Modulation of Ca2+-gated
cardiac muscle Ca2+-release
channel (ryanodine receptor) by mono- and divalent ions
Wei
Liu,
Daniel A.
Pasek, and
Gerhard
Meissner
Departments of Physiology and Biochemistry and Biophysics,
University of North Carolina, Chapel Hill, North Carolina 27599-7260
 |
ABSTRACT |
The effects of mono- and divalent ions on
Ca2+-gated cardiac muscle
Ca2+-release channel (ryanodine
receptor) activity were examined in [3H]ryanodine-binding
measurements. Ca2+ bound with the
highest apparent affinity to Ca2+
activation sites in choline chloride medium, followed by KCl, CsCl,
NaCl, and LiCl media. The apparent
Ca2+ binding affinities of
Ca2+ inactivation sites were lower
in choline chloride and CsCl media than in LiCl, NaCl, and KCl media.
Sr2+ activated the ryanodine
receptor with a lower efficacy than
Ca2+. Competition studies
indicated that Li+,
K+,
Mg2+, and
Ba2+ compete with
Ca2+ for
Ca2+ activation sites. In 0.125 M
KCl medium, the Ca2+ dependence of
[3H]ryanodine binding
was modified by 5 mM Mg2+ and 5 mM
,
-methyleneadenosine 5'-triphosphate (a nonhydrolyzable ATP
analog). The addition of 5 mM glutathione was without appreciable effect. Substitution of Cl
by 2-(N-morpholino)ethanesulfonic acid ion caused an
increase in the apparent Ca2+
affinity of the Ca2+ inactivation
sites, whereas an increase in KCl concentration had the opposite
effect. These results suggest that cardiac muscle ryanodine receptor
activity may be regulated by 1)
competitive binding of mono- and divalent cations to
Ca2+ activation sites,
2) binding of monovalent cations to
Ca2+ inactivation sites, and
3) binding of anions to anion
regulatory sites.
sarcoplasmic reticulum
 |
INTRODUCTION |
IN MUSCLE, AN ACTION potential activates intracellular
Ca2+-release channels to cause the
release of Ca2+ from an
intracellular Ca2+ compartment,
the sarcoplasmic reticulum (SR), into the myofibrillar space (for
review, see Refs. 5, 26). The SR
Ca2+-release channels are also
known as ryanodine receptors (RyRs) because they can bind the plant
alkaloid ryanodine with high affinity and specificity (for review, see
Refs. 3, 15). The cardiac muscle (RyR2) and skeletal muscle (RyR1) RyRs
have been purified as 30S protein complexes composed of four large
(relative molecular weight of ~560,000) (3, 15) and four small
(FK-506-binding protein, relative molecular weight of ~12,000) (24)
subunits. Physiological and biochemical evidence suggests that RyR1 and RyR2 are ligand-gated channels with
Ca2+ as a major regulator.
Activation by micromolar Ca2+ and
inhibition by millimolar Ca2+
suggest at least two classes of
Ca2+ binding sites, high-affinity
activation sites and low-affinity inactivation sites. Various other
endogenous and exogenous effectors have been identified, including
Mg2+, ATP, calmodulin, caffeine,
and ryanodine (3, 15, 30).
The neutral plant alkaloid ryanodine binds with nanomolar affinity and
high specificity to RyRs and has been used as a sensitive probe in
assessing the activity of RyRs (1, 16, 29). As a general rule,
conditions that open the channel, such as the presence of micromolar
Ca2+ or millimolar adenine
nucleotide increase the affinity of
[3H]ryanodine binding.
RyR activity is sensitive to ionic strength and composition, as
assessed in
[3H]ryanodine binding,
vesicle Ca2+ efflux, and
single-channel measurements. This phenomenon has been especially
observed for RyR1. Inorganic phosphate and anions often classified as
chaotropic ions (C
,
SCN
,
I
,
) stimulated RyR1 activity,
whereas replacement of Cl
by gluconate, propionate, and buffer anions such as
2-(N-morpholino)ethanesulfonic acid (MES) anion and
piperazine-N,N'-bis(2-ethanesulfonic
acid) (PIPES) anion was inhibitory (6, 7, 13, 18). An increase in KCl
or NaCl concentration increased RyR1 activity (1, 9, 16, 18, 19, 21).
In contrast, RyR2 activity has been reported to be only little affected
by ionic composition (6, 19).
It is well established that there exist major differences in the
regulation of the skeletal muscle and cardiac muscle receptor isoforms
by Ca2+,
Mg2+, ATP, and other endogenous
and exogenous effectors (3, 15). Here, we show that the two receptors
are regulated in a similar, but not identical, manner by mono- and
divalent ions. Our results indicate that cations regulate
Ca2+-gated RyR2 activity by at
least two different mechanisms: 1) competitive binding to high-affinity
Ca2+ activation sites and
2) binding to low-affinity
Ca2+ inactivation sites.
Cl
had an activating
effect.
 |
EXPERIMENTAL PROCEDURES |
Preparation of SR vesicles.
