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J Biol Chem, Vol. 274, Issue 47, 33341-33347, November 19, 1999
From the To test the hypothesis that interactions among
several putative domains of the ryanodine receptor (RyR) are involved
in the regulation of its Ca2+ release channel, we
synthesized several peptides corresponding to selected
NH2-terminal regions of the RyR. We then examined their
effects on ryanodine binding and Ca2+ release activities of
the sarcoplasmic reticulum isolated from skeletal and cardiac muscle.
Peptides 1-2s, 1-2c, and 1 enhanced ryanodine binding to cardiac RyR
and induced a rapid Ca2+ release from cardiac SR in a
dose-dependent manner. The order of the potency for the
activation of the Ca2+ release channel was 1-2c > 1 > 1-2s. Interestingly, these peptides produced significant
activation of the cardiac RyR at near zero or subactivating
[Ca2+], indicating that the peptides enhanced the
Ca2+ sensitivity of the channel. Peptides 1-2c, 1-2s, and
1 had virtually no effect on skeletal RyR, although occasional and
variable extents of activation were observed in ryanodine binding
assays performed at 36 °C. Peptide 3 affected neither cardiac nor
skeletal RyR. We propose that domains 1 and 1-2 of the RyR, to which
these activating peptides correspond, would interact with one or more
other domains within the RyR (including presumably the
Ca2+-binding domain) to regulate the Ca2+ channel.
The ryanodine receptor
(RyR)1 is a large
homotetrameric molecule that contains the calcium release channel of
the sarcoplasmic reticulum (SR) (1-4). The carboxyl-terminal region of
the RyR is composed of 4-12 (5, 6) transmembrane helices, which form
the calcium release channel. The major portion of the RyR molecule
protrudes into the cytoplasm, spanning the gap between the plasma
membrane and the SR (7). This bulky cytoplasmic domain of the RyR,
often called the junctional foot, presumably serves as a receptor for
various types of stimuli, effectors (8-13), and regulatory proteins
such as calmodulin (14-16) and FK506-binding protein (17, 18). Thus,
there must be an intricate communication network within the receptor
molecule to send the signal from various cytoplasmic effector/regulator
binding domains to the Ca2+ release channel domain.
However, only very limited information is available regarding the
actual mechanism for channel regulation.
Several pieces of information suggest the hypothesis that the near
NH2-terminal region of the RyR may be involved in its
channel regulation. First, this is one of the two regions where most
mutations have been found in patients with malignant hyperthermia and
central core disease (19-26), and experimental models of these
mutations in fact produced abnormalities in Ca2+ release
channel regulation (27-31). An antibody raised against this
NH2-terminal region altered the Ca2+ dependence
of channel gating (32). Chemical modification of the
NH2-terminal region of the RyR affected intramolecular
cross-linking under conditions that would produce oxidation-induced
activation of the Ca2+ channel (33, 34). Furthermore, this
region has a primary structure that is highly homologous to the
IP3 binding region of the IP3 receptor, which
is clearly involved in the regulation of its channel gating
(35-38).
The goal of the present study is to test the above hypothesis. For this
purpose, we synthesized peptides corresponding to two selected domains
of the near NH2-terminal region, designated as domain 1-2
and 3 (see Table I). We then investigated the effects of these
synthetic peptides on ryanodine binding and SR Ca2+ release
using SR vesicles isolated from both skeletal and cardiac muscles. The
peptides corresponding to domain 1-2 activated the RyR2 and induced a
rapid Ca2+ release from cardiac SR. These peptides produced
no activation of the RyR1 as long as the temperature during the assay
does not exceed a certain level. The other peptide corresponding to
domain 3 affected neither RyR2 nor RyR1. As shown in the
[Ca2+] dependence of activation, the peptides
corresponding to domain 1-2 activate the RyR2 in zero or near zero
[Ca2+]. Furthermore, they serve as an amplifier of
Ca2+-dependent activation of the RyR2. The
present results suggest that the domain corresponding to these
peptides, namely domain 1-2, is involved in the mechanism of
activating the SR Ca2+ channel by mediation of
intramolecular domain-domain interaction.
