(Received for publication, May 26, 1995; and in revised form, August 17, 1995)
From the
Hydrogen peroxide (HO
) at millimolar
concentrations induces Ca
release from actively
loaded sarcoplasmic reticulum vesicles and induces biphasic
[
H]ryanodine binding behavior. High affinity
[
H]ryanodine binding is enhanced at
concentrations from 100 µM to 10 mM (3-4-fold). At H
O
concentrations
greater than 10 mM, equilibrium binding is inhibited.
H
O
decreased the k
for [
H]ryanodine binding by increasing
its association rate, while having no effect on the rate of
dissociation of [
H]ryanodine from its receptor.
H
O
(1 mM) also reduced the EC
for Ca
activation from 632 nM to 335
nM. These effects were completely abolished in the presence of
catalase, ruthenium red, and/or Mg
(mM).
H
O
-stimulated
[
H]ryanodine binding is not further enhanced by
either doxorubicin or caffeine. The direct interaction between
H
O
and the Ca
release
mechanism was further demonstrated in single-channel reconstitution
experiments. Peroxide, at submillimolar concentrations, activated the
Ca
release channel following fusion of a sarcoplasmic
reticulum vesicle to a bilayer lipid membrane. At millimolar
concentrations of peroxide, Ca
channel activity was
inhibited. Peroxide stimulation of Ca
channel
activity was reversed by the thiol reducing agent dithiothreitol.
Paralleling peroxide induced activation of ryanodine binding,
Ca
transport, and single Ca
channel
activity, it was observed that the ryanodine receptor formed large
disulfidelinked protein complexes that dissociated upon addition of
dithiothreitol.
Reactive oxygen species (ROS) have been shown to
mediate various pathological conditions in a variety of
tissues(1) . These molecular oxygen-derived intermediates may
be generated either by electron reduction or energy activation (i.e. light). Among these ROS include hydrogen peroxide
(H
O
), singlet oxygen (
O
), hypochlorous acid (HOCl), superoxide
radical (O
), and the hydroxyl radical (
OH).
In various muscle types, ROS have been
implicated in alteration of normal Ca homeostasis via
disruption of normal sarcoplasmic reticulum (SR) function. This may be
accomplished by inhibiting the Ca
ATPase pump and/or
by activating the Ca
release channel. A number of
studies have detailed the effects of one or more ROS on whole muscle
tissue or on isolated sarcoplasmic reticulum (SR) derived from smooth,
cardiac, and/or skeletal
muscles(2, 3, 4, 5, 6, 7, 8, 9, 10) .
Treatment of these tissues with ROS attempt to induce oxidative stress
similar to that experienced during ischemia and/or reperfusion. In
smooth muscle, O
has been shown to
inhibit both Ca
-ATPase activity and Ca
uptake into the SR while stimulating inositol
1,4,5-trisphosphate-induced Ca
release (3, 4) . In cardiac muscle, HOCl reduced contractile
function (7) and inhibited SR Ca
uptake(11) . On a more microscopic level,
H
O
activated cardiac SR Ca
channel gating activity at mM concentrations(9) . Superoxide also decreased
Ca
uptake into cardiac SR vesicles by increasing the
Ca
permeability of the SR via opening the SR
Ca
release channel(2) . These studies
indicate that, while it is not the only intracellular organelle
effected, the SR is a likely target for ROS in these tissues.
Reduced oxygen species may also interfere with skeletal muscle
function. Reperfusion of ischemic muscle, vitamin E deficiency, and
muscular dystrophy are important pathological conditions where ROS have
been implicated in cytotoxic damage(12) . Reactive oxygen
intermediates generated during repetitive exercise have also been shown
to promote muscle fatigue(13) . Despite this evidence, only a
few studies have examined the direct effect of ROS on skeletal muscle
and SR function. The oxidant peroxydisulfate and the OH radical both inhibited Ca
uptake
into isolated SR vesicles, while peroxydisulfate also increased the
Ca
permeability of the SR membrane(8) . In
other work describing the effect of sulfhydryl oxidizing reagents on SR
Ca
release, HOCl, and the highly reactive
O
, were shown to stimulate Ca
release from actively loaded SR
vesicles(10, 14) . Calcium release induced by ROS was
shown to be blocked by SH reducing agents and ruthenium red, indicating
the SR Ca
release channel was a primary target for
their interaction.