Cardiac SR vesicles enriched in
[3H]ryanodine binding
activity were prepared from canine hearts in the presence of protease inhibitors (100 nM aprotinin, 1 µM leupeptin, 1 µM pepstatin, 1 mM
benzamidine, and 0.2 mM phenylmethylsulfonyl fluoride) using a
discontinuous sucrose gradient of 20, 30, and 40% sucrose (17). Membranes at the 20%/30% sucrose interface were recovered,
quick-frozen in small aliquots, and stored at
135°C before
use. "Heavy" rabbit skeletal muscle SR vesicles were prepared in
the presence of the above protease inhibitors as described previously
(14). The maximum number of high-affinity
[3H]ryanodine binding
sites determined under optimal binding conditions (11) ranged from 2.5 to 8 pmol/mg protein for cardiac SR vesicles and from 11 to 23 pmol/mg
protein for skeletal muscle SR vesicles, depending on the preparations.
[3H]ryanodine binding
measurements.
Unless otherwise indicated, cardiac muscle and skeletal muscle SR
vesicles were incubated for 40 and 96 h, respectively, at 12°C in
20 mM imidazole, pH 7.2, 0.2 mM Pefabloc SC, 20 µM leupeptin, 1 nM
[3H]ryanodine, and the
indicated salts and free Ca2+
concentrations. Nonspecific
[3H]ryanodine binding
was determined using a 1,000-fold excess of unlabeled ryanodine.
Unbound [3H]ryanodine
was separated from the protein-bound
[3H]ryanodine by
filtration of sample aliquots through Whatman GF/B filters presoaked
with 2% polyethyleneimine, followed by washing with three 5-ml volumes
of ice-cold 0.1 M KCl and 1 mM potassium PIPES (pH 7.0) medium. The
radioactivity remaining with the filters was determined by liquid
scintillation counting to obtain bound [3H]ryanodine.
Determination of free
[Ca2+]
and
[Sr2+].
Free Ca2+ concentrations of >1
µM were determined with a
Ca2+-selective electrode (World
Precision Instruments, Sarasota, FL). Free
Ca2+ concentrations of <1 µM
were obtained by including in the solutions the appropriate amounts of
Ca2+ and ethylene
glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA) [or
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid and nitriloacetic acid] as determined using the stability constants and the mixed solution program published by Schoenmakers et
al. (22). Effects of Sr2+ were
determined in assay media containing contaminating levels of
Ca2+ (2-4 µM) and 1 mM
EGTA. Free Sr2+ concentrations
were calculated using the
EGTA-Sr2+
complexation constants published by Smith and Martell (23) and verified with a Ca2+-selective
electrode for Sr2+ concentrations
>100 µM.
Data analysis.
The Ca2+ dependence of
[3H]ryanodine binding
was analyzed with the assumption that the RyR2 possesses cooperatively
interacting high-affinity Ca2+
activation and low-affinity Ca2+
inactivation binding sites. A simple scheme used to describe the
Ca2+ dependence of channel
activity was
|
(Scheme 1)
|
In scheme 1, the RyR ion
channel is present in its closed
Ca2+-free form (R) at
[Ca2+] of <0.1 µM,
and its Ca2+-activated
(ACa) and
Ca2+-inactivated
(CaICa)
forms are present at micro- and millimolar Ca2+ concentrations, respectively.
The tetrameric RyR contains cooperatively interacting
Ca2+ activation sites and
Ca2+ inactivation sites (see
RESULTS); however, only one
Ca2+ activation and one
Ca2+ inactivation site are shown.
Ryanodine binding (and, by extension,
Ca2+-release channel activity) was
fitted by the product of an activation and an inactivation variable,
each related to [Ca2+]
by the Hill
formula
|
(1)
|
where B is the
[3H]ryanodine binding
value at a given
[Ca2+],
Bo is the binding maximum,
Ka and
Ki are Hill
activation and inactivation constants, respectively, and
na and
ni are the
respective Hill coefficients. In the calculations,
Bo was included as one of the variables.
In the competition studies,
[3H]ryanodine binding
was fitted with the
equations
|
(2a)
|
|
(2b)
|
where B is the
[3H]ryanodine binding
value at a given
[Ca2+],
Bo is the binding maximum in the
absence of the inhibitor (I), KCa is the
Ca2+ activation constant,
Ki is the
inhibition constant of the inhibitor, and
na and
ni are the
respective Hill coefficients.
Results are given as means ± SD, with the number of experiments in
parentheses. Unless otherwise indicated, significance of differences of
data was analyzed with Student's unpaired
t-test. Differences were regarded as
statistically significant at P < 0.05.
Materials.
[3H]ryanodine was
purchased from DuPont NEN. Unlabeled ryanodine was obtained from
Calbiochem, and leupeptin and Pefabloc SC (a protease inhibitor) were
from Boehringer Mannheim. All other chemicals were of analytical grade.
 |
RESULTS |
Determination of the effects of ionic composition on the
Ca2+ dependence
of [3H]ryanodine binding.
In the present study, a majority of the binding experiments was
performed at a relatively low incubation temperature of 12°C to
minimize loss of RyR activity during the
[3H]ryanodine-binding
reaction. The Ca2+ dependence of
[3H]ryanodine binding
to RyR2 was determined in 0.25 M KCl medium at four different time
points to obtain the time required for equilibrium binding (Fig.
1).
