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INTRODUCTION |
According to a widely accepted hypothesis (1-7), skeletal
muscle-type E-C coupling1 is
triggered by the voltage-dependent binding of one of the
cytoplasmic loops (II-III loop) of the dihydropyridine (DHP) receptor
1 subunit to the SR Ca2+ release channel protein, also
referred as ryanodine receptor (RyR). The critical role of the II-III
loop was recognized first by an earlier finding of Tanabe et
al. (8, 9) that replacement of the II-III loop of the
cardiac DHP receptor with the skeletal muscle-type sequence conferred
the skeletal muscle-type E-C coupling activity in dysgenic myotubes
expressing chimeric DHP receptors. This concept was further supported
by the finding that a recombinant peptide corresponding to the II-III
loop activated ryanodine binding and Ca2+ channel activity
(10).
Further studies with shorter peptides corresponding to various regions
of the II-III loop have permitted new insight into functionally
important subdomains of the loop. A 61-residue recombinant peptide
corresponding to the Glu666-Glu726 region
activated the RyR, suggesting that the ability of activating E-C
coupling is localized in this region (11). According to our studies
with a series of synthetic peptides corresponding to different regions
of the loop (12), only one peptide corresponding to the
Thr671-Leu690 region (designated peptide A)
activated ryanodine binding and induced Ca2+ release from
SR (12, 13). Activating functions of peptide A are retained in a
truncated form of peptide A, corresponding to
Arg681-Leu690, but further truncation
abolished its activity (13). These findings indicate that an essential
domain for the activation of E-C coupling (designated the
"activator" of E-C coupling) is localized in the
Arg681-Leu690 region of the II-III loop. The
concept that there is another important domain of the II-III loop that
may be involved in the regulation of E-C coupling has emerged from the
findings as follows. As reported in our recent paper (12), a synthetic
peptide corresponding to the Glu724-Pro760
region of the II-III loop (designated peptide C), but not other peptides, inhibited peptide A-induced activation of the RyR. This suggests that the Glu724-Pro760 region of the
loop may serve as an antagonist of the activator described above. The
idea that this portion is critical for E-C coupling has also emerged
from the recent report by Nakai et al. (14). According to
the report, chimeric replacement of the
Phe725-Pro742 region of the II-III loop (the
region corresponding to the N-terminal half of peptide C) from cardiac
type to skeletal muscle type conferred the E-C coupling properties of
skeletal muscle-type to dysgenic myotubes expressing cardiac-type DHP
receptor. Thus, there appear to be at least two important domains of
the II-III loop that are required for skeletal muscle-type E-C coupling.
The main purpose of the present study is to gain new insight into the
mechanism by which E-C coupling is regulated by these two domains of
the II-III loop. As shown in the present study, peptide A produces a
rapid change of the RyR conformation from a resting state to an
activated state, and peptide C reverses this process. Interestingly,
these changes induced by peptide A and peptide C correspond to the
conformational changes induced by T-tubule depolarization and
polarization, respectively. This predicted that peptide C would
counteract not only peptide A-dependent activation of the
RyR but also depolarization-induced activation of E-C coupling. To test
this, we investigated the effects of peptide C on
depolarization-induced conformational change in the RyR and
Ca2+ release from the SR. As shown here,
depolarization-induced changes in the RyR to an active conformational
state and Ca2+ release from SR were blocked or reversed by
peptide C. These results suggest a new concept, that
depolarization-dependent activation of E-C coupling and
polarization-dependent repriming of the system are
modulated by the voltage-dependent alternative binding of the activator domain (the in situ counterpart of peptide A)
and the blocker/primer domain (the in situ counterpart of
peptide C), respectively, to the E-C coupling site(s) of the RyR.
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EXPERIMENTAL PROCEDURES |
Preparation--
The triad-enriched microsomal fraction was
prepared from rabbit leg and back muscles by differential
centrifugation as described previously (15). After the final
centrifugation, the sedimented fraction was homogenized in a solution
containing 0.3 M sucrose, 0.15 M gluconate,
proteolytic enzyme inhibitors (0.1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 0.8 µg/ml antipain, 2 µg/ml trypsin inhibitor) and 20 mM MES, pH 6.8 (PI buffer), to a final
protein concentration of 20-30 mg/ml. The preparation was quickly
frozen in liquid nitrogen and stored at
70 °C.
