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INTRODUCTION |
Ryanodine receptor
(RyR)1 is a large
(~2.3-MDa) homotetrameric Ca2+ release channel in the
sarcoplasmic reticulum (SR) membrane in vertebrate skeletal muscles and
plays a critical role in excitation-contraction coupling (1-4). The
RyR channel is mainly activated by two distinct modes:
depolarization-induced Ca2+ release (DICR) and
Ca2+-induced Ca2+ release (CICR). DICR, which
is the primary mechanism in skeletal muscle contraction, is triggered
directly or indirectly by the conformational change of the voltage
sensor, the dihydropyridine receptor (DHPR), on depolarization of the
transverse tubule membrane. On this occasion, extracellular
Ca2+ entry is not necessarily required. In contrast, CICR
is caused by activation of the RyR channel by micromolar or greater
concentrations of Ca2+, which is attained by the
Ca2+ influx through DHPR, although tight association with
DHPR is not required for this mode.
Adult mammalian skeletal muscles predominantly express the type 1 isoform of RyR (RyR1). Some specific muscles, e.g. diaphragm and soleus, however, also have a minuscule amount (<1-4%) of the type 3 isoform (RyR3; Refs. 5-7). Recent studies using gene-targeted mice revealed that RyR1 could mediate both DICR and CICR, whereas RyR3
showed CICR but not DICR (8, 9). Frog and other nonmammalian vertebrate
skeletal muscles, in contrast, express nearly equal amounts of the two
isoforms of RyR, referred to as
- and
-RyR (10, 11), which are
homologues of RyR1 and RyR3, respectively (12, 13). Because of an RyR1
homologue in the primary structure,
-RyR is believed to mediate DICR
in nonmammalian skeletal muscles. This is supported by the fact that a
Crooked Neck Dwarf mutant of chicken that lacks normal
-RyR fails to exhibit DICR (14). The role and significance of
-RyR in these muscles, however, are still unclear.
- and
-RyR purified from frog skeletal muscle demonstrated the
CICR channel activity, and the Ca2+ release appeared to be
a simple summation of each contribution (11, 15). Further
investigations on [3H]ryanodine binding activity revealed
that these isoforms were very similar in activity and that their
responses to CICR modulators were indistinguishable under conditions
simulating the myoplasm, suggesting an equal assignation of
- and
-RyR to CICR in situ in frog skeletal muscle (16, 17).
These experiments were conducted using purified proteins that were
solubilized with a detergent such as CHAPS to separate them from each
other. It is well known that the RyR channel activity is modulated by
several accessory proteins (e.g. 12-kDa FK506-binding
protein (FKBP12) and calmodulin; Refs. 4, 18, 19). However, no or only
a minor amount of such accessory proteins was detected in the purified
RyR preparations (20, 21), probably because of dissociation of these
proteins from RyR in the presence of CHAPS during the solubilization
and purification procedure; this, in turn, might cause changes in properties of these isoforms. Recent investigation revealed that CHAPS
greatly enhanced [3H]ryanodine binding to RyRs in SR
vesicles of rabbit and frog skeletal muscles (22). In addition, the
affinities for divalent cations of the activating and inactivating
Ca2+ sites of the purified
- and
-RyR were found to
be lower than those of the Ca2+ release channel of RyR
in situ (17). Determination of the activity of each of
-
and
-RyR in situ are therefore required for better understanding of their roles.
In the present study, we established a method for separating the total
[3H]ryanodine binding activity into those of
- and
-RyR in frog skeletal muscle SR vesicles where the native
organization of the Ca2+ release channel including RyR and
accessory proteins was still maintained. This success is attributable
entirely to two findings: first, an extremely slow dissociation of
ryanodine from RyR, practically irreversible at a low temperature (23,
24); and second, separation of the two isoforms without loss of their
binding activity by immunoprecipitation with a specific monoclonal
antibody after solubilization of the SR with CHAPS (6, 25). The
results, contrary to our expectations, demonstrate that
[3H]ryanodine binding activity of
-RyR is much lower
than that of
-RyR in the SR vesicles, suggesting a selective
suppression of the CICR activity of
-RyR in frog skeletal muscle.
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EXPERIMENTAL PROCEDURES |
Materials--
[3H]Ryanodine (60-90 Ci/mmol) was
purchased from NEN Life Science Products, Inc. Goat anti-rat
IgG-agarose, nonimmune rat IgG, and monoclonal anti-calmodulin antibody
(C-7055) were obtained from Sigma. Anti-FKBP12 antibody (PA1-026) was
from Affinity Bioreagents Inc. Egg lecithin (egg total phosphatide
extract) was from Avanti Polar Lipids. All other reagents were of
analytical grade.
Isolation of SR Vesicles--
SR vesicles were prepared from
bullfrog leg muscle (11). The isolated vesicles were quickly frozen in
liquid N2 and stored at
80 °C until used. Membrane
protein was measured by the biuret method using bovine serum albumin as
a standard.
SDS-Polyacrylamide Gel Electrophoresis (PAGE) and Western
Blotting--
SDS-PAGE was performed with 2-12% or 5-15% linear
gradient gels (21). Gels were stained with Coomassie brilliant blue.
For Western blotting, gels were electrophoretically transferred onto polyvinylidene difluoride membranes. Western blotting was
performed with rabbit anti-FKBP12 antibody (1:1000 dilution), rat
anti-
-RyR antibody (1H7, 1:100 dilution), and mouse anti-calmodulin
antibody (1:100 dilution), and positive bands were detected by an ECL
system using peroxidase-conjugated secondary antibodies (21).