[3H]ryanodine binding
had a Ca2+ threshold of ~1 µM,
was optimal at 10-100 µM, and showed some inactivation in the
1-10 mM Ca2+ range. In
agreement with previous studies (6, 12, 17, 27, 29), these results
suggest that the cardiac receptor has both high-affinity
Ca2+ activation sites and
low-affinity Ca2+ inactivation
sites. The
[3H]ryanodine binding
curves of Fig. 1 show that an incubation time of 40 h was sufficient to
obtain close to equilibrium binding conditions.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 1.
Time course of
[3H]ryanodine binding.
Cardiac sarcoplasmic reticulum vesicles were incubated for the
indicated times in medium containing 20 mM imidazole, pH 7.2, 0.25 M
KCl, protease inhibitors (0.2 mM Pefabloc, 20 µM leupeptin), 1 nM
[3H]ryanodine, and the
indicated concentrations of free
Ca2+. Specific
[3H]ryanodine binding
was determined as described under EXPERIMENTAL
PROCEDURES. Continuous lines were obtained by fitting
the data according to Eq. 1 in
EXPERIMENTAL PROCEDURES.
|
|
Figure 2 compares the
Ca2+ dependence of
[3H]ryanodine binding
to rabbit skeletal muscle and canine cardiac muscle SR vesicles in 0.25 M KCl and 0.25 M choline chloride media. In the KCl medium, two
bell-shaped Ca2+
activation/inactivation curves were obtained. Equation 1, which describes the binding of
Ca2+ to cooperatively interacting
receptor activation and inactivation sites, provided a reasonable fit
(lines) to the
[3H]ryanodine binding
data in the KCl medium. Values for the Hill activation constant for
Ca2+
(KCaa) of 1.0 and 2.4 µM
were obtained for RyR1 and RyR2, respectively. In agreement with
previous reports (6, 17, 19), a substantially higher
Ca2+ concentration was required
for the inactivation of RyR2 than RyR1 [Hill inhibition constant
for Ca2+
(KCai) = 5,860 and 300 µM,
respectively]. Table 1 shows for
RyR2 the averaged Hill constants and coefficients of several
experiments.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 2.
Ca2+ dependence of
[3H]ryanodine binding
to skeletal and cardiac muscle ryanodine receptors (RyRs) in KCl and
choline chloride (CholCl) media. Specific
[3H]ryanodine binding
was determined as described in EXPERIMENTAL
PROCEDURES in medium containing the indicated
salts and concentrations of free
Ca2+. Continuous lines for data in
KCl (skeletal and cardiac RyRs) and choline chloride media (RyR2) were
obtained by fitting data with Eq. 1 in
EXPERIMENTAL PROCEDURES. Continuous
line for skeletal RyR in choline chloride medium was obtained by
assuming that choline ion is a weak
Ca2+ agonist of the
Ca2+-release channel and fitting
data with Eq. 2 of Meissner et al.
(18). Data from 1 representative experiment are shown. Averaged Hill
constants and coefficients of indicated number of experiments with RyR2
are shown in Table 1.
|
|
Replacement of K+ with choline ion
affected RyR2 differently than RyR1 in that a decrease rather than
increase in the maximal level of
[3H]ryanodine binding
was observed (Fig. 2). Two other differences were that only close to
background levels of binding to RyR2 could be detected at
<10
7 M
Ca2+ and
[3H]ryanodine binding
showed little inhibition at high
Ca2+ concentrations. For RyR1,
substantial levels of binding were observed at
<10
8 M
Ca2+. These could be described
assuming that the choline ion is a weak, noncooperative
Ca2+ channel agonist of RyR1
(Eq. 2 of Ref. 18) but not RyR2. Table 1 lists the apparent KCaa and
na values for RyR2 in 0.25 M choline chloride medium.
Bell-shaped Ca2+
activation/inactivation curves were obtained for RyR2 in medium
containing the chloride salts of
Li+,
Na+,
K+, and
Cs+ at a concentration of 0.25 M
(Fig. 3). The binding levels were highest in KCl medium,
intermediate in CsCl and NaCl media, and lowest in LiCl medium.
Equation 1 provided a reasonable fit
(lines) to the
[3H]ryanodine-binding
data. Table 1 summarizes the Hill constants and coefficients in the
different media. The averaged
KCaa in
Li+,
Na+,
K+, and
Cs+ media were 32, 7.8, 1.7, and
3.6 µM, respectively. The highest apparent
Ca2+ binding affinities to the
Ca2+ inactivation sites were
obtained in LiCl and KCl media followed by NaCl and CsCl media. An
na of close to
two indicated that Ca2+ activated
[3H]ryanodine binding
by cooperative interactions involving at least two
Ca2+ binding sites. These results
suggest that monovalent cations affect RyR2 activity by interacting
with receptor Ca2+ activation and
inactivation sites.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 3.
Ca2+ dependence of
[3H]ryanodine binding
to RyR2 in medium containing different inorganic monovalent cations.
Specific [3H]ryanodine
binding was determined as described in EXPERIMENTAL
PROCEDURES in medium containing the indicated salts and
concentrations of free Ca2+.
Continuous lines were obtained by fitting data with
Eq. 1 in EXPERIMENTAL
PROCEDURES. One representative experiment is shown.
Averaged Hill constants and coefficients of indicated number of
experiments are shown in Table 1.
|
|
The [3H]ryanodine
binding level and Ca2+ dependence
of [3H]ryanodine
binding were affected by the ionic strength and anionic composition of
the assay medium (Fig.