Synthesis of Peptides--
Peptides were synthesized on an
Applied Biosystems model 431 A synthesizer employing Fmoc
(N-(9-fluorenyl)methoxycarbonyl) as the
-amino protecting
group. Amidated peptides were cleaved and deprotected with 95%
trifluoroacetic acid. Purification was carried out by reversed-phase
high pressure liquid chromatography using a Rainin Instruments
preparative C8 column. Purified peptides were then dialyzed against
water using a dialysis membrane with a cutoff Mr
of 500. Five millimolar solutions of peptides were prepared in 10 mM HEPES, pH 7.2.
[3H]Ryanodine Binding Assay--
Triad vesicles
(0.5 mg/ml) were incubated in 0.1 ml of a reaction solution containing
8-10 nM [3H]ryanodine (68.4 Ci/ml, NEN Life
Science Products), 0.3 M KCl, 100 µM EGTA,
64.4 µM CaCl2, 20 mM MOPS, pH 7.2 for 2 h at 36 °C in the absence or in the presence of various
concentrations of peptides. Specific binding was calculated as the
difference between the binding in the absence (total binding) and in
the presence (nonspecific binding) of 10 µM
nonradioactive ryanodine (16). Experiments were carried out in
duplicate, and each data point was obtained by averaging the duplicates.
Site-specific Fluorescent Labeling of the RyR Moiety of the
Triad--
Site-specific fluorescent labeling of the RyR moiety of the
triad was performed using the cleavable hetero-bifunctional
cross-linking reagent sulfosuccinimidyl
3-((2-(7-azido-4-methylcoumarin-3-acetamido)ethyl)dithio)propionate (SAED) (17, 18) with the aid of neomycin as a carrier in the following
way. First, neomycin-SAED conjugates were formed by incubating 0.4 mM neomycin with 0.2 mM SAED in 20 mM HEPES (pH 7.5) for 15 min at 22 °C in the dark. The
reaction was quenched by a 10-fold dilution with 10 mM
lysine. Twenty ml of the neomycin-SAED conjugate (final neomycin
concentration, 20 µM) was mixed in the dark with 300 mg
of triad protein, brought to 300 ml with PI buffer, and photolyzed with
UV light in a Pyrex tube at 4 °C for 10 min.
-Mercaptoethanol was
added (final concentration, 100 mM) to cleave the disulfide
bond of SAED. After incubation on ice for 1 h, the mixture was
centrifuged for 15 min at 100,000 × g, and the
sedimented triads were resuspended in PI buffer to a final protein
concentration of ~20 mg/ml. The neomycin-mediated incorporation
resulted in the specific incorporation of the MCA into the RyR moiety
of the triad as determined by fluorometry of electrophoretically
separated protein bands (cf. Ref. 17). Although there was a
small decrease in the activity of the RyR after MCA incorporation, the
labeled triads retained sufficient activities of ryanodine binding
(77.3 ± 9.7% of the unlabeled preparation) and
depolarization-induced Ca2+ release (cf. Ref.
19).
Assays of Protein Conformational Changes in the RyR Moiety of the
Triad Induced by Peptide A and T-tubule Depolarization--
To induce
conformational changes in the RyR by peptide A, a base solution (150 mM potassium gluconate, 15 mM NaCl, 2.5 mM EGTA, 1.61 mM CaCl2, 20 mM imidazole, pH 6.8; [Ca2+] = 1.0 µM) containing the MCA-labeled triads (2.0 mg/ml) was mixed with an equal volume of the base solution containing various concentrations of peptide A using a stopped-flow apparatus (BioLogic SFM4).