Antibody Production and Immunoprecipitation--
A monoclonal
antibody against frog
-RyR, 1H7, was produced in rats according to
the method of Kishiro et al. (26) using the purified
-RyR
as an antigen. This antibody selectively recognized
-RyR among
proteins of SR vesicles prepared from frog skeletal muscle and did not
react with
-RyR (see "Results"). It also did not react with any
RyR isoforms of mammals (RyR1-3) or
-RyR of chicken or fish (data
not shown).
Immunoprecipitation was performed using 1H7-bound agarose beads, which
were prepared from anti-rat IgG-agarose beads (6, 25). Briefly, an
aliquot of 100 µl of the antibody solution was incubated for 2 h
at 4 °C with 30 µl of goat anti-rat IgG-agarose beads in a buffer
containing 0.5 M NaCl, 20 mM Tris-HCl, pH 7.5, and 0.05% Tween 20. Nonimmune rat IgG was similarly adsorbed to the
beads for control experiments. These beads were washed three times with
the buffer and stored at 4 °C until use. SR vesicles were
solubilized with 1% CHAPS and 0.5% egg lecithin in the above buffer
and incubated with the antibody beads for 2 h at 4 °C. After
washing three times with the buffer, proteins bound to the beads were
subjected to SDS-PAGE.
[3H]Ryanodine Binding Assay--
SR vesicles were
usually incubated with 8.5 nM [3H]ryanodine
for 5 h at 25 °C in 200 µl of a buffer containing 0.17 M NaCl, 20 mM
3-(N-morpholino)-2-hydroxypropanesulfonic acid/NaOH, pH 6.8, and 2 mM dithiothreitol. 1 mM
,
-methylene
adenosine 5'-triphosphate (AMPPCP) and various concentrations of
Ca2+ buffered with 10 mM EGTA were supplemented
unless otherwise indicated. Free Ca2+ concentrations were
calculated using the value of 8.79 × 105
M
1 as the apparent binding
constant for Ca2+ of EGTA (27). After 5 h of
incubation when the [3H]ryanodine binding reached nearly
steady state, the vesicles were supplemented with 20 µM
nonradioactive ryanodine to terminate further incorporation of
[3H]ryanodine, followed by immediate cooling to 4 °C,
and solubilized with 1% CHAPS and 0.5% egg lecithin. For separation
of the total binding into that for
- and
-RyR,
-RyR was
immunoprecipitated by incubating for 2 h at 4 °C with the
1H7-bound agarose beads, which had been washed by the reaction medium
containing 1% CHAPS and 0.5% egg lecithin. The precipitated agarose
beads were washed three times with the same washing buffer, and the
radioactivity responsible for
-RyR was recovered by incubating the
beads with 0.1 M glycine-HCl, pH 1.5, which was used to
disrupt the antigen-antibody complex. The resultant supernatant, on the
other hand, gave [3H]ryanodine binding to
-RyR which
was determined by the gel filtration method using a small-scale column
in a centrifuge (21). Total binding to SR vesicles was separately
determined in a similar way from the supernatant after
immunoprecipitation using nonimmune rat IgG-agarose beads instead of
1H7-agarose beads. Nonspecific radioactivity was determined in the
presence of 20 µM unlabeled ryanodine at the onset of the
binding reaction.
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RESULTS |
Rationale for Separation of Total
[3H]Ryanodine Binding to Frog SR Vesicles into
Contributions of
- and
-RyR--
In previous experiments (6), we
successfully determined [3H]ryanodine binding to
RyR3 in the mixture of solubilized RyR1 and RyR3. In the present
experiments, however, total binding to SR vesicles must be separated
into
- and
-RyR by immunoprecipitation after solubilization of SR
vesicles by CHAPS and phospholipids. These reagents, however, enhanced
[3H]ryanodine binding at steady state as shown in Fig. 6
(also see Ref. 22). Because the immunoprecipitation procedure requires 3-4 h of additional incubation, [3H]ryanodine binding
might increase during this period. Resources to avoid this are required.
Fig. 1, inset, shows the time
course of [3H]ryanodine binding to frog skeletal muscle
SR vesicles at 25 °C in an isotonic medium containing 0.17 M NaCl, 8.5 nM [3H]ryanodine, and
the optimal Ca2+ (pCa 4.0; see "Experimental
Procedures"). Because frog SR vesicles show very low
[3H]ryanodine binding activity without added ligands
other than Ca2+ (24), 1 mM AMPPCP, a
nonhydrolyzable ATP analog, was supplemented to the medium to stimulate
the binding. The binding followed an apparently exponential time course
(24) and was close to near steady state at 4 h (Fig. 1.,
inset). After 5 h of incubation (Fig. 1, upward
arrow), incorporation of [3H]ryanodine was
terminated by addition of 20 µM unlabeled ryanodine, and
the vesicles were solubilized with 1% CHAPS and 0.5% egg lecithin to
separate
- and
-RyR as described below. The following change of
bound [3H]ryanodine after solubilization is shown in Fig.
1. The radioactivity gradually decreased with time at 25 °C
(open circles) because of replacement of the bound
[3H]ryanodine by the unlabeled one. At 4 °C, in
contrast, it was unchanged up to 3.5 h, because the bound
[3H]ryanodine hardly dissociated from RyR at the low
temperature (filled circles; Refs. 23, 24). Thus, by
incubating at 4 °C, we were able to hold the
[3H]ryanodine binding at the steady state under a
specified condition for several hours after solubilization. This
allowed us to separate
- and
-RyR retaining their
[3H]ryanodine binding.