4). An increase
in KCl concentration from 0.1 to 1.0 M increased the maximal
[3H]ryanodine-binding
level about threefold, from 0.3 to 0.9 pmol/mg protein. Replacement of
Cl
by
MES
in the 0.25 M medium
decreased the maximal binding level about twofold.
Equation 1 provided a good fit (lines)
to the
[3H]ryanodine-binding
data determined in the five media shown in Fig. 4. Ionic strength and
anionic composition primarily affected the binding of
Ca2+ to the
Ca2+ inactivation sites (Table 1).
The KCai increased ~10-fold
as the KCl concentration was increased from 0.1 to 0.5 M KCl. A linear
correlation coefficient of 0.96 (n = 15) indicated that the increase in
KCai was highly significant. Replacement of Cl
by
MES
decreased
KCai 7.5-fold. Together, the
data of Figs. 2-4 suggest that both the actions of monovalent
cations and anions must be taken into account to understand the way in which Ca2+ activates and
inactivates RyR2.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 4.
Effects of ionic strength and anionic composition on
Ca2+ dependence of
[3H]ryanodine binding.
Specific [3H]ryanodine
binding was determined as described in EXPERIMENTAL
PROCEDURES in medium containing the indicated
concentrations of KCl, K+-MES, and
free Ca2+. Continuous lines were
obtained by fitting experimental data according to Eq. 1 in EXPERIMENTAL
PROCEDURES. One representative experiment is shown.
Averaged Hill constants and coefficients of indicated number of
experiments are shown in Table 1.
|
|
Scatchard analysis indicated the presence of a single high-affinity
[3H]ryanodine binding
site. Changes in binding affinity without a major change in maximal
binding value (3.0 ± 0.5 pmol/mg protein) were
observed in 0.25 M KCl, 0.25 M choline chloride, and 0.25 M
K+-MES media (all at 100 µM free
Ca2+; not shown). This result
suggests that the different binding levels of Figs. 2-4 reflect
changes in binding affinities rather than the number of high-affinity
binding sites.
Dependence of [3H]ryanodine
binding on Sr2+
and Ba2+.
Figure 5 compares the effects of
Ca2+,
Sr2+, and
Ba2+ on
[3H]ryanodine binding
in 0.25 M KCl medium. Contaminating levels of
Ca2+ (2-4 µM) were kept
below 0.1 µM free Ca2+ by
including 1 mM EGTA in the assay media.
[3H]ryanodine binding
had a higher threshold for Sr2+
than for Ca2+ (~10 µM vs. ~1
µM), was optimal at ~1 mM
Sr2+, and showed some inactivation
at 5 mM Sr2+.
Ba2+ did not noticeably activate
RyR2. The KCaa for Sr2+ (283 ± 21 µM,
n = 3) and
Ca2+ (1.7 ± 0.7 µM,
Table 1) differed by more than a factor of 100. The Hill
inactivation constant and Hill coefficients for
Sr2+ did not significantly differ
from those shown for Ca2+ in Table
1.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 5.
Dependence of
[3H]ryanodine binding
on divalent cations. Specific
[3H]ryanodine binding
was determined as described in EXPERIMENTAL
PROCEDURES in 0.25 M KCl medium containing the
indicated concentrations of free divalent cations
(X2+). Continuous lines were
obtained by fitting data with Eq. 1 in
EXPERIMENTAL PROCEDURES. One
representative experiment is shown. Averaged Hill constants and
coefficients for Sr2+ were as
follows: activation constant = 283 ± 21, activation coefficient = 2.4 ± 0.6, inhibition constant = 7,021 ± 1,258, and inhibition
coefficient = 1.5 ± 0.4 (n = 3).
|
|
Effects of AMP and caffeine on
Ca2+ dependence
of [3H]ryanodine binding.
The effects of two channel activators, AMP and caffeine (3, 15), on the
Ca2+ dependence of
[3H]ryanodine binding
were determined in 0.25 M KCl medium. In these studies, AMP rather than
ATP or a nonhydrolyzable ATP analog was used because AMP, in contrast
to adenosine triphosphates, binds Ca2+ with negligible affinity. AMP
(5 mM) did not significantly affect the
Ca2+ binding affinity of the
Ca2+ activation sites (Table 1).
Ca2+ binding affinity to the
Ca2+ inactivation sites of the
receptor decreased approximately twofold. Caffeine (20 mM) increased
the apparent Ca2+ affinity of the
Ca2+ activation sites of the
receptor by more than 15-fold.
KCai increased approximately
twofold (Table 1). Similar changes in the Hill constants and
coefficients were obtained when the effects of 5 mM AMP and 20 mM
caffeine were examined in 0.25 M LiCl, 0.25 M NaCl, or 0.25 M CsCl
media (not shown).
Interaction of mono- and divalent cations with high-affinity
Ca2+ activation
sites of RyR2.
We determined whether monovalent cations inhibit RyR2 activity by
competing with Ca2+ for the
Ca2+ activation sites, as observed
for RyR1 (18). In these studies, we took advantage of the observation
that, at low Ca2+ concentrations,
the
[3H]ryanodine-binding
level was the highest in choline chloride medium (Fig. 2) by assessing
the inhibitory effects of K+ in
0.5 M choline chloride medium containing varying low concentrations of
free Ca2+ (Fig.