To induce conformational changes in the RyR induced by T-tubule
depolarization, we used the K+ to Na+
replacement protocol, which was originally devised in the skinned fiber
system by Lamb and Stephenson (20, 21) and was adopted to our triad
system (19, 22). The MCA-labeled triads were first polarized by
incubating the triads (2.0 mg/ml) within the base solution (see above)
containing 5.0 mM Mg·ATP and an ATP-regenerating system (2.5 mM phosphoenolpyruvate and 10 units/ml pyruvate
kinase) for 10-15 min. Then, the T-tubule moiety was depolarized by
mixing 15 µl of the solution containing the polarized triads with 135 µl of depolarization solution (150 mM sodium gluconate,
15 mM NaCl, 2.5 mM EGTA, 1.61 mM
CaCl2, 20 mM imidazole, pH 6.8;
[Ca2+] = 1.0 µM) using a stopped-flow
apparatus (BioLogic SFM4).
The time courses of fluorescence change of the protein-bound MCA
(excitation at 368 nm, emission at 440 nm using an interference filter
with a 70-nm bandwidth) induced by peptide A or T-tubule depolarization
were monitored with the stopped-flow fluorometer (BioLogic SFM-4 with
MOS-200 optical system) as described previously (19).
Assays of Ca2+ Release Induced by T-tubule
Depolarization--
The triads were first polarized by incubating the
triads (2.0 mg/ml) as described above. To depolarize the T-tubule, 15 µl of the polarization solution containing the polarized triads was mixed with 135 µl of depolarization solution (see above) containing 2.5 µM fluo-3, as described previously (22). The time
course of Ca2+ release was monitored in a stopped-flow
fluorometer (BioLogic SFM4) using fluo-3 as the Ca2+
indicator (excitation at 437 nm, emission at 530 nm with a 510-nm cut-off filter).
 |
RESULTS AND DISCUSSION |
Counteraction between Peptide A and Peptide C in the Regulation of
Conformational States of the RyR--
Peptide A, corresponding to the
Thr671-Leu690 region of the II-III loop,
mimics some features of a physiologic activation of E-C coupling in
skeletal muscle, whereas peptide C, corresponding to the
Glu724-Pro760 region of the loop, blocks the
activation by peptide A (12). This has led us to the hypothesis that
the RyR is regulated by these two portions of the II-III loop in a
reciprocal fashion. Fig. 1A
shows the effects of various concentrations of peptide C on the
ryanodine binding activity in the absence or in the presence of peptide
A. In agreement with our recent report (12), peptide C alone has
virtually no effect on ryanodine binding in the range of the
concentrations tested. In the presence of 30 µM peptide A, which produced a near maximal enhancement of ryanodine binding (see
Fig. 1A, inset), peptide C blocked peptide
A-dependent enhancement with an IC50 (the
concentration for half maximal inhibition) of about 50 µM. At 200 µM, peptide C produced an almost
complete blocking of the peptide A-dependent activation.
Thus, peptide C reverses the activation of the RyR by peptide A,
although peptide C alone has no direct effect on the
RyR. In other words, peptide C seems to regulate the RyR by serving as
an antagonist of the activating peptide A.

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Fig. 1.
A, peptide A-dependent
enhancement of ryanodine binding is suppressed by peptide C, but
suppression by chimeric peptide C (chimeric C) is much less extensive.
Inset, magnitude of ryanodine binding enhancement as a
function of the concentration of peptide A added. B, effects
of shorter peptides corresponding to N-terminal and C-terminal halves
of peptide C (peptide C1 and peptide C2,
respectively). The reference curve (dotted line) represents
the plot of 30 µM peptide A + peptide C shown in
A. C, lack of effect of peptide C on caffeine-
and polylysine-induced enhancement of ryanodine binding. Data represent
the mean ± S.D. of four to six experiments.
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As described above, the N-terminal half of peptide C (see the
underlined portion of peptide C in the sequence shown below) corresponds to the so-called "determinant of skeletal muscle-type E-C
coupling" (the Glu726-Pro742 region of the
II-III loop) (23), the skeletal muscle-type sequence of which seemed to
be essential for the skeletal muscle-type E-C coupling.
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(Eq. 1)
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In order to test the importance of the type of sequence in this
segment, we synthesized a chimeric peptide C by changing this segment
to cardiac type sequence (see the segment of chimeric peptide C with
italic letters). As shown in Fig. 1A and Table I, the extent of blocking of
peptide A-dependent activation by chimeric peptide C
(chimeric C) was significantly less than that by peptide C, indicating
that the skeletal muscle-type sequence in this region is important for
the blocking function of peptide C. In light of the suggested
importance of the skeletal muscle-type sequence of this segment for E-C
coupling (l.c.), it is suggested that blocking function residing
in the peptide C region of the II-III loop is in fact required for E-C
coupling besides the activating function located in the peptide A
region.