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Fig. 1.
Time course of [3H]ryanodine
binding to frog skeletal muscle SR vesicles and resources for
maintaining [3H]ryanodine binding at the steady
state value after solubilization. 100 µg of SR vesicles was
incubated with 8.5 nM [3H]ryanodine at
25 °C in medium containing 0.17 M NaCl, 20 mM 3-(N-morpholino)-2-hydroxypropanesulfonic
acid/NaOH, pH 6.8, 2 mM dithiothreitol, 1 mM
AMPPCP, and 0.1 mM free Ca2+. Inset,
time course of the binding reaction. After 5 h of incubation
(arrow), 20 µM nonradioactive ryanodine, 1%
CHAPS, and 0.5% egg lecithin were added to the medium to solubilize
the SR with cessation of further incorporation of
[3H]ryanodine. Further incubation at 25 °C (open
circles) or 4 °C (filled circles) was continued for
a period as indicated on the abscissa. The radioactivity
derived from bound [3H]ryanodine was determined as
described under "Experimental Procedures" and plotted against
incubation time. The data are mean ± S.E. (n = 3). Symbols without bars mean that the magnitude
of S.E. was within the size of the symbols. Note that the bound
[3H]ryanodine was gradually decreased at 25 °C,
whereas it was unchanged at 4 °C.
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The immunoprecipitation method with a monoclonal antibody against
-RyR, 1H7, was used to separate
- and
-RyR but to retain their
[3H]ryanodine binding activity as shown previously (21,
28). Fig. 2A shows an SDS-PAGE
pattern for the immunoprecipitation experiments. In frog skeletal
muscle SR vesicles,
- and
-RyR were detected as two bands of
nearly equal intensity (Fig. 2A, SR). The ratio of intensity
of these bands (
:
) was estimated by densitometry to be 45:55.
When SR proteins after solubilization with CHAPS were
immunoprecipitated with control nonimmune IgG, both isoforms remained
in the supernatant, and neither was detected in the precipitated beads
(Fig. 2A, IgG). In contrast, when immunoprecipitated with
1H7,
-RyR was recovered all in a single band in the beads and was
not found in the supernatant, whereas
-RyR remained in the
supernatant (Fig. 2A, 1H7). These indicate that 1H7
specifically and completely immunoprecipitated
-RyR from the
solubilized SR proteins. This complete separation of the two RyRs was
consistently observed, irrespective of the presence or absence of the
CICR modulators (Ca2+, AMPPCP, caffeine, and ryanodine)
that were used in the [3H]ryanodine binding experiments
(data not shown).

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Fig. 2.
Separation of -
and -RyR by immunoprecipitation with an
-RyR-specific monoclonal antibody, 1H7.
A, SDS-polyacrylamide gel electrophoresis of - and
-RyR separated. 15 µg of frog SR vesicles was solubilized
(SR), followed by immunoprecipitation with nonimmune IgG
(IgG) or 1H7, a monoclonal antibody against -RyR
(1H7), as described under "Experimental Procedures." The
resultant supernatant (lane S) and precipitate (beads;
lane B) were subjected to SDS-PAGE on a 2-12% linear
gradient gel together with 15 µg of unprocessed SR vesicles
(SR) and stained with Coomassie brilliant blue. H
and L, bands for heavy and light chains of IgG,
respectively. Molecular masses (in kDa) of standards are
indicated on the left. Note that the supernatant
(S) showed the band for -RyR ( ) without the
contaminating band of -RyR ( ), and that the beads (B)
showed the reverse when immunoprecipitated with 1H7. With IgG, in
contrast, both - and -RyR were retained in the supernatant.
B, model experiments for determination of
[3H]ryanodine binding to - and -RyR by
immunoprecipitation. 1 µg of each purified - and -RyR and a
mixture of them (0.5 µg each) were incubated with
[3H]ryanodine as in Fig. 1 in the presence of 1% CHAPS
and 0.5% egg lecithin and then processed to immunoprecipitation with
1H7. The radioactivity of protein-bound ryanodine in the resultant
supernatant (open columns) and beads (filled
columns) were determined. Total binding (hatched
columns) was separately determined from the supernatant of
immunoprecipitation with nonimmune IgG instead of 1H7. The data are
mean ± S.E. (n = 3).
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Model experiments using the purified
- and
-RyR clearly proved
that the method of immunoprecipitation with 1H7 as described under
"Experimental Procedures" properly determined
[3H]ryanodine binding of individual isoforms (Fig.
2B). The purified
- and
-RyR were prelabeled with
[3H]ryanodine up to steady state and then
immunoprecipitated with 1H7 (see "Experimental Procedures"). The
[3H]ryanodine binding activity of the supernatant after
immunoprecipitation with nonimmune IgG, which was separately
determined, is referred to as total binding, because the supernatant
should contain both isoforms (Fig. 2B, hatched columns).
Total binding of
- and
-RyR was 134 ± 6 and 131 ± 3 pmol/mg protein, respectively, and they were similar as shown
previously (16, 17). By immunoprecipitating with 1H7, almost all of the
radioactivity for
-RyR was recovered in the beads (Fig. 2B,
filled columns), whereas all the binding for
-RyR remained in
the supernatant (Fig. 2B, open columns). With each isoform,
the sum of radioactivity in the supernatant and the precipitate (beads)
was consistent with the total binding, indicating no significant loss
of bound [3H]ryanodine during immunoprecipitation (Fig.