6). To interpret the
[3H]ryanodine-binding
data, three simple alternative types of inhibition were considered,
namely, that K+ was a competitive,
noncompetitive, or uncompetitive inhibitor. Choline ion was assumed to
be without effect on RyR2 activity. As observed for RyR1 (18), the RyR2
binding data could not be fitted assuming non- or uncompetitive
inhibition (not shown) but could be well fitted when it was assumed
that K+ inhibited
[3H]ryanodine binding
by a competitive mechanism (Eqs. 2a and 2b, EXPERIMENTAL
PROCEDURES) (Fig. 6). Table
2 summarizes the derived Hill
Ca2+ activation and inactivation
constants and coefficients for K+,
as well as for Li+,
Mg2+, and
Ba2+. Similar
Ca2+ activation constants were
obtained for the four cations.
Mg2+ was the most effective in
inhibiting
[3H]ryanodine binding
(Ki = 0.007 mM).
Ba2+ was ~100-fold less
effective than Mg2+ in competing
with Ca2+ for the
Ca2+ activation sites. Of the two
monovalent cations tested, Li+ was
more effective than K+ in
inhibiting
[3H]ryanodine binding
(Ki = 21 and 31 mM, respectively). The latter data are consistent with the observation
that Ca2+ concentrations required
to half-maximally activate
[3H]ryanodine binding
were higher for LiCl than for KCl medium (Fig. 3 and Table 1).

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 6.
Inhibition of
[3H]ryanodine binding
by K+. Specific
[3H]ryanodine binding
was determined in 0.5 M choline chloride medium containing the
indicated concentrations of K+ and
free Ca2+. Total
Cl concentration was kept
constant by adjusting choline ion concentration so that choline ion
concentration + K+ concentration = 0.5 M. In A and
B, continuous lines were obtained with
Eqs. 2a and 2b in EXPERIMENTAL
PROCEDURES, using a single set of parameters for all
data. In B, data were plotted using
derived Hill inactivation coefficient of 1.6. One representative
experiment is shown. Averaged Hill constants and coefficients of 3 separate experiments are shown in Table 2.
|
|
Effects of magnesium
,
-methyleneadenosine
5'-triphosphate and glutathione on
Ca2+ dependence
of [3H]ryanodine binding.
Attempts were made to determine the
Ca2+ dependence of
[3H]ryanodine binding
under more physiologically relevant conditions by performing
experiments at room temperature in 0.125 M KCl medium in the presence
of magnesium
,
-methyleneadenosine 5'-triphosphate (AMP-PCP)
and/or glutathione. In rat heart muscle, the myofibrillar concentrations of K+,
Na+, and
Cl
were estimated to be
74-128, 4-11, and 11-25 mM, respectively (25). The total
ATP and free Mg2+ concentrations
in intact hearts ranged from 5 to 10 mM (8, 10) and from 0.7 to 1.0 mM
(20), respectively. Figure 7 compares the
Ca2+ dependence of
[3H]ryanodine binding
at room temperature (~24°C) in 0.125 M KCl medium in the absence
and presence of 5 mM AMP-PCP and 5 mM
Mg2+ (~0.7 mM free
Mg2+ at ~1 µM
Ca2+). Addition of 5 mM
MgAMP-PCP shifted the Ca2+
activation curve to the right and rendered the channel less sensitive to inactivation at Ca2+
concentrations of up to 1 mM. Free
Ca2+ concentrations in excess of 1 mM were not tested because of difficulties of keeping
Ca2+ in solution in 5 mM AMP-PCP
medium. RyRs contain sulfhydryl groups that are important to their
function (30). These may be in a reduced state in vivo because cells
maintain a reducing environment by containing thiol-reducing compounds,
the most abundant being glutathione (4). Figure 7 shows that the
addition of a relatively high concentration of glutathione (5 mM) had a
minimal effect on the levels and
Ca2+ dependence of
[3H]ryanodine binding
in the 0.125 M KCl medium containing and lacking 5 mM MgAMP-PCP.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 7.
Effects of magnesium , -methyleneadenosine 5'-triphosphate
(MgAMP-PCP) and glutathione on
Ca2+ dependence of
[3H]ryanodine binding.
Specific [3H]ryanodine
binding was determined at room temperature (~24°C) in 0.125 M KCl
medium containing the indicated concentrations of MgAMP-PCP,
glutathione (GSH), and free Ca2+.
Continuous lines were obtained by fitting data with
Eq. 1 in EXPERIMENTAL
PROCEDURES. One representative experiment is shown.
Averaged Hill constants and coefficients of indicated number of
experiments are shown in Table 1.
|
|
 |
DISCUSSION |
In the present study, the effects of mono- and divalent ions on the
cardiac muscle RyR (Ca2+ release)
channel were investigated by determining the
Ca2+ dependence of
[3H]ryanodine binding.
These studies led to two new observations that may have important
implications for the mechanism of SR
Ca2+ release in cardiac muscle.
First, the results of this study show that the affinity of
Ca2+ binding to the
Ca2+ activation sites and
inactivation sites of RyR2 is dependent on ionic composition. Second,
competition studies indicate that physiologically relevant mono- and
divalent cations such as K+ and
Mg2+ inhibit
[3H]ryanodine binding
by competitive binding to Ca2+
activation sites.