We previously found that shorter peptides of peptide C,
viz. peptide C1 and peptide C2
(corresponding to the Phe725-Gly743 and
Asp740-Pro760 regions of the II-III loop,
respectively; see the above sequence), had no appreciable effect on
peptide A-dependent activation up to 50 µM
(12). Further studies with higher concentrations of these shorter
peptides revealed new features, shown in Fig. 1B. As seen,
peptide C1 had again virtually no blocking effect even at
higher concentrations, indicating that the N-terminal half of peptide C
alone is not capable of competing with peptide A. Interestingly,
however, peptide C2 produced a small but significant competition with peptide A at high concentrations. The addition of
equimolar concentrations of peptide C1 to peptide
C2 produced little or no additional change in the extent of
blocking by peptide C2. From these findings, we propose
that a relatively large region (presumably the whole length of peptide
C) is required for an effective blocking of peptide
A-dependent activation. As seen in the above scheme,
peptide C2 contains a cluster of negatively charged
residues (DDEEDE). Because the cluster of positively charged residues
located in peptide A seems to play a key role for its activating
function (13), the cluster of acidic residues located in the peptide
C2 region likely plays a key role in the blocking function
of peptide C. We tentatively propose that the blocking function is
localized in the C2 region, but the C1 region
with the skeletal muscle-type sequence is required for some other
functions, such as the binding of peptide C to the RyR.
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Table I
The concentrations of peptide C and its derivatives for their
IC50 of peptide A (30 µM)-induced enhancement of
ryanodine binding
Number of each experiment = 5. Data are mean ± S.D.
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As seen in Fig. 1 and Table I, the extent of blocking by chimeric
peptide C (Fig. 1A) is about the same as that by peptide C2, or an equimolar mixture of C1 and
C2 (Fig. 1B). This indicates that the partial
blocking of peptide A-dependent activation by chimeric
peptide C described above is solely due to the inhibitory function
localized in the C-terminal half of chimeric peptide C.
In the experiments shown in Fig. 1C, we investigated the
effects of increasing concentrations of peptide C on enhancement of
ryanodine binding by the generally used Ca2+
release-inducing reagents caffeine (24, 25) and polylysine (26, 27). As
seen, peptide C had no effect on both caffeine- and
polylysine-dependent enhancement, even at the high
concentrations investigated. Thus, peptide C is a specific blocker of
peptide A. This is consistent with the view that peptide C is competing with peptide A at the specific E-C coupling site(s) of the RyR.
Although ryanodine binding assays provide a convenient method for the
assessment of conformational states of the RyR, the assay is not
suitable for monitoring rapid conformational changes occurring in the
RyR. Stopped-flow fluorometry of the fluorescent conformational probe,
MCA, specifically attached to the RyR moiety (17, 19, 27) permits us to
study rapid conformational changes in the RyR with a high temporal
resolution. In the experiment shown in Fig.
2A, we investigated the
effects of peptide A on the conformational state of RyR by means of the
stopped-flow fluorometry. As seen, peptide A induced a rapid increase
in the MCA fluorescence (
F) in a
dose-dependent manner. An increase of the peptide
concentration up to 50 µM resulted in an increase in both
the magnitude (A) and the rate constant (k) of
F. Upon further increase of peptide A concentration, however, the
activation by peptide A was somewhat suppressed. Fig. 2B
shows the [peptide A] dependence of the initial rate of
F
(i.e. A·k value). Importantly, the
general pattern of the dose-dependent activation seen here
(Fig. 2B) shows a striking resemblance to that obtained from
the ryanodine binding assay (cf. Fig. 1A, inset).
This indicates that ryanodine binding and MCA fluorescence assays can
provide us with the essentially identical information about the
functional/conformational state of the RyR, although only the MCA
fluorometry permits a sufficient temporal resolution for the studies of
the activator-induced rapid conformational changes.