2B, columns in
-RyR and
-RyR). With a mixed preparation of equal amounts of
-
and
-RyR, furthermore, approximately half of the total binding was detected in each of the supernatant and the precipitate (Fig. 2B,
+
). Thus, solubilization with supplement of
nonradioactive ryanodine followed by immunoprecipitation with the 1H7
antibody works well to determine the individual
[3H]ryanodine binding of
- and
-RyR in the SR
vesicles where both isoforms occur.
[3H]Ryanodine Binding Activity of Two RyR Isoforms in
Frog Skeletal Muscle SR--
A three-step procedure was used to
determine the binding of
- and
-RyR in SR vesicles of frog
skeletal muscles. First, the SR vesicles were labeled with
[3H]ryanodine to determine total
[3H]ryanodine binding at the steady state under a
specified condition. Second, they were supplemented with nonradioactive
ryanodine, cooled down to 4 °C, and solubilized with CHAPS and
phospholipids. Finally, the solubilized specimen was incubated at
4 °C with 1H7 to separate
- and
-RyR into the precipitate of
agarose beads and the supernatant, respectively, and the
[3H]ryanodine binding of each isoform was determined.
Total [3H]ryanodine binding to SR vesicles was separately
determined using nonimmune IgG-agarose beads as mentioned above,
confirming the validity of our results. Fig.
3 shows the stimulatory effect of various
amounts of AMPPCP on
- and
-RyR in SR vesicles. The ryanodine
binding reaction was carried out at the optimal concentration of pCa
4.0 in an isotonic medium containing 0.17 M NaCl in the presence of 0.2 and 1 mM AMPPCP. Surprisingly, the
[3H]ryanodine bound to
-RyR was much less than that
bound to
-RyR; the radioactivities in the beads were <1 and 4% of
those in the supernatant at 0.2 mM and 1 mM
AMPPCP, respectively. This low binding is not attributable to loss of
the bound [3H]ryanodine from the beads during the
separation procedure, because the sum of the binding in the supernatant
and the beads was almost equal to the total binding. This reduced value
of
-RyR (~4% of
-RyR) was consistently observed at a longer
incubation period (over 15 h) of the binding reaction, excluding
the possibility of a shorter incubation time for this reason.

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Fig. 3.
[3H]Ryanodine
binding activity of - and
-RyR in the SR vesicles.
[3H]Ryanodine binding to the SR vesicles was carried out
at 25 °C as in Fig. 1 in the presence of 0.2 and 1 mM
AMPPCP. Then SR vesicles were rapidly cooled to 4 °C and solubilized
with 1% CHAPS and 0.5% egg lecithin in the presence of 20 µM supplemented nonradioactive ryanodine. The two
isoforms ( - and -RyR) were separated by immunoprecipitation with
1H7 as described under "Experimental Procedures." The radioactivity
in the precipitate (beads; filled columns) and the
supernatant (open columns) represents the ryanodine binding
activity of - and -RyR, respectively. Total binding
(hatched columns), which was obtained separately from the
supernatant of immunoprecipitation with nonimmune IgG, represents the
binding of the sum of the two isoforms. Inset, results for
-RyR redrawn on an extended scale. The data are mean ± S.E.
(n = 4). Note that -RyR shows much lower
[3H]ryanodine binding activity than -RyR, although
-RyR is as sensitive to AMPPCP as -RyR. The results in the
presence of 5 mM AMPPCP were similar.
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The increase in AMPPCP from 0.2 to 1 mM enhanced the total
binding ~3-fold, which was primarily ascribed to an increase in the
binding to
-RyR. It should be noted, however, that
-RyR is also
sensitive to AMPPCP in this stimulation of [3H]ryanodine
binding (Fig. 3, inset). The enhancement factor for
-RyR
appeared to be somewhat greater than that for
-RyR with the increase
from 0.2 to 1 mM AMPPCP. This is also the case with 5 mM AMPPCP (data not shown). Similar results were also
obtained by immunoprecipitation using a polyclonal antibody against
-RyR (25) instead of 1H7 (data not shown). Consequently, these
results indicate that
-RyR intrinsically has much lower ryanodine
binding than
-RyR in the SR vesicles.
Ca2+ dependence of the [3H]ryanodine binding
to each isoform is shown in Fig. 4. For
clear observation, 1 mM AMPPCP was supplemented to the
reaction medium to enhance the binding.
-RyR showed a biphasic
Ca2+ dependence, with apparent EC50 values of
~16 µM and ~0.8 mM for Ca2+
activation and Ca2+ inactivation, respectively (Fig. 4,
triangles).
-RyR also showed a similar biphasic
Ca2+ dependence, although the relationship seemed to be
slightly shifted to a lower Ca2+ concentration range (Fig.
4, squares, also see inset). There was only a
slight difference, however, between
- and
-RyR in the
Ca2+ sensitivity in either Ca2+ activation or
Ca2+ inactivation.
-RyR, in contrast, displayed much
less binding at all of the Ca2+ concentrations examined
(Fig. 4, squares). The peak value for
-RyR (0.036 pmol/mg
protein) at pCa 4.0 was <4% of that for
-RyR (0.95 pmol/mg).

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Fig. 4.
Ca2+ dependence of the
[3H]ryanodine binding for -
and -RyR in SR vesicles.
[3H]Ryanodine binding experiments were carried out in the
presence of 1 mM AMPPCP with varied free Ca2+
concentrations and processed as in Fig. 3. Inset, results
for -RyR redrawn on an extended scale. The data are mean ± S.E. (n = 3). Note that -RyR showed biphasic
Ca2+ dependence similar to that of -RyR, although the
amount of binding was much smaller.