The Ca2+ dependence of
Ca2+-release channel activity was
examined by measuring
[3H]ryanodine binding
to SR vesicles. A low
[3H]ryanodine
concentration was used to limit binding to a single high-affinity
receptor site. Under these conditions,
[3H]ryanodine binding
is a sensitive method in assessing RyR activity (3, 15). Although
single-channel measurements provide more direct information on
Ca2+-release channel activity than
[3H]ryanodine binding
measurements and channels gate on a much faster time scale than they
bind [3H]ryanodine
(µs to ms vs. min to h), an advantage of
[3H]ryanodine binding
is that it allows the study of a large number of ionic conditions.
One goal of this study was to find out how mono- and divalent ions
affect the cardiac RyR. A second goal was to compare their effects on
the cardiac and skeletal RyRs. Such a comparison is of interest because
the two receptor isoforms are differently regulated by
Ca2+ and other effectors. Because
our studies with the skeletal muscle RyR were mostly performed with
0.25 M salt solutions (18), we chose 0.25 M solutions for most of our
studies with the cardiac RyR as well. The binding parameters of RyR1
and RyR2 are summarized in Table 3 and are
compared below.
The effects of monovalent cations on the
Ca2+ dependence of
[3H]ryanodine binding
were determined by using the chloride salts of
Li+,
Na+,
K+, and
Cs+. The same order of apparent
Ca2+ binding affinities was
obtained for the two receptor isoforms, however, with RyR1 displaying
higher apparent Ca2+ binding
affinities than RyR2 (Table 3). Values of
na of 1.9 and
greater indicate that Ca2+
activated RyR2 by a cooperative interaction involving at least two
Ca2+ activation sites. In choline
chloride medium, the Ca2+
dependence of
[3H]ryanodine binding
differed in that, at free Ca2+
<10
8 M, significant
levels of
[3H]ryanodine binding
were observed for RyR1 but not RyR2, which suggests an apparent agonist
action of choline ion for RyR1 (18) but not RyR2. Another difference
was that at micromolar Ca2+
concentrations, the
[3H]ryanodine binding
level of RyR2 was higher in KCl than choline chloride medium, whereas
the opposite was observed for RyR1 (Fig. 2). This result suggests that
monovalent cations may influence the activity of RyR2 in additional
ways, not indicated by the scheme in EXPERIMENTAL
PROCEDURES.
Differences were observed in apparent binding affinities of
Ca2+ to the inactivation sites of
the cardiac and skeletal muscle RyR and the order of the affinities in
the various monovalent cation media (Table 3). For RyR2, the
Ca2+ affinities were similar in
LiCl, NaCl, and KCl media. In CsCl and choline chloride media,
decreased affinities were observed. Values for
ni of
Ca2+ binding to the cardiac
Ca2+ inactivation sites ranged
from 0.7 to 1.4, suggesting that
Ca2+ bound with no or low
cooperativity to the Ca2+
inactivation sites. Our results are consistent with previous SR vesicle
Ca2+ flux (2, 17), single-channel
(12, 27), and
[3H]ryanodine binding
(2, 6, 27) measurements; these studies also showed an activation and
inactivation of RyR2 by Ca2+ in
K+ or
Cs+ media, but they are at
variance with a study that failed to show an inhibition of
single-channel activities by 10 mM
Ca2+ (2).
Major differences were observed in the action of divalent cations.
Ca2+ and
Sr2+ activated RyR2, whereas
Mg2+ and
Ba2+ inhibited
[3H]ryanodine binding
by competing with Ca2+ for the
Ca2+ activation sites. Divalent
cations similarly affected
[3H]ryanodine binding
to RyR1 (D. A. Pasek and G. Meissner, unpublished observations) (Table
3). The results of the present study also indicate competitive binding
of monovalent cations to the Ca2+
activation sites of RyR2 to affect the receptor's
Ca2+ sensitivity. The inhibition
constants of K+ and
Mg2+ were 42 and 0.013 mM for RyR1
and 31 and 0.007 mM for RyR2, respectively (Table 3). The intracellular
K+ and free
Mg2+ concentrations in
cardiomyocytes have been estimated to be 74-128 mM (25) and
0.4-0.8 mM (20), respectively. Therefore, in cardiomyocytes a
small fraction of the Ca2+-release
channel's Ca2+ activation sites
may be occupied by K+ instead of
Mg2+ at rest. Occupation of some
sites by K+ may be of
physiological significance because
Ca2+ may bind faster to channel
sites occupied by K+ than sites
occupied by more tightly bound
Mg2+.
The activity of RyR1 is greatly affected by the anionic composition of
assay medium (6, 7, 13, 18). In 0.25 M
K+-MES, RyR1 bound only close to
baseline levels of
[3H]ryanodine (18).
Substitution of Cl
by
MES
also reduced the
[3H]ryanodine binding
levels of RyR2 but to a lesser extent. Substitution of
Cl
by MES
appeared to primarily affect
[3H]ryanodine binding
to RyR2 by causing a decrease in
KCai. Increase
of KCl concentration from 0.1 to 0.5 M had an opposite effect by
increasing
KCai of RyR2.
It therefore appears that RyR2 is also sensitive to regulation by
anions.