Background and Terminology of the Domain Peptides Used
The use of synthetic peptides corresponding to key domains of
the RyR to examine their effects on RyR function would be a useful
approach to characterize putative functions of these domains. Since
several pieces of evidence suggest that the NH2-terminal region of the RyR may serve as an important regulatory domain of
channel function (see Introduction), we selected some subdomains from
this region of the RyR1 and RyR2 as illustrated in Table I. Domain
1-2s and its cardiac counterpart, domain 1-2c, consist of two
subdomains: domain 1, which is composed of an identical sequence for
both RyR1 and RyR2; and domain 2, where there is some difference
between both isoforms (cf. underlined residues). The
synthesized peptides will be identified by names identical to their
corresponding in situ domains just defined.
Preparation
Cardiac Microsomes--
Dog ventricular cardiac muscle was
homogenized in four volumes of 0.3 M sucrose, 40 mM imidazole, pH 6.8, 5 µg/ml leupeptin, 0.1 mM phenylmethanesulfonyl fluoride, in a Warning blender at high speed. During the homogenization the pH was adjusted to 6.8. The
homogenate was then centrifuged at 5,500 × g for 10 min. The pellet was discarded and the supernatant filtered through
Whatman filter paper. The filtrate was centrifuged at 12,000 × g for 20 min. The pellet was discarded and supernatant
filtered through Whatman filter paper. The filtrate was centrifuged at
143,000 × g for 30 min. The supernatant was discarded,
and the pellet was homogenized in 0.3 M sucrose, 0.15 M potassium gluconate, proteolytic enzyme inhibitors (0.1 mM phenylmethanesulfonyl fluoride, 10 µg/ml aprotinin,
0.8 µg/ml antipain, 2.0 µg/ml soybean trypsin inhibitor), 20 mM MES, pH 6.8 (PI buffer), with a glass/glass homogenizer,
and centrifuged again at 143,000 × g for 45 min. The
resultant pellet was resuspended in PI buffer to a final concentration of 20-30 mg/ml, frozen immediately in liquid N2, and
stored at Skeletal Microsomes--
Triad-enriched microsomal fractions
were prepared from the rabbit back paraspinous and hind leg skeletal
muscle by a method of differential centrifugation as described
previously (39). Microsomes from the final centrifugation were
homogenized in PI buffer to a final concentration of 20-30 mg/ml,
frozen immediately in liquid N2, and stored at
Synthesis of Peptides
Peptides were synthesized on an Applied Biosystems model 431 A
synthesizer employing Fmoc (N-(9-fluorenyl)methoxycarbonyl) as the [3H]Ryanodine Binding Assay
Dog cardiac (1.5 mg/ml) or rabbit skeletal (0.5 mg/ml)
microsomes were incubated in 0.1 ml of a reaction solution containing 10 nM (dog cardiac) or 8 nM (rabbit skeletal)
[3H]ryanodine (68.4 Ci/ml, NEN Life Science Products),
0.3 M KCl, 1 mM EGTA, and various amounts of
CaCl2 to create various levels of [Ca2+], 20 mM MOPS, pH 7.2, for 2 h at 36 °C, 16 h at
22 °C, or 72 h at 4 °C in the presence of various
concentrations of peptides and/or modulators. Samples were filtered
onto glass fiber filters (Whatman GF/B) and washed twice with 5 ml of
distilled water. Filters were then placed in scintillation vials
containing 10 ml of the scintillation mixture Ecoscint A and counted in
a Beckman LS 3801 counter. Specific binding was calculated as the
difference between the binding in the absence (total binding) and in
the presence (nonspecific binding) of 10 µM
non-radioactive ryanodine. Experiments were carried out in duplicate,
and each datum point was obtained by averaging the duplicates (40).