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Fig. 5 demonstrates the effect of
caffeine on
-RyR (Fig. 5A) and
-RyR (Fig.
5B) in the SR vesicles. At pCa 5.6, which is near the
threshold for Ca2+ activation without caffeine, 10 mM caffeine enhanced the binding several 10-fold. At the
optimal concentration of pCa 4.0, however, the enhancement by caffeine
was 3-fold at most. In the presence of caffeine, the binding at pCa 5.6 was significantly higher than that at pCa 4.0. These findings were
common to the two isoforms and consistent with the well-known
modification of the RyR channel activity by caffeine: increased
sensitivity to Ca2+ in the Ca2+ activation and
enhancement of the peak activity of CICR and
[3H]ryanodine binding (17, 22). It should be noted that
-RyR showed [3H]ryanodine binding 10-30 times as
great as that of
-RyR under the same conditions. These results
indicate that
-RyR is sensitive to caffeine, as is the case with
-RyR, in SR vesicles, although the former is much lower than the
latter in ryanodine binding activity even in the presence of
caffeine.

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Fig. 5.
Effect of caffeine on the
[3H]ryanodine binding to -
and -RyR in SR vesicles. Determinations
were carried out with or without 10 mM caffeine as in Fig.
4. A, -RyR; B, -RyR. The data are mean ± half the range of deviation of duplicate determinations. Note that
the enhancement by caffeine was more marked at pCa 5.6 than at pCa 4.0. In the presence of 10 mM caffeine, the binding at pCa 5.6 was much higher than that at pCa 4.0, although these Ca2+
concentrations corresponded to those near the threshold and the optimum
for Ca2+ activation, respectively, in the absence of
caffeine. These findings are consistent with the following conclusions
on the effect of caffeine: increase in the sensitivity to
Ca2+ in Ca2+ activation and enhanced peak
value.
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Effects of High Salt and CHAPS on [3H]Ryanodine
Binding to
- and
-RyR in SR Vesicles--
In isotonic media,
-RyR had [3H]ryanodine binding activity much lower
than
-RyR, although the sensitivity to Ca2+, AMPPCP, and
caffeine was still retained. The effects of high salt and CHAPS, both
of which potently stimulate [3H]ryanodine binding to SR
vesicles (22, 29), were examined (Fig.
6). In medium containing 1 M
NaCl, the total [3H]ryanodine binding was nearly 5-fold
higher than that in medium containing 0.17 M NaCl. A marked
enhancement in binding was observed not only with
-RyR but also with
-RyR. The nonequivalence of the two RyRs, however, was still
remarkable:
-RyR showed 6-fold higher [3H]ryanodine
binding than
-RyR. The addition of 1% CHAPS with 0.5% egg lecithin
into the isotonic medium increased the total binding 4-fold. Under this
condition, the [3H]ryanodine binding to
-RyR was more
enhanced, reaching nearly half of the binding to
-RyR. Thus,
[3H]ryanodine binding activity of
-RyR is effectively
increased by solubilization of the SR vesicles with CHAPS. These
findings indicate that some molecular interaction that is weakened by
CHAPS may selectively suppress the CICR activity of
-RyR.

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Fig. 6.
Effects of high salt and CHAPS on the
[3H]ryanodine binding to -
and -RyR in SR vesicles.
[3H]Ryanodine binding to SR vesicles was carried out as
in Fig. 1 under the conditions indicated. The following procedures were
performed as in Fig. 3. The data are mean ± S.E.
(n = 3-6). Note that the presence of 1 M
NaCl (center) instead of 0.17 M
(left) greatly enhanced the binding of -RyR as well as
-RyR, but nonequivalence of the two isoforms was still prominent.
Addition of 1% CHAPS with 0.5% egg lecithin (right) more
markedly increased the binding of -RyR, reaching approximately half
that of -RyR.
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FKBP12 Homologue Cannot Be a Candidate for Selective
Suppression--
It has been reported that FKBP12 selectively inhibits
the Ca2+ release channel activity of RyR1 among three RyR
isoforms in mammals (30, 31). Possible involvement of FKBP12 in the
suppressed activity of
-RyR was therefore tested using FK506, which
specifically removed the FKBP12 (32). Fig.
7A demonstrates Western blots of the fraction immunoprecipitated with 1H7 of solubilized frog SR
vesicles. A small protein of ~15 kDa was positively reacted with
anti-FKBP12 antibody (Control lane). This band disappeared from the SR vesicles treated with 100 µM FK506 before
immunoprecipitation, without change in the amount of precipitated
-RyR (+FK506 lane). The reacted band was closer in its
partial N-terminal amino acid sequence to human FKBP12.6 than to
FKBP12.0 (Fig. 7B). The [3H]ryanodine binding
of SR vesicles treated with 100 µM FK506 was compared
with that of nontreated ones (Fig. 7C). The selective suppression of
-RyR was consistently observed even after the SR
vesicles were pretreated with FK506. These results suggest that
although a homologue of mammalian FKBP12 is tightly associated with
-RyR in frog skeletal muscle, it cannot be responsible for the
selective suppression of
-RyR.

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Fig. 7.