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Fig. 2.
Peptide A induces a rapid increase of the
RyR-bound MCA fluorescence (i.e. formation of an
active conformational state of the RyR). A, time courses of
MCA fluorescence increase induced by various concentrations of peptide
A. Each trace was obtained by signal averaging a total of 75 traces
from three experiments. B, initial rates of peptide
A-induced conformational change as a function of the concentration of
peptide A. Traced line was obtained by fitting a single
exponential function (y = yo + A(1 e kt)). The
initial rate (A·k) was calculated from the
A and k values obtained from the fitting. Data
represent the mean ± S.D. of three experiments.
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Fig. 3A depicts time courses
of conformational changes of the RyR when the vesicles were incubated
first with various concentrations of peptide C and then 30 µM peptide A was added to activate the RyR. As seen,
peptide A-induced
F was blocked significantly by peptide C in a
concentration-dependent manner. The [peptide C] dependence of the reduction of the initial rate of peptide A-induced fluorescence change is shown in Fig. 3B. The [peptide C]
dependence of the blockage is similar to that shown in the ryanodine
binding assay (Fig. 1), although the IC50 in the
stopped-flow assay (approximately 30 µM, Fig.
3B) is somewhat lower than that in the ryanodine binding assay (approximately 50 µM, Fig. 1). These results
indicate that peptide C blocks the peptide A activation by interfering
with the binding of peptide A to the RyR.

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Fig. 3.
A, Peptide A-dependent
formation of the active conformational state of the RyR is suppressed
by peptide C in a concentration-dependent manner. Each
trace was obtained by signal averaging a total of 75-90 traces
from three experiments. B, the initial rates of the peptide
A-induced MCA fluorescence increase as a function of the concentration
of peptide C added. The initial rate was calculated as described in the
legend to Fig. 2. Data represent the mean ± S.D. of three
experiments.
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The isolated triad preparation contains two classes of RyRs: coupled
RyRs, which are linked with the T-tubule DHP receptors, and free RyRs,
which are not linked with the DHP receptors. In this preparation, the
population of free RyRs must be significantly larger than that of the
coupled RyRs due to the fact that a significant portion of the
previously coupled RyRs (which had been about 50% of the total RyR
population (28-30)) must have been dissociated from T-tubules during
fragmentation and isolation of the vesicles. Furthermore, the rapid
accessibility of the added peptide A to the activating site(s), which
seems to be critical for rapid activation of the RyR in those
stopped-flow experiments, would be much higher in the free RyRs than
the coupled RyRs. Therefore, these data in Fig. 3 seem to represent
almost primarily the conformational response of the free RyRs to the
added peptides, although peptide A may also be accessible to the
coupled RyRs. Diagram D1A
summarizes our interpretation of the above data. Binding of peptide A
to the putative E-C coupling site(s) of the RyR produces a rapid conformational change from the state with low MCA fluorescence to the
new state with high MCA fluorescence, which leads to the enhancement of
ryanodine binding and Ca2+ release from the SR
(cf. Refs. 19 and 27). Peptide C by itself produces
virtually no change in the RyR conformation. However, activation of the
RyR by peptide A, viz. the increase in the MCA fluorescence
level, was blocked by peptide C owing to the competition between
peptide A and peptide C to the specific E-C coupling site(s).

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Diagram 1.
Proposed models to account for the present
results. A, regulation of the RyR by peptide A and peptide C
in the SR that is not linked with the T-tubule. The activated state of
the RyR (with high MCA fluorescence) and the inactivated state of the
RyR (with low MCA fluorescence) are produced by a competitive binding
of peptide A and peptide C to the RyR, respectively. B,
regulation of the RyR by the activator domain (the region
corresponding to peptide A) and the blocker/primer domain (the
region corresponding to peptide C) of the DHP receptor II-III
loop in the coupled triads. The activated state of the RyR is
produced by depolarization-dependent binding of the activator
domain of the loop to the E-C coupling site(s), whereas the inactivated
(resting) state of the RyR is produced by
polarization-dependent binding of the blocker domain to the
RyR site(s).