The Ca2+ dependence of
[3H]ryanodine binding
was analyzed under conditions that were thought to approximate those in
the myocardium. These included the use of a 0.125 M KCl medium
containing 5 mM MgAMP-PCP and/or 5 mM glutathione (Fig. 7). The
addition of 5 mM MgAMP-PCP modified the
Ca2+ dependence of RyR2 by
rendering the channel less sensitive to activation at
Ca2+ concentrations of <10 µM
and less sensitive to Ca2+
inactivation at Ca2+
concentrations of >100 µM. Glutathione has recently been shown to
reduce the affinity and maximal binding values but not
Ca2+ dependence of
[3H]ryanodine binding
to skeletal muscle RyR (28). At variance with this result, the addition
of 5 mM glutathione had a minimal effect on both the level and
Ca2+ dependence of
[3H]ryanodine binding
to cardiac RyR.
In conclusion, the results of this study show that monovalent ions
affect in a similar, but not identical, manner the in vitro regulation
of the cardiac and skeletal muscle
Ca2+-release channels by
Ca2+. In the absence of other
activating ligands, Ca2+ activates
the cardiac channel to a greater extent than the skeletal channel (17).
Two possible explanations for this difference that we considered in our
experiments were an increased Ca2+
affinity of the Ca2+ activation
sites and decreased Ca2+ affinity
of the Ca2+ inactivation sites. A
consequence of a widened "Ca2+
window" is that higher levels of receptor activation can be
achieved. Our results indicate that
Ca2+ bind to the
Ca2+ inactivation sites of the
cardiac receptors with a lower apparent affinity than those in the
skeletal muscle receptor, whereas
Ca2+ bind with a somewhat higher
apparent affinity to the Ca2+
activation sites of the skeletal than cardiac muscle RyR. Accordingly, differences in Ca2+ binding
affinity to the Ca2+ inactivation
sites may be primarily responsible for the different activity levels of
the two receptors.
 |
ACKNOWLEDGEMENTS |
Support by National Institutes of Health Grants HL-27430 and AR-18687
is gratefully acknowledged.
 |
FOOTNOTES |
Address for reprint requests: G. Meissner, Dept. of Biochemistry
and Biophysics, Univ. of North Carolina, Chapel Hill, NC 27599-7260.
Received 14 May 1997; accepted in final form 12 September 1997.
 |
REFERENCES |
1.
Chu, A.,
M. Diaz-Munoz,
M. J. Hawkes,
K. Brush,
and
S. L. Hamilton.
Ryanodine as a probe for the functional state of the skeletal muscle sarcoplasmic reticulum calcium release channel.
Mol. Pharmacol.
37:
735-741,
1990[Abstract].
2.
Chu, A.,
M. Fill,
E. Stefani,
and
M. L. Entman.
Cytoplasmic Ca2+ does not inhibit the cardiac muscle sarcoplasmic reticulum ryanodine receptor Ca2+ channel, although Ca2+-induced Ca2+ inactivation of Ca2+ release is observed in native vesicles.
J. Membr. Biol.
135:
49-59,
1993[Medline].
3.
Coronado, R.,
J. Morrissette,
M. Sukhareva,
and
D. M. Vaughan.
Structure and function of ryanodine receptors.
Am. J. Physiol.
266 (Cell Physiol. 35):
C1485-C1504,
1994[Abstract/Free Full Text].
4.
Deneke, S. M.,
and
B. L. Fanburg.
Regulation of cellular glutathione.
Am. J. Physiol.
257 (Lung Cell. Mol. Physiol. 1):
L163-L173,
1989[Abstract/Free Full Text].
5.
Fabiato, A.
Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum.
Am. J. Physiol.
245 (Cell Physiol. 14):
C1-C14,
1983[Abstract/Free Full Text].
6.
Fruen, B. R.,
P. K. Kane,
J. R. Mickelson,
and
C. F. Louis.
Chloride-dependent sarcoplasmic reticulum Ca2+ release correlates with increased Ca2+ activation of ryanodine receptors.
Biophys. J.
71:
2522-2530,
1996[Abstract].
7.
Hasselbach, W.,
and
A. Migala.
Modulation by monovalent anions of calcium and caffeine induced calcium release from heavy sarcoplasmic reticulum vesicles.
Z. Naturforsch. [C]
47:
440-448,
1992[Medline].
8.
Hohl, C. M.,
A. A. Garleb,
and
R. A. Altschuld.
Effects of simulated ischemia and reperfusion on the sarcoplasmic reticulum of digitonin-lysed cardiomyocytes.
Circ. Res.
70:
716-723,
1992[Abstract].
9.
Kasai, M.,
N. Yamaguchi,
and
T. Kawasaki.
Effect of KCl concentration on gating properties of calcium release channels in sarcoplasmic reticulum vesicles.
J. Biochem. (Tokyo)
117:
251-256,
1995[Abstract].
10.
Koretsune, Y.,
M. C. Corretti,
H. Kusuoka,
and
E. Marban.
Mechanism of early ischemic contractile failure: inexcitability, metabolic accumulation, or vascular collapse?
Circ. Res.
68:
255-262,
1991[Abstract].
11.
Lai, F. A.,
M. Misra,
L. Xu,
H. A. Smith,
and
G. Meissner.
The ryanodine receptor-Ca2+ release channel complex of skeletal muscle sarcoplasmic reticulum. Evidence for a cooperatively coupled, negatively charged homotetramer.
J. Biol. Chem.
264:
16776-16785,
1989[Abstract/Free Full Text].