Effect of FK506 on the dissociation of the
FKBP12 homologue from -RyR and the suppressed
binding of -RyR. A, Western
blots of the fraction of frog SR vesicles that was immunoprecipitated
with 1H7. Immunoprecipitation was carried out as shown in Fig. 2 in the
presence (+FK506) and absence (Control) of 100 µM FK506. Molecular masses (in kDa) of standards are
indicated on the left. Note that a band of ~15 kDa, which
reacted with anti-human FKBP12, specifically disappeared after
incubating the SR with FK506 (lower panel). FK506 did not
affect the amount of -RyR immunoprecipitated (upper
panel). B, N-terminal partial amino acid sequence of
frog FKBP12 homologue and its alignment with those of human FKBP12.0
and FKBP12.6. C, [3H]ryanodine binding of -
and -RyR in the SR vesicles with (+FK506) or without
(Control) 100 µM FK506. Assays were carried
out as in Fig. 3. The data are mean ± S.E. (n = 3). The binding of each isoform was normalized with the total binding
under respective conditions. 100% denotes 0.81 and 2.3 pmol/mg protein for Control and +FK506,
respectively. Note that FK506 did not eliminate the suppression of
-RyR in contrast to its marked removal of the ~15-kDa
protein.
|
|
It is known that calmodulin is associated with RyR and affects its
Ca2+ release channel activity (1-3). We therefore tested
the presence of calmodulin in our SR vesicles by Western blotting with
commercial anti-calmodulin antibody. No positive bands were detected,
indicating the undetectable amount of calmodulin in the SR (data not
shown). This suggests a minor possibility of involvement of calmodulin in the suppression of
-RyR.
Scatchard Plot Analysis of [3H]Ryanodine Binding to
- and
-RyR in SR Vesicles--
Fig.
8 demonstrates dose-dependent
[3H]ryanodine binding to
- and
-RyR in the SR
vesicles together with total binding in medium containing 1 M NaCl. In isotonic medium, the binding to
-RyR was too
little to enable us to carry out reliable analysis. The reaction was
therefore made under a more favorable condition for ryanodine binding
(16, 29); the medium contained 1 M NaCl, 1 mM
AMPPCP, and 10 mM caffeine, and not only
-RyR but also
-RyR showed more enhanced binding (see Fig. 6). The amounts of
[3H]ryanodine bound to
-RyR (Fig. 8,
squares) and
-RyR (Fig. 8, triangles)
increased with increase in free [3H]ryanodine
concentration (Fig. 8A). Scatchard plot analysis revealed that the results could be expressed by a linear line in either case,
indicating a single class of homogeneous binding sites (Fig. 8B). The best fit binding parameters (Kd
and Bmax) were obtained by fitting the data to
an equation B = Bmax × [free
ryanodine]/(Kd + [free ryanodine]) according to
the Marquardt-Levenberg algorithm using SigmaPlot for Macintosh,
version 5. The obtained sets of binding parameters were as follows:
Kd = 12.8 ± 1.3 nM, and
Bmax = 3.9 ± 0.3 pmol/mg protein for
-RyR; and Kd = 2.3 ± 0.1 nM,
and Bmax = 4.9 ± 0.2 pmol/mg protein for
-RyR (expressed as mean ± S.E. for duplicate determinations at
each of six different ligand concentrations).
-RyR
(Kd = 12.8 ± 1.3 nM) showed 6-fold
lower affinity than
-RyR (Kd = 2.3 ± 0.1 nM). The Kd values of the purified
-
and
-RyR under similar conditions were 2-4 nM (11).
Thus, the affinity of
-RyR for [3H]ryanodine was
significantly lower in the SR vesicles than in the purified
preparations, whereas
-RyR showed similar affinities between native
and purified preparations. The maximal binding sites of
- and
-RyR (Bmax = 3.9 ± 0.3 and 4.9 ± 0.2 pmol/mg SR protein, respectively) were consistent with the content
of each isoform in the SR vesicles (45:55 for
- and
-RyR; see
Fig. 2). These results indicate that the suppressed
[3H]ryanodine binding of
-RyR is primarily
attributable to a reduced affinity but not to a decreased
Bmax value.

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Fig. 8.
Dose-dependent
[3H]ryanodine binding to -
and -RyR in SR. Frog skeletal muscle SR
vesicles (100 µg) were incubated with 0.5-21 nM
[3H]ryanodine for 5 h at 25 °C in 1 M
NaCl, 20 mM
3-(N-morpholino)-2-hydroxypropanesulfonic acid/NaOH, pH 6.8, 2 mM dithiothreitol, 1 mM AMPPCP, 10 mM caffeine, and 0.1 mM free Ca2+.
The results for total SR (circles) and individual isoforms
(squares, -RyR; triangles, -RyR) were
determined as in Fig. 3. A, presentation in common
coordinates to show the range of deviation. The data are mean ± half the range of deviation of duplicate determinations. Symbols
without bars mean that the deviation was within the
size of the symbol. B, Scatchard plot for the average at
each ligand concentration. Linear Scatchard plots indicate that each
isoform had a single class of [3H]ryanodine binding
sites. Binding parameters (Kd and
Bmax) were obtained by fitting the data in Fig.
8A to the equation B = Bmax × [free ryanodine]/(Kd + [free ryanodine]);
12.8 ± 1.3 nM and 3.9 ± 0.3 pmol/mg protein for
-RyR and 2.3 ± 0.1 nM and 4.9 ± 0.2 pmol/mg
protein for -RyR, respectively. The results of total binding to SR
vesicles ( + ) could also be fitted by a single class of binding
sites with Kd of 3.8 ± 0.5 nM and
Bmax of 7.6 ± 0.3 pmol/mg protein. The
continuous lines in A and B were drawn
using these values. The dashed lines represent the
calculated sum using the parameters determined for - and -RyR.