Although the mechanism underlying
excitation-contraction coupling in skeletal muscle is poorly
understood, oxidation of thiols has been shown to regulate the SR
Ca release channel, which mediates Ca
release preceding muscle contraction(14) .
Oxidation-induced opening of the SR Ca
channel has
also been confirmed at the single-channel level following the fusion of
SR vesicles to planar lipid bilayer membranes (BLM)(15) . In
this report, we show that the cellular oxidant,
H
O
, modifies the SR Ca
release channel. Moreover, activation of the Ca
release channel by H
O
appears to result
from an oxidation of key thiol groups to a disulfide. These results may
be significant in understanding muscle cell dysfunction under oxidative
stress and may provide insight into the molecular regulation of the
Ca
release channel.
Figure 1:
The effect of
HO
on Ca
-dependent ATPase
activity, A23187-stimulated ATPase activity, the rate of Ca
uptake, and the amount of Ca
uptake. SR (1
mg/ml) was incubated at different H
O
concentrations for 1 min prior to conducting the various assays. The
final assay conditions for uptake experiments were: SR (0.2 mg/ml);
buffer, 100 mM KCl and 20 mM Hepes at pH 7.0, 1
mM Mg
, 0.5 mM Mg-ATP. For ATPase
experiments, all concentrations were similar except SR concentrations
(0.1 mg/ml). The data are the average of three independent experiments
and are expressed as a percentage of control. In the absence of
H
O
, Ca
-dependent ATPase
activity = 0.45 µmol/mg/min; A23187-stimulated ATPase
activity = 1.20 µmol/mg/min; total amount of Ca
uptake = 111 nmol/mg; and the maximal rate of
Ca
uptake = 12.98
nmol/mg/s.
In Fig. 2, it was demonstrated
that HO
directly stimulated Ca
release from actively loaded SR vesicles. Calcium was released at
concentrations of H
O
as low as 1 mM,
while the release rates appeared to saturate between 50 and 80
mM H
O
. The maximal release rate
derived from the kinetic evaluation using the program ENZFITTER
(Elsvier-Biosoft) was 7.28 ± 0.51 nmol/mg/min, while the
EC
was 12.9 ± 2.8 mM.
Figure 2:
HO
-stimulated
Ca
release from SR vesicles. SR vesicles, at 0.2
mg/ml, were actively loaded in a buffer containing 200 µM arsenazo III (ASIII), 100 mM KCl, 20 mM Hepes, 1
mM MgCl
, 20 µM CaCl
, pH
7.0, by addition of 0.5 mM Mg-ATP. Upon completion of
Ca
uptake, the indicated concentration of
H
O
was added. The Ca
concentration was continuously monitored (at 675-685 nm)
using a dual wavelength spectrophotometer. The Ca
release rates were calculated from the initial slope of free
Ca
concentration versus time following
addition of H
O
. Identical experiments were also
performed in the presence of 10 µM ruthenium red. The data
shown are the average of representative experiments performed in
duplicate.
When
Ca release was measured in the presence of the
Ca
channel inhibitor, ruthenium red (10
µM), no release of Ca
was observed with
up to 80 mM H
O
. The ability to block
Ca
release with a specific channel inhibitor suggests
that H
O
was affecting the Ca
release mechanism of SR, and was not causing an increase in the
SR Ca
permeability by nonspecific effects such as
lipid peroxidation.
Figure 3:
HO
stimulated high
affinity [
H]ryanodine binding in a
concentration-dependent manner. [
H]ryanodine
binding was carried out as follows. SR membranes (0.1 mg/ml) were
incubated at 37 °C for 3 h in a medium containing 250 mM KCl, 15 mM NaCl, 15 nM
[
H]ryanodine, and 20 mM Hepes, pH 7.1.
Assay buffer contained 50 µM Ca
(free)
and various amounts of H
O
. An identical set of
experiments were performed using H
O
pretreated
with 10 units of catalase prior to incubation. The binding was quenched
by rapid filtration. The filters were rinsed twice with 5 ml of buffer
and counted. The data shown are the average of representative
experiments performed in duplicate. EC
= 0.650
mM and IC
= 19.0
mM.
