increased SR Ca2+ leakage caused by extremely high
Ca2+ channel blocker concentrations that produce a
stimulated activity of the Ca2+-ATPase. Another consequence
of the increased Ca2+ permeability at 320 µM of
D-600 is a decrement in the EA of the Ca2+-uptake activity (Fig. 5). These increments in the
ATPase activity produced by these Ca2+ channel blockers are
not consistent with the fact that at the same doses, these
Ca2+ channel blockers inhibit Ca2+ uptake in SR
(5, 19-22, 24). We recently
found that 10-160 M diltiazem does not have any effect on the
Ca2+-ATPase activity in SR of fast-twitch rabbit skeletal
muscle (12). Another consequence of the increased
passive Ca2+ permeability at 320 µM of D-600 is a
decrement in the activation energy, EA, of the
Ca2+-uptake activity (Fig. 5).
Based on the observations described in this paper, we propose that the
effect of <80 µM D-600 on skeletal muscle fiber contractility can be
explained by a direct interaction with the LSR Ca2+-ATPase,
specifically with the transmembrane Ca2+-binding domain.
This is in agreement with the fact that D-600, similar to other
Ca2+ channel blockers, is hydrophobic and capable of being
incorporated into the membrane matrix (15). Our results,
however, cannot distinguish whether D-600 binds to the transmembrane
helices of the ATPase or disrupts the lipid bilayer neighboring the
transmembrane domain, subsequently interfering with Ca2+
translocation. Our results also show that D-600, at concentrations up
to 160 µM, does not have any effect on SR Ca2+ leakage.
This strongly suggests that D-600 does not affect the lipid bilayer of
the SR membrane, but rather affects the area closely associated with
the transmembrane domain.
We cannot exclude the possibility that, in addition to its effect on
Ca2+-uptake inhibition (22), D-600 may also
inhibit the T-tubular membrane voltage sensor with a mechanism similar
to that described for Ca2+ channel blockage in single
dialyzed heart cells (17). Thus the effect of D-600 in
skeletal muscle appears to be complex. In addition to its
Ca2+ channel-blocking effect, which has been shown to play
no role in excitation-contraction coupling (2,
13), D-600 also inhibits the SR Ca2+ transport
system. The inhibition of the Ca2+- ATPase by D-600 could
by itself explain the effects of this drug on the
excitation-contraction coupling mechanism.
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ACKNOWLEDGEMENTS |
We gratefully acknowledge Miriam Gitler of NOVA Research for the
review of this manuscript and her comments.
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FOOTNOTES |
This work was supported by Grant DGAPA IN218397, Universidad Nacional
Autónoma de Mexico, Mexico (A. Ortega), by the Natural Sciences
and Engineering Research Council of Canada (J. R. Lepock), and by
National Institute of Neurological Disorders and Stroke Grant
R01-NS-17098 (H. Gonzalez-Serratos).
Address for reprint requests and other correspondence: A. Ortega, Dept. de Bioquímica, Facultad de Medicina,
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Received 14 April 1999; accepted in final form 24 January 2000.
 |
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