Note that the directly determined and calculated results at the lowest
concentration of the ligand were actually indistinguishable
(A), although they appeared very diverse in the Scatchard
plot (B). See "Results" for details.
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|
The total [3H]ryanodine binding to the SR vesicles (Fig.
8, circles) corresponded well to the calculated sum of
binding amounts of
- and
-RyR as shown by the dashed
line (Fig. 8A). In Scatchard plot analysis, the results
could also be fitted well by an assumption of a single class of
homogeneous binding sites for ryanodine (Fig. 8B).
Kd and Bmax values were
similarly calculated by the results in Fig. 8A to be
3.8 ± 0.5 nM and 7.6 ± 0.3 pmol/mg SR protein,
respectively. This Bmax value was slightly lower
than the sum (8.8 ± 0.4 pmol/mg protein) of the two
Bmax values for
- and
-RyR. The upper
limit of available concentrations of [3H]ryanodine used
here was 21 nM, which may be insufficient for saturable
binding of
-RyR. If higher concentrations of the ligand were
feasible, the Scatchard plot would be downward convex with an intercept
at ~8.8 pmol/mg protein, as shown by the dashed line (Fig.
8B).
 |
DISCUSSION |
In the present study, we investigated the
[3H]ryanodine binding to the isolated SR vesicles as an
index of the CICR activity of the two RyR isoforms (
- and
-RyR)
in frog skeletal muscle, because ryanodine can bind only the open form
of RyR. After the binding reached the steady state, solubilization of
SR vesicles by CHAPS and phospholipids at a low temperature in the
presence of an excess amount of nonradioactive ryanodine and
immunoprecipitation with the monoclonal antibody against
-RyR (1H7)
enabled us to identify each contribution of
- and
-RyR to the
total [3H]ryanodine binding. This is the first report to
demonstrate the properties of the individual RyR isoforms of
nonmammalian skeletal muscles under conditions in which these proteins
exist in the intact SR membranes. Lipid bilayer experiments with SR
vesicles from skeletal muscles of nonmammalian vertebrates including
frog reported two distinct types of channel activity (3, 33-35): low
peak open probability with marked Ca2+ inactivation
and high peak open probability with no or slight Ca2+
inactivation. No evidence, however, can identify which isoform shows
which type of channel activity. Marengo et al. (35) showed that the two types of channel activity were convertible to each other by sulfhydryl oxidation and reduction of channels. Note that the
experiments presented here were carried out in the presence of 2 mM dithiothreitol.
We demonstrated that the [3H]ryanodine binding to
-RyR
is much lower (only ~4% at most) than that of
-RyR in SR vesicles in isotonic medium. Neither isoform, however, showed a difference in
its Ca2+ dependence (Fig. 4) or sensitivity to AMPPCP (Fig.
3) and caffeine (Fig. 5), which are well known stimulants of CICR.
These findings lead us to conclude that
-RyR may have CICR activity
selectively suppressed to a great extent in SR vesicles from frog
skeletal muscle. This is in marked contrast to the results obtained
with the purified
- and
-RyR, which showed nearly equal
[3H]ryanodine binding in the physiological milieu (Refs.
16, 17; see Fig. 2). Consistently, the affinity for
[3H]ryanodine of
-RyR was considerably lower
(Kd = 12.7 nM) than that of
-RyR
(Kd = 2.3 nM) in the SR in the medium
containing 1 M NaCl, 1 mM AMPPCP, and 10 mM caffeine (Fig. 8), whereas the purified
- and
-RyR
showed similar Kd values of 2-4 nM
(11), which are close to the value of
-RyR in the SR. The selective
suppression of
-RyR may also be true with the native
Ca2+ release channel in SR of intact or skinned fibers.
Scatchard plot analysis revealed that the suppression of
-RyR is
primarily caused by a marked decrease in affinity for ryanodine, but
not a reduction in Bmax (Fig. 8). This excludes
the possibility that some populations of
-RyR would be completely
inactive or silent, whereas others would be active. Alterations of
-RyR in sensitivity to CICR modulators, such as Ca2+,
AMPPCP, and caffeine, also cannot be the cause for the reduced activity
of
-RyR in view of the results shown in Figs. 3-5. All of the
-RyRs are potentially viable, but they may be in a suppressed state.
We have recently demonstrated that the CICR activity can be defined by
at least three independent parameters: activation by Ca2+,
inactivation by Ca2+ or Mg2+, and the potential
peak activity (17). An adenine nucleotide dose-dependently
increased the potential peak activity that determined the upper limit
of CICR activity at the optimum Ca2+ concentration, but the
stimulating reagent did not change affinities for Ca2+ or
Mg2+ of RyR in Ca2+ activation or inactivation
(17). Caffeine showed dual effects of Ca2+ sensitization in
Ca2+ activation and an increase in the peak activity in
CICR. Separate experiments showed that these stimulators also
dose-dependently increased affinity for
[3H]ryanodine of purified RyR and SR vesicles. These
effects, although in the reverse direction, may be reflected in the low
affinity for [3H]ryanodine of
-RyR in the present
case. This type of inhibition seems more suitable for stable
suppression of the activity, because the extent of inhibition will be
consistent regardless of free Ca2+ concentrations.