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Effects of Peptide C on E-C Coupling Phenomena in the Coupled
Triads--
As described above, peptide A increases MCA fluorescence,
and peptide C reverses the peptide A-induced increase of MCA
fluorescence. This is reminiscent of similar fluorescence changes
occurring in the coupled triads. Namely, the fluorescence intensity of
the RyR-bound MCA increases upon T-tubule depolarization, whereas it
decreases upon T-tubule polarization (31). Such a tight correlation between the two experiments, one in the free RyRs and the other in the
coupled RyRs, suggests the following hypothesis (cf. Diagram D1B). Upon T-tubule depolarization, the region of the II-III
loop corresponding to peptide A (activator of E-C coupling) will bind to the E-C coupling site(s) of the RyR, leading it to an active conformational state with a high MCA fluorescence (cf. Refs.
19 and 31). Upon T-tubule polarization, the RyR-bound activator will be
replaced by the region of the II-III loop corresponding to peptide C,
leading to a blocked (or primed) state of the RyR with a low MCA fluorescence.
In order to test this model, we monitored MCA fluorescence increase
induced by T-tubule depolarization (at a level of G10, cf.
Ref. 22) in the presence of various concentrations of peptide C. The
important feature of this experiment is that only the RyRs linked with
the T-tubule DHP receptor show voltage-dependent
conformational (MCA fluorescence) changes. This is in sharp contrast to
the activation by general activators of the RyR (Ca2+,
caffeine, polylysine, etc.), which will react with both free and
coupled RyRs. This provides us with a straightforward method for
testing the effect of peptide C on the voltage-dependent
operation of the II-III loop. In the experiment shown in Fig.
4A, the vesicles were first
incubated with various concentrations of peptide C, and after priming
the system (viz. after polarizing the T-tubule and loading
the SR with Ca2+ (19, 22)), the T-tubule moiety was
depolarized (see "Experimental Procedures"). As seen,
depolarization-induced MCA fluorescence increase (i.e. the
voltage-dependent formation of an active conformational state of the RyR) was blocked by peptide C in a
concentration-dependent manner. The IC50 was 20 µM as determined from the d[F]/dt
versus [peptide C] plot (Fig. 4B), which is
essentially identical with the IC50 for the peptide C
inhibition of the peptide A-dependent activation (Fig.
3B). We also carried out the same type of experiments as in
Fig. 4A by adding equivalent concentrations of peptide C simultaneously with T-tubule depolarization (rather than depolarizing after preincubation with peptide C). The degree of inhibition by
peptide C without preincubation was almost identical with that after
preincubation, although the former was somewhat smaller than the
latter. These results suggest that the occupancy of peptide C at the
E-C coupling site(s) of the RyR interfered with the
voltage-dependent binding of the activator domain of the
II-III loop to the site(s), although the voltage-dependent
changes in the position of the II-III loop domains may be occurring in
a normal way.

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Fig. 4.
A, time courses of the formation of the
active conformational state (increase of the RyR-bound MCA
fluorescence) by T-tubule depolarization in the presence of various
concentrations of peptide C. Each trace was obtained by signal
averaging a total of 100-125 traces from four experiments.
B, the initial rates of depolarization-induced
conformational change as a function of the concentration of peptide C
added. Data represent the mean ± S.D. of four experiments.
C, suppression of depolarization-induced RyR conformational
change by chimeric peptide C is significantly less compared with that
by peptide C. Each trace was obtained by signal averaging a total of
75-90 traces from three experiments.
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The magnitude of the relative MCA fluorescence increase
(
F/Fo) induced by T-tubule
depolarization (about 4%, Fig. 4A) was significantly larger
than that induced by a maximally activating concentration of peptide A
(about 1%, Fig. 3A). This is rather surprising, because the
population of the coupled RyRs responsible for the former event is
significantly smaller than that of the free RyRs responsible for the
latter, as described above. The most reasonable explanation for this
would be that the changes in the fluorescence intensity are greatly
enhanced in the coupled RyRs because of their location in the space
between the T-tubule and SR membranes, which is presumably a more
hydrophobic environment than that of the free RyRs.