12.
Laver, D. R.,
L. D. Roden,
G. P. Ahern,
K. R. Eager,
P. R. Junankar,
and
A. F. Dulhunty.
Cytoplasmic Ca2+ inhibits the ryanodine receptor from cardiac muscle.
J. Membr. Biol.
147:
7-22,
1995[Medline].
13.
Ma, J.,
K. Anderson,
R. Shirokov,
R. Levis,
A. Gonzalez,
M. Karhanek,
M. M. Hosey,
G. Meissner,
and
E. Rios.
Effects of perchlorate on the molecules of excitation-contraction coupling of skeletal and cardiac muscle.
J. Gen. Physiol.
102:
423-448,
1993[Abstract].
14.
Meissner, G.
Adenine nucleotide stimulation of Ca2+-induced Ca2+ release in sarcoplasmic reticulum.
J. Biol. Chem.
259:
2365-2374,
1984[Abstract/Free Full Text].
15.
Meissner, G.
Ryanodine receptor/Ca2+ release channels and their regulation by endogenous effectors.
Annu. Rev. Physiol.
56:
485-508,
1994[Medline].
16.
Meissner, G.,
and
A. El-Hashem.
Ryanodine as a functional probe of the skeletal muscle sarcoplasmic reticulum Ca2+ release channel.
Mol. Cell. Biochem.
114:
119-123,
1992[Medline].
17.
Meissner, G.,
and
J. S. Henderson.
Rapid calcium release from cardiac sarcoplasmic reticulum vesicles is dependent on Ca2+ and is modulated by Mg2+, adenine nucleotide, and calmodulin.
J. Biol. Chem.
262:
3065-3073,
1987[Abstract/Free Full Text].
18.
Meissner, G.,
E. Rios,
A. Tripathy,
and
D. A. Pasek.
Regulation of skeletal muscle Ca2+ release channel (ryanodine receptor) by Ca2+ and monovalent cations and anions.
J. Biol. Chem.
272:
1628-1638,
1997[Abstract/Free Full Text].
19.
Michalak, M.,
P. Dupraz,
and
V. Shoshan-Barmatz.
Ryanodine binding to sarcoplasmic reticulum membrane; comparison between cardiac and skeletal muscle.
Biochim. Biophys. Acta
939:
587-594,
1988[Medline].
20.
Murphy, E.,
C. Steenbergen,
L. A. Levy,
B. Raju,
and
R. E. London.
Cytosolic free magnesium levels in ischemic rat heart.
J. Biol. Chem.
264:
5622-5627,
1989[Abstract/Free Full Text].
21.
Ogawa, Y.,
and
H. Harafuji.
Osmolarity-dependent characteristics of [3H]ryanodine binding to sarcoplasmic reticulum.
J. Biochem. (Tokyo)
107:
894-898,
1990[Abstract].
22.
Schoenmakers, J. M.,
G. J. Visser,
G. Flik,
and
A. P. R. Theuvene.
CHELATOR: an improved method for computing metal ion concentrations in physiological solutions.
Biotechniques
12:
870-879,
1992[Medline].
23.
Smith, R. M.,
and
A. E. Martell.
Critical Stability Constants. New York: Plenum, 1974, vol. 1.
24.
Timerman, A. P.,
H. Onoue,
H. B. Xin,
S. Barg,
J. Copello,
G. Wiederrecht,
and
S. Fleischer.
Selective binding of FKBP12.6 by the cardiac ryanodine receptor.
J. Biol. Chem.
271:
20385-20391,
1996[Abstract/Free Full Text].
25.
Von Zglinicki, T.,
and
M. Bimmler.
The intracellular distribution of ions and water in rat liver and heart muscle.
J. Microsc.
146:
77-85,
1987[Medline].
26.
Wier, W. G.
Cytoplasmic Ca2+ in mammalian ventricle: dynamic control by cellular processes.
Annu. Rev. Physiol.
52:
467-485,
1990[Medline].
27.
Xu, L.,
G. Mann,
and
G. Meissner.
Regulation of cardiac Ca2+ release channel (ryanodine receptor) by Ca2+, H+, Mg2+, and adenine nucleotides under normal and simulated ischemic conditions.
Circ. Res.
79:
1100-1109,
1996[Abstract/Free Full Text].
28.
Zable, A. C.,
T. C. Favero,
and
J. J. Abramson.
Glutathione modulates ryanodine receptor from skeletal muscle sarcoplasmic reticulum. Evidence for redox regulation of the Ca2+ release mechanism.
J. Biol. Chem.
272:
7069-7077,
1997[Abstract/Free Full Text].
29.
Zimanyi, I.,
and
I. N. Pessah.
Comparison of [3H]ryanodine receptors and Ca2+ release from rat cardiac and rabbit skeletal muscle sarcoplasmic reticulum.
J. Pharmacol. Exp. Ther.
256:
938-946,
1991[Abstract].
30.
Zucchi, R.,
and
S. Ronca-Testoni.
The sarcoplasmic reticulum Ca2+ channel/ryanodine receptor: modulation by endogenous effectors, drugs and disease states.
Pharmacol. Rev.
49:
1-51,
1997[Abstract/Free Full Text].
AJP Cell Physiol 274(1):C120-C128
0363-6143/98 $5.00
Copyright © 1998 the American Physiological Society