It is interesting to consider the mechanism of how
-RyR is
suppressed in SR vesicles. Because the suppression is not observed in
the purified proteins or their mixtures (Fig. 2), it is likely that
some factors in tight association with
-RyR may suppress its CICR
activity in these vesicles. This is strongly supported by the fact that
addition of CHAPS, which solubilizes the SR to weaken protein-protein
or protein-lipid interactions, more selectively enhanced the
[3H]ryanodine binding activity of
-RyR than that of
-RyR (Fig. 6). The putative factors should be associated with
-RyR alone or should suppress the activity of this isoform even if
they bind to both isoforms, because the suppression was effective only
on
-RyR. To date, many proteins have been proposed to interact with the RyRs to modulate their channel activity, including calmodulin, FKBP12, triadin, and calsequestrin (4, 18, 19). Among them, FKBP12
appeared to be a probable candidate as a suppressive factor, because it
was reported to selectively inactivate RyR1, although it can bind to
both RyR1 and RyR3 (21, 30). Consistently, our results indicate that a
homologue of mammalian FKBP12 may be tightly associated with
-RyR
(Fig. 7). However, this does not seem to be the case, because addition
of FK506, which effectively purged the protein from the
immunoprecipitated
-RyR, did not eliminate the suppression of the
[3H]ryanodine binding activity of
-RyR. Because
calmodulin was not detected in our SR vesicles by Western blotting with
anti-calmodulin antibody, involvement of calmodulin in the suppression
also may be unlikely.
Alternatively, it is also possible that the lowered activity of
-RyR
in the SR membrane may be an inherent nature of
-RyR itself. It has
been demonstrated that the purified RyR1 channels displayed
heterogeneous populations of low open probability (<0.1) and high open
probability (~ 0.3), whereas the purified RyR3 channels showed a
homogeneous population of open probability (~1) in an almost
all-or-none Ca2+-dependent manner in planar
lipid bilayers (21). Both isoforms, in contrast, showed similar
activity in [3H]ryanodine binding in the presence of
CHAPS (21). This different behavior of RyR isoforms has generally been
explained by the difference in vulnerability to some oxidative
conditions. However, this might reflect an association state of the RyR
monomer in a planar lipid bilayer, which is different from that in an
aqueous solution containing a detergent, because tetramer formation is
indispensable for activity. Then,
-RyR might be inclined to be at
the lowered state in CICR activity in the SR membrane, although the
relationship between the extent of association and activity has yet to
be determined. Further experiments are necessary to evaluate this possibility.
It has been reported that the CICR activity may be under suppressive
control by DHPR in mammalian skeletal muscles (36, 37). Suda (36)
observed that caffeine-induced Ca2+ release in rat skeletal
muscle cells was terminated on repolarization of the membrane, probably
through DHPR. Shirokova et al. (37) recently demonstrated
with cultured mouse myotubes that discrete Ca2+ release
events, which were probably activated by Ca2+, occurred
primarily at locations where depolarization of the membrane did not
elicit the continuous Ca2+ release, irrespective of the
presence of RyR3. These findings suggest that the CICR through RyR1
could be under the negative control of DHPR. On the basis of the
homology between RyR1 and
-RyR in the structure and function, it can
reasonably be presumed that
-RyR might also be selectively
suppressed by interaction with DHPR in frog skeletal muscle. Sucrose
density gradient ultracentrifugation revealed that SR vesicles used in
this study were hardly associated with the transverse tubule (data not
shown). This may exclude the possibility of direct suppressive control
of the CICR of
-RyR by DHPR. Furthermore, it should be noted that
there has been no evidence to date that indicates the occurrence of
suppressed RyR1 in SR vesicles from rabbit skeletal muscles, unlike
-RyR.
Recent studies demonstrated that Ca2+ sparks, discrete
Ca2+ release events probably activated by Ca2+,
are clearly observed in frog skeletal muscle at rest and on depolarization (38, 39). Shirokova et al. (40) reported that
no or only sparse sparks were detected in adult mammalian skeletal
muscles, which primarily express RyR1 with very little RyR3 at most
(40, 41), whereas they were easily detectable in frog skeletal muscle
where
-RyR occurred in an amount almost equal to that of
-RyR.
They proposed that
-RyR might be essential for the production of the
Ca2+ sparks in frog skeletal muscle. This appears to be
compatible with our results; however, there is one essential
difference. They assumed an intrinsic difference between RyR1
homologues (including
-RyR) and RyR3 homologues (including
-RyR),
whereas we showed that there was no difference in CICR activity between
the two isoforms.
-RyR, however, is selectively suppressed in SR
vesicles but not in the purified state. They also indicated that RyR3
might inactivate RyR1, because the time course of the discrete
Ca2+ release was prolonged by knockout of RyR3 (37).
Bertocchini et al. (42), on the contrary, proposed possible
amplification by RyR3 of CICR through RyR1 in mammalian neonate
skeletal muscle because of reduction greater than that expected from
the RyR3 content on its knockout. Cooperative interaction between the
two isoforms is an attractive hypothesis, and further investigations are required.
The results of the present study provide important information
regarding the roles of the two RyR isoforms in skeletal muscles. The
[3H]ryanodine binding activity of
-RyR is much lower
than that of
-RyR in SR vesicles in an isotonic milieu, regardless
of the presence or absence of CICR modulators. This suggests that the CICR may be mainly mediated by
-RyR in situ in frog
skeletal muscle.
-RyR is thought to function primarily as a DICR
channel, at least in nonmammalian skeletal muscles (2, 3). Thus, the
two RyR isoforms may play distinct roles in Ca2+ release
from SR:
-RyR as a DICR channel and
-RyR as a CICR channel.
Further investigations will clarify the significance of the two RyR
isoforms in nonmammalian vertebrate skeletal muscles.