In the experiment shown in Fig. 4C, we compared the extent
of inhibition of depolarization-induced MCA fluorescence by peptide C
and that by chimeric peptide C at 200 µM. In good
agreement with the results of ryanodine binding assay (cf.
Fig. 1), the potency of blocking depolarization-induced conformational
change is significantly less in chimeric peptide C compared with
peptide C. This indicates again that the skeletal muscle-type sequence of the region corresponding to the
Glu726-Pro742 region of the II-III loop is
important for peptide C to exert an efficient reversal of the in
situ mechanism of activation.
Because the formation of active conformational state of the RyR
(i.e. high MCA fluorescence state) is a prerequisite for the channel opening and Ca2+ release (cf. Refs. 19,
27), inhibition of this process by peptide C should also inhibit
Ca2+ release from the SR. In the experiment shown in Fig.
5A, we investigated the
effects of various concentrations of peptide C on
depolarization-induced Ca2+ release from the SR. As seen,
peptide C in fact blocked depolarization-induced Ca2+
release in a concentration-dependent manner. Fig.
5B shows the [peptide C] dependence of the reduction of
the initial rate of Ca2+ release. The
dose-dependent reduction of the initial Ca2+
release rate (Fig. 5B) is similar to that of
depolarization-induced conformational change. We also carried out the
same type of experiment with some control peptides. As described
previously (12), peptide B and peptide D (which represent the
Arg694-Val722 and
Pro760-Val790 regions, respectively, of the
II-III loop in the neighborhood of the peptide C region) had no effect
on peptide A-induced Ca2+ release. As seen in Table
II, neither peptide B nor peptide D had
any appreciable effect on depolarization-induced Ca2+
release, even at 200 µM.

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Fig. 5.
A, Time courses of Ca2+
release induced by T-tubule depolarization in the presence of various
concentrations of peptide C. Each trace was obtained by signal
averaging a total of 100-125 traces from five experiments.
B, the initial rates of depolarization-induced calcium
release as a function of the concentration of peptide C added. Data
represent the mean ± S.D. of five experiments.
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Table II
The initial rates of Ca2+ release induced by T-tubule
depolarization in the presence of 200 µM peptide C and
the equivalent concentration of control peptides (peptides B and D)
Peptides B, C, and D correspond to the Arg694-Val722,
Glu724-Pro760, and Pro760-Val790
regions of the II-III loop, respectively. Number of experiments = 5. Data are mean ± S.D.
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Relationship of the Present Findings to Those by Others--
In a
recent paper, Nakai et al. (14) described that there is a
considerable amount of inconsistency in the literature concerning the
proposed locations of critical domain(s) of the II-III loop. However,
many (if not all) of those apparently inconsistent findings, together
with the present findings, may be explained by a general hypothesis as
elaborated below. First, the finding by Nakai et al. (14)
that replacement of a short segment of the cardiac loop with the
corresponding skeletal residues Phe725-Pro742
was sufficient to produce skeletal type E-C coupling has led to their
suggestion that this region may serve as an "agonist," rather than
the peptide A region being the activator. This suggestion was based
upon the assumption that the cardiac sequence of the peptide A region
of the loop would have no capability of activating the RyR1 (14). As a
matter of fact, as shown in our recent report, both skeletal and
cardiac sequences of the critical 10-residue portion of peptide A
(peptide A-10) can activate the RyR1, although cardiac peptide A-10 is
somewhat weaker than its skeletal counterpart (cf. Figs. 3 and 6 of Ref.13). We propose that both skeletal and cardiac loops have
a common activating domain in the peptide A-10 region, which has a
similar amino acid sequence (skeletal, RKRRKMSGRL; cardiac, KERKKLARTA,
cf. Ref. 13). This concept can well explain an earlier
finding that both skeletal and cardiac constructs of the II-III loop
activated the RyR1 (10). This concept is also in accord with the recent
report that the II-III loop construct subjected to mutations within the
peptide A-10 region became incapable of interacting with the 37-residue
construct of the RyR (32).