When catalase, an enzyme that detoxifies
HO
by converting it to H
O and
O
, was present at 5 units/ml in the incubation medium, no
stimulation or reduction in binding was observed (Fig. 3). In
addition, when assayed in the presence of 5 µM ruthenium
red, H
O
failed to enhance
[
H]ryanodine binding (Table 2). These data
further suggest that H
O
interacts directly with
the SR Ca
release channel.
In order to more fully
describe the interaction between HO
and the
ryanodine receptor, time-dependent association and dissociation
experiments were conducted. Increasing H
O
concentrations stimulated the rate of association of
[
H]ryanodine to its receptor (Fig. 4a and Table 1). A linear transformation of the association
data highlighted this more clearly (Fig. 4b).
Interestingly, at 100 mM H
O
when
equilibrium measurements (3-h incubation) indicated only a small amount
of ryanodine binding, time-dependent measurements show a rapid
acceleration in the rate of ligand binding followed by a rapid
dissociation.
Figure 4:
The effect of HO
on binding kinetics: linear transformation. This graph depicts
the linear transformation of time-dependent association, where B
represents the amount of
[
H]ryanodine bound at equilibrium, and B
represents the amount bound at any given time.
The data shown are the average of representative experiments performed
in duplicate.
Experiments (at 1 and 10 mM
HO
) were also carried out to determine the
influence of H
O
on the dissociation of bound
[
H]ryanodine from its receptor ( Fig. 5and Table 1). SR vesicles were labeled with
[
H]ryanodine in the absence of
H
O
and allowed to equilibrate for 2 h at 37
°C. Aliquots were subsequently diluted 100-fold into a buffer that
contained 0, 1, or 10 mM H
O
without
ryanodine. Peroxide in the dissociation buffer had no effect on the
dissociation of [
H]ryanodine from its receptor ( Fig. 5and Table 1). These data coupled with the
H
O
-dependent increase in the rate of
association suggested that the increase in
[
H]ryanodine binding caused by
H
O
(Fig. 3) was due solely to an
enhancement of the ryanodine association kinetics.
Figure 5:
The effect of HO
on dissociation kinetics. SR membranes were incubated with 1
nM [
H]ryanodine for 2 h at 37 °C.
Aliquots were then assayed for bound ryanodine (B
). Dissociation of bound ryanodine was initiated
by a 100-fold dilution into a medium (without ryanodine) containing 0,
1, or 10 mM H
O
. The residual bound
ryanodine at the indicated time (B
) was determined
by filtration. Each data point represents the average of three
measurements.
An increase in
association rate with little or no change in the dissociation rate
should result in a decrease in the equilibrium dissociation constant, K. In equilibrium binding studies (Fig. 6),
we observed that 1 mM H
O
reduced the K
, from 10.19 to 7.17 nM, a result in
agreement with the K
calculated from the ratio of K
/K
(Table 1). With either method of calculating the K
, there is a small increase in the affinity of
the receptor for binding ryanodine in the presence of peroxide (1
mM). As shown in Fig. 6, there is also an increase in
the number of high affinity ryanodine binding sites (B
:control = 4.2; 1 mM peroxide,
5.4 pmol/mg).
Figure 6:
Scatchard analysis of HO
(1 mM)-stimulated [
H]ryanodine
binding. Equilibrium binding measurements were carried out for 4 h in
the presence of 1 nM [
H]ryanodine and
varying concentrations (0.5 to 64 nM) of unlabeled ryanodine.
The data were fit to a one-site model using the nonlinear regression
program ENZFITTER.
Several activators, including caffeine, have been
shown to alter the Ca-dependent binding of ryanodine
to its receptor. In Fig. 7, it is demonstrated that
H
O
sensitized the ryanodine receptor to
activation by Ca
. In the absence of
H
O
, the EC
for Ca
binding was determined to be 632 nM, while in the
presence of 1 mM H
O
, the EC
was 335 nM Ca
. It was also observed
that known activators and inhibitors of Ca
release
similarly affect peroxide's ability to stimulate high affinity
[
H]ryanodine binding to SR (Table 2).
Activation of ryanodine binding by H
O
was
considerably decreased by two Ca
channel inhibitors,
Mg
and ruthenium red. H
O
had
no effect on the ability of doxorubicin to stimulate binding, since
binding had already been maximally activated by doxorubicin. However,
H
O
stimulated binding well beyond that induced
by 5 mM caffeine.