Assays of Peptide-induced Ca2+ Release
Dog cardiac microsomes (0.4 mg/ml) were incubated in a solution
containing 0.15 M potassium gluconate, 1 mM
MgATP, an ATP-regenerating system, 20 mM MES, pH 6.8 (Solution A) for 5 min to load the SR moiety with Ca2+.
Then one volume of Solution A was mixed with one volume of Solution B
containing 0.15 M potassium gluconate, 20 mM
MES, pH 6.8, and various concentrations of peptides. The
Ca2+ concentration in both solutions was ~0.25
µM. The time course of SR Ca2+ release was
monitored in a stopped-flow apparatus (Bio-Logic SFM-4) using 2.5 µM fluo-3 as a Ca2+ indicator as described
previously (41). Fifteen to 20 traces (each representing 1,000 data
points) of the fluo-3 signal were averaged for each experiment.
Statistical Analysis
Statistical analysis for the comparison of mean values was
performed using an unpaired Student's t test.
p Effects of Domain Peptides on Ryanodine Binding--
We examined
two groups of synthetic peptides corresponding to the two domains of
the RyR1, domain 1-2 and domain 3 (cf. Table I) for their effects on ryanodine binding
to the RyR1 and RyR2. Fig. 1 depicts the
results of the ryanodine binding assay carried out at 36 °C. As seen
in Fig. 1, peptide 1-2s produced a significant enhancement of
ryanodine binding to the RyR2 in a concentration-dependent manner. In many experiments, peptide 1-2s produced no appreciable enhancement of ryanodine binding to the RyR1 in the same concentration range. In some other experiments carried out at 36 °C, however, peptide 1-2s produced various extents of enhancement of ryanodine binding in RyR1. Fig. 2A is a
histogram illustrating the relationship between the frequency of
occurrence and the extent of enhancement of ryanodine binding to the
RyR2 and RyR1 produced by the maximally activating concentration of
peptide 1-2s (200 µM) at 36 °C. As shown, a
significant amount of enhancement (151-210%) occurred in all
experiments in case of the RyR2. In case of the RyR1, however, the
level of enhancement varied over a wide range, from no effect to levels
comparable to the RyR2. We carried out the same type of ryanodine
binding assay at 22 °C (the temperature at which all stopped-flow
experiments described below were carried out), and the effects on the
RyR2 and the RyR1 were compared. As shown, peptide 1-2s produced a
significant and consistent activation of the RyR2 (Fig. 2B).
Interestingly, the peptide produced no appreciable effect on the RyR1
in all experiments. Furthermore, peptide 1-2s produced significant
activation of ryanodine binding to RyR2, even in ryanodine binding
experiments performed at 4 °C assay (data not shown). These results
indicate that the domain peptide 1-2s activates the RyR in a
RyR2-specific manner. However, RyR1 also becomes responsive to peptide
activation above a transitional temperature, which appears to be in the
vicinity of 36 °C. Another domain peptide, peptide 3s, did not
produce any appreciable effect on either the RyR1 or the RyR2, as
determined by the ryanodine binding assay at 36 °C (Fig.
3), providing a negative control for the
above experiments.
The amino acid sequence of the region of the RyR1 corresponding to
peptide 1-2s (i.e. domain 1-2s) and that of the
corresponding region of the RyR2 (domain 1-2c) are essentially
identical, but are different in four residues located in their
subdomain 2 (cf. Table I). We compared the effects of both
isoforms of peptide 1-2 on ryanodine binding to the RyR2 at 36 °C
and at different Ca2+ concentrations in the assay solution.
The synthetic peptide corresponding to the domain 1-2c, namely peptide
1-2c, enhanced ryanodine binding to the RyR2 (Fig.
4). The concentration dependence of
activation was indistinguishable between peptides 1-2s and 1-2c at 10 µM Ca2+ (Fig. 4) This suggests that the
activating function of these peptides may be localized within their
subdomain 1 where both peptides have a common amino acid sequence. As
shown in Fig. 4, peptide 1 corresponding to subdomain 1 in fact
activated RyR2 with the same concentration dependence of activation as
the longer peptides 1-2s and 1-2c.