Then what makes the region of skeletal residues
Phe725-Pro742 essential for E-C coupling
without having an agonist function? The present study provides some
clues to this question. As shown (cf. Fig. 3A),
peptide C blocked MCA fluorescence increase induced by both peptide A
and T-tubule depolarization. Furthermore, according to our preliminary
data, the addition of peptide C to the depolarized triads resulted in a
decrease of MCA fluorescence (data not shown). These results would
indicate that upon T-tubule polarization, the peptide C region of the
II-III loop binds to the RyR in replacing the activator domain, which
results in a blocked or resting conformational state of the RyR
(cf. Diagram D1B). The binding of the blocker to
the RyR to form its resting state is essential for E-C coupling presumably for the purpose of repriming the system. This is because without it, a new cycle of E-C coupling cannot take place
(cf. Diagram D1B). The
Phe725-Pro742 region corresponds to the
N-terminal half of peptide C (i.e. peptide C1),
and replacement of this portion to the cardiac sequence produced a
significant reduction in the blocking (or priming) ability of peptide
C, as shown in the present study. Furthermore, as suggested from the
present data (see Fig. 1B), the peptide C1
region may serve as a mechanism to link the inhibitory peptide C2 region to the RyR. Thus, it is not unreasonable to
speculate that the Phe725-Pro742 segment may
serve as a link of the whole loop to the RyR as well.
Some of the discrepancies in the literature may be at least partly
ascribable to the difference in the method of approach. Synthetic or
constructed peptides would be accessible to their designated sites in
an unrestricted manner. On the other hand, in chimeras, all of those
domains under discussion are parts of the II-III loop; hence, their
accessibility to the RyR must be restricted and controlled in a
voltage-dependent manner. Therefore, it is likely that the
activator located in the Arg681-Leu690 region
of the II-III loop becomes accessible to the RyR only after the two
events have occurred: first, binding of the
Phe725-Pro742 segment to the RyR, and second,
depolarization of the T-tubule membrane.
Thus, the accumulated pieces of information in the literature, although
apparently inconsistent, can be explained by a unified scheme. Namely,
the regulation of skeletal muscle-type E-C coupling by the II-III loop
is mediated not by a single particular domain, but by multiple domains
with different roles. We tentatively propose three such domains:
activator, linker, and blocker/primer, located in the A,
C1, and C2 regions of the II-III loop, respectively.
Conclusion--
The hypothesis that E-C coupling in skeletal
muscle may be regulated by at least two domains of the II-III loop
(activator and blocker, located in the
Thr671-Leu690 and
Glu724-Pro760 regions, respectively) was
tested. Several pieces of new evidence shown here support this concept.
First, peptide C (synthetic peptide corresponding to the blocker
region) blocked T-tubule depolarization-induced conformational
change in the RyR as well as Ca2+ release from the SR.
Second, peptide C also blocked conformational changes in the RyR
induced by peptide A (synthetic peptide corresponding to the activator
region). The [peptide C] dependence of inhibition of
depolarization-induced conformational change was similar to that of
peptide A-induced conformational change, supporting the notion that
voltage-dependent activation of the RyR is mediated by the
region of the II-III loop corresponding to peptide A. Third, replacement of the portion of peptide C, which corresponds to the
determinant of skeletal muscle-type E-C coupling (14), from the
skeletal muscle-type sequence to the cardiac type produced a
significant reduction of the blocking ability of peptide C. This
suggests that skeletal-type E-C coupling requires not only the
activating function localized in the peptide A region of the II-III
loop but also the blocking function residing in the peptide C region.
Based upon these findings, we propose the following mechanism
(cf. Diagram D1B). Depolarization-induced
activation of E-C coupling is mediated by the binding of the activator
(located in the region of the II-III loop corresponding to peptide A)
to the specific E-C coupling site(s) of the RyR. The binding of the blocker (located in the other region of the II-III loop corresponding to peptide C) to the E-C coupling site(s) removes the activator from
the site(s) in a competitive manner. The binding of the blocker/primer is mediated by polarization of the T-tubule, and this step is a
prerequisite to the next cycle of depolarization-induced activation. Therefore, this segment is one of the domains that are essential for
E-C coupling. For the sake of simplicity of the model, we tentatively
assume that the activator and the blocker bind to the same regulatory
site of the RyR in a competitive manner. However, it is also possible
that they bind to different sites of the RyR.