Figure 7:
HO
-stimulated high
affinity [
H]ryanodine binding in a
Ca
-dependent manner. The free Ca
concentration was determined by titrating Ca
with various amounts of EGTA. The data shown are an average of
three independent determinations.
Peroxide's interaction with the
Ca release mechanism from SR could result from an
oxidation of critical thiols on the receptor, which might be expected
to be reversible upon addition of reducing agents. To determine if the
stimulatory effect of H
O
on
[
H]ryanodine binding required
H
O
to be continually present, time-dependent
association experiments were conducted following
H
O
, catalase, and DTT treatment (Fig. 8). SR vesicles were incubated with 1 mM
H
O
in the [
H]ryanodine
binding medium for 30 min preceding the addition of either DTT (0.5
mM) or catalase or both. We observed that the addition of DTT
to the binding medium inhibited the H
O
-induced
stimulation of ryanodine binding whether H
O
was
present or had been converted to H
O and O
by
catalase (Fig. 8). This suggested that
H
O
-stimulated binding was due to an oxidation
of sulfhydryl groups, associated with the ryanodine receptor, to
disulfides. When catalase alone was added following the initial 30-min
incubation period, the stimulatory effect of H
O
persisted. The oxidation-induced stimulatory effect of
H
O
on [
H]ryanodine
binding occurred during the initial incubation period and continued
throughout the course of the binding experiment. This effect was
persistent unless DTT reduced the oxidized SH groups to their native
state.
Figure 8:
The effects of DTT and catalase on
HO
-stimulated
[
H]ryanodine binding. SR membranes were incubated
with [
H]ryanodine, and aliquots were assayed for
binding as a function of time. Thirty minutes after the start of the
binding experiment (t = 30), DTT (0.5 mM),
catalase (100 units), or both catalase and DTT were added to the
reaction medium. The binding assay then continued until the indicated
time when binding was terminated by rapid
filtration.
Figure 9:
Polyacrylamide gel electrophoresis of SR
membranes following treatment with HO
and
reducing agents as described in the methods. H
O
treatment: lanes 1 and 5, no
H
O
; lanes 2 and 6, 1
mM H
O
; lanes 3 and 7, 10 mM H
O
; lanes 4 and 8, 100 mM H
O
; lanes 1-4, solubilized with Laemmli buffer without DTT; lanes 5-8, solubilized with Laemmli buffer + 2
mM DTT.
Figure 10:
Peroxide modification of Ca release channel activity. Following fusion of an SR vesicle to a
BLM, the cis chamber was perfused with standard buffer (500 mM CsCl, no added Ca
) and current was recorded as a
function of time (A). B, 0.1 mM
H
O
was added to the cis side; C,
H
O
was raised to 1.0 mM; D,
H
O
raised to 10 mM; E, 100
µM Ca
cis added. The open state
probabilities (P
) for traces A-E are as follows: A, P
= 0.05; B, P
= 0.85; C, P
= 0.10; D, P
= 0.01. The time for each trace was 500 ms/trace. Current
traces were recorded at a holding potential of +25 mV with respect
to the trans side (ground) of the bilayer. These observations were made
in five independent bilayer experiments using two separate SR
preparations.
In the range of HO
concentrations (0.1-0.5 mM) for Ca
channel activation, addition of the reducing agent DTT inhibited
channel activity by decreasing the open probability (P
). Neither the H
O
or DTT
had any effect on the single-channel conductance. As shown in Fig. 11, at 0.2 mM H
O
, DTT
inhibited H
O
-stimulated Ca
channel activity. Activation of the Ca
release
channel by H
O
was completely reversed by
addition of high concentrations of either DTT or
-mercaptoethanol.
Figure 11:
DTT inhibited HO
stimulation of Ca
release channel activity.
Following fusion of an SR vesicle to a BLM, perfusion of the cis
chamber, and addition of 0.2 mM peroxide, increasing
concentrations of DTT were added to the cis chamber. Following 2 min of
stirring, traces were recorded and open channel probabilities (P
) were calculated. These experiments were
repeated in four independent bilayer
experiments.
Recently, ROS have drawn attention for their potential to
disrupt normal muscle function by targeting specific proteins for
modification. HO
is reportedly produced
physiologically in muscle cells undergoing oxidative stress (ischemia,
reperfusion, exercise, etc.). Modification of the Ca
release mechanism of sarcoplasmic reticulum by
H
O
may promote muscle dysfunction during such
pathophysiological conditions. We have examined the interaction between
hydrogen peroxide and the Ca
transport mechanisms
from skeletal muscle SR.
When ATPase activity and Ca uptake were assayed as a function of H
O
concentration with intact SR vesicles, a biphasic concentration
dependence was observed. Peroxide concentrations up to 20 mM increased the permeability of intact SR vesicles, which resulted
in an increased Ca
-dependent ATPase activity and a
decreased net accumulation of Ca
. This was not due to
an interaction with the Ca
pump, since peroxide did
not affect the ability of the Ca
-ATPase to hydrolyze
ATP when analyzed in the presence of the Ca
ionophore, A23187. Exposure of the SR to
H
O
concentrations greater than 20 mM apparently decreased the Ca
permeability of the
SR, which stimulated Ca
uptake into SR vesicles and
inhibited the steady-state ATPase activity.
An apparent discrepancy
between the biphasic effect of HO
on ATPase
activity and active Ca
uptake (Fig. 1) and the
monophasic stimulation of Ca
release (Fig. 2)
can be explained by differences in the manner in which these assays
were carried out. In Fig. 2, the SR vesicles were first actively
loaded with Ca
, and then exposed to
H
O
. The initial Ca
release
rate is a measure of the initial rate at which H
O
reacts with the Ca
release mechanism and opens
the Ca
release channel. In contrast, the data
presented in Fig. 1were taken over a period of several minutes
and represent measurements of ATPase activity and net Ca
uptake following exposure of SR vesicles to
H
O
. As shown in Fig. 4a, high
concentrations of H
O
(10-100 mM)
first activated the release mechanism and then inhibited the
Ca
channel as a function time. Thus, the effect of
high concentrations of H
O
was to stimulate
rapid initial release of Ca
from SR vesicles.
However, the Ca
ATPase activity and Ca
uptake, which was measured on the time scale of several minutes,
displayed a biphasic H
O
concentration
dependence (Fig. 1).
While activation of Ca release by H
O
(EC
13
mM) and modification of Ca
-dependent ATPase
activity occurs at [H
O
] of
approximately 15 mM, submillimolar concentrations of
H
O
were observed to activate high affinity
ryanodine binding (EC
0.65 mM). A higher
concentration of H
O
is required to modify
Ca
fluxes across SR vesicles, than to alter ryanodine
binding. A likely explanation for this discrepancy is that the
ryanodine binding measurements, which were carried out over a time
scale of hours, represent equilibrium measurements. SR thiols were
oxidized in less than 30 min (Fig. 8). The transport assays,
however, do not reflect equilibium measurements. A relatively large
excess of H
O
was required to oxidize thiols and
modify transport on the time scale of the assays shown in Fig. 1and Fig. 2.
The mechanism underlying
HO
-induced Ca
release appears
to involve a direct interaction with the ryanodine receptor from the
terminal cisternae. Peroxide had a biphasic effect on high affinity
[
H]ryanodine binding to the receptor (Fig. 3). As an activator, H
O
increased
the apparent affinity of the Ca
binding site
responsible for activating [
H]ryanodine binding (Fig. 6) and induced rapid Ca
release (Fig. 2). H
O
slightly decreased the K
for [
H]ryanodine binding (Fig. 6) by accelerating the rate at which ryanodine binds to
its receptor (Fig. 4, a and b). The failure of
H
O
to stimulate
[
H]ryanodine binding in the presence of the
Ca
channel inhibitors ruthenium red and
Mg
( Fig. 3and Table 2) further supports
the hypothesis that H
O
directly interacts with
the Ca
release protein from SR.
At high
concentrations, HO
demonstrated a
time-dependent activation and inactivation of ryanodine binding (Fig. 4). This time-dependent loss of ryanodine binding led to
complete inactivation of the receptor, which suggests that the receptor
was damaged via intermolecular cross-linking, extensive protein
oxidation, or destruction of its supporting membrane by lipid
peroxidation.
Peroxide-induced stimulation of Ca release, [
H]ryanodine binding, and
single-channel activity appears to be mediated by sulfhydryl
interactions on the Ca
release protein. In Fig. 8and Fig. 11, it is demonstrated on the level of
ryanodine binding and single-channel activity that the stimulatory
effect of H
O
was reversible upon addition of
thiol reducing agents. Following incubation with
H
O
, the oxidant may be removed (by the addition
of catalase) without altering its stimulatory effect (Fig. 8).
Only when DTT was added did the stimulation of
[
H]ryanodine binding return to normal levels. It
appears that activation of the Ca
release channel via
peroxide was mediated by a direct SH oxidation, and that reduction of
this disulfide restores the receptor to its native state. This was also
supported by the data derived from the electrophoresis experiments (Fig. 9).
Analysis of SR proteins by SDS-PAGE strongly
suggests that activation of the release mechanism by
HO
induced the formation of disulfide-linked
high molecular mass aggregates or multi-protein complexes. As the
concentration of H
O
was increased from 1 to 100
mM, we observed a progressive decrease in the density of
protein bands at 450 kDa (ryanodine receptor) and 170 kDa. At 100
mM H
O
, both bands completely
disappeared. These data parallel the concentration-dependent increase
in association of [
H]ryanodine to its receptor (Fig. 4, a and b), peroxide-stimulated
Ca
release rate (Fig. 2), and activation of
Ca
channel activity by peroxide (Fig. 10).
These data directly demonstrate that during activation of the SR
Ca
release channel by H
O
,
several proteins form a complex via oxidation of protein thiols
localized on the SR Ca
release channel.
It has
been previously demonstrated that oxidation of SH groups to disulfides
activates the Ca release mechanism of SR, while
reduction of the disulfides inhibits Ca
release(14) . As shown in Fig. 8, addition of the
reducing agent DTT to peroxide-treated SR reduced ryanodine binding to
a level equal to that observed under control conditions. The addition
of DTT also restored the high molecular mass complex containing the
450-kDa junctional foot protein to its native state (Fig. 9).
Recently, it has been shown that during activation of Ca
release by a number of known channel activators
(Ca
, caffeine, ATP, and ryanodine), hyperreactive
thiols associated with the ryanodine receptor and triadin no longer
interact with nanomolar concentrations of the fluorescent maleimide,
7-diethylamino-3(4`-maleimidyl phenyl)-4-methycoumarin(21) .
Moreover, activation leads to the formation of a high molecular weight
complex linked via disulfide bridges(22) . These results
strongly suggest that during physiological activation of the
Ca
release process, the Ca
release
channel is disulfide-linked to other key SR proteins, and that
reduction of these disulfide linkages results in channel closure. The
formation of a disulfide-linked protein complex induced by the addition
of H
O
may reflect a normal protein-protein
interaction that occurs during Ca
release channel
activation.
Lower concentrations of HO
were
required to activate and inhibit Ca
channel activity
in single-channel measurements ( Fig. 10and Fig. 11) than
were needed to modify [
H]ryanodine binding (Fig. 3) and Ca
release from SR vesicles (Fig. 2). Although the reasons for this higher sensitivity of
the reconstituted channel are not well understood, this phenomenon has
been previously observed with other agonists in this and other
laboratories(23) . Interestingly, the concentrations of
peroxide required to activate single-channel gating in skeletal SR are
substantially lower than those required to activate Ca
channel gating in single cardiac Ca
channels.
At micromolar Ca
concentrations, 3-5 mM peroxide was required to activate single-channel
activity(9) . Consistent with our results, cardiac SR
Ca
channel activation was also inhibited by the
addition of the disulfide reducing agent DTT.
Oxidation of protein
thiols by ROS may be relevant in various pathophysiological conditions.
Harmful oxygen species have been shown to disrupt normal cell
Ca concentrations during reperfusion of cardiac (24) and skeletal muscle tissue (25) following an
extended period of ischemia(24) . In heart muscle, this may
also be accompanied by a modification of the Ca
transient(26) . The increase in resting Ca
is
most likely mediated by Ca
entry via a damaged
sarcolemma and/or by the disruption of the internal SR membrane
systems.
While it has not been conclusively established that
endogenous sulfhydryl reactivity regulates the skeletal muscle
Ca release channel in vivo, our current
understanding of excitation-contraction coupling from experiments
performed in vitro strongly suggest that modification of SH
groups can control the gating pattern of the SR Ca
release channel. The continued use of SH probes may provide us
with more detailed information regarding SR Ca
channel function.