We have carried out the same type of experiment at lower concentrations
of Ca2+; at 0.2 µM and nominal zero
Ca2+. Table II depicts the
AC50 values for each peptide at three different [Ca2+] values including 10 µM described
above. Interestingly, at lower Ca2+ concentrations, some
differences in the potency of activation among these domain peptides
became visible, i.e. the extent of activation by peptide
1-2s is much smaller than the extents of activation by peptide 1-2c
and peptide 1. This indicates that, at this submaximally activating
concentration of Ca2+, the apparent affinity of peptide
1-2s to the RyR2 is somewhat lower than peptides 1-2c and peptide 1. It also appears that peptide 1-2c produces a slightly larger magnitude
of activation than peptide 1 at high peptide concentrations (data not
shown). Thus, the order of the strength of activation of these peptides
at 0.2 µM and nominal zero Ca2+ is 1-2c
It should be noted that, at nominal zero Ca2+, all of these
three peptides produced a small but significant increase in the ryanodine binding to the RyR2. This suggests that there is a unique feature of Ca2+-independent activation, which is not
usually seen with many known activating reagents. As seen from the
comparison of the AC50 values at three different
[Ca2+] values (Table II), the AC50 values are
significantly larger for all peptides at lower [Ca2+]
values. These results indicate that the domain peptides bind preferentially to the calcium-bound RyR2, although the presence of
Ca2+ is not an essential requirement for the activation by
these peptides.
Domain Peptides Induce Ca2+ Release from Cardiac
SR--
Fig. 5 depicts the time courses
of Ca2+ release from cardiac SR induced by various
concentrations of peptides 1-2s, 1-2c, and 1. In these experiments, a
solution containing cardiac microsomes that had been loaded with
calcium by MgATP-dependent Ca2+ transport
(Solution A) was mixed with a solution containing various concentrations of peptides (Solution B). Then, the time course of the
changes in the Ca2+ concentration was monitored using
fluo-3 at a starting [Ca2+] of 0.25 µM. As
seen, these peptides produced a rapid Ca2+ release in a
dose-dependent manner mirroring the effect of these peptides on ryanodine binding experiments with the RyR2. In Fig. 6, the initial rates of Ca2+
release are plotted as a function of the concentration of these three
peptides added. The order of the strength of inducing Ca2+
release estimated from this plot is 1-2c > 1 > 1-2s,
consistent with ryanodine binding assays carried out at equivalent
[Ca2+].
An interesting feature revealed in these Ca2+ release
experiments is the biphasic change in the Ca2+ signal. As
seen in Fig. 5, the rapid Ca2+ release produced by higher
concentrations of peptide 1-2c (
None of these peptides induced Ca2+ release from skeletal
muscle SR even at high concentrations, in agreement with the ryanodine binding data obtained at the equivalent temperature, 22 °C.
Furthermore, peptide 3s, which had no effect on ryanodine binding to
the RyR1 or the RyR2, did not produce any Ca2+ release from
either cardiac or skeletal muscle SR.
Ca2+ Dependence of Domain Peptide Activation of the
RyR2--
Fig. 7 shows the
Ca2+ dependence of ryanodine binding to the RyR2 in the
absence or presence of 100 µM peptide 1. As seen, peptide 1 produces significant enhancement of ryanodine binding to RyR2 not
only in the activating [Ca2+] range, but also in the zero
to subactivating [Ca2+] range. The extent of activation
by the peptide is about 200% of control at maximally activating
[Ca2+], while it increases considerably at submaximally
activating [Ca2+]. This indicates that these domain
peptides not only serve as an amplifier of the
Ca2+-dependent activation of the RyR2, but also
as a Ca2+-independent activator.
As shown in Fig. 8, at zero
[Ca2+] peptide 1 produced a significant enhancement of
ryanodine binding to the RyR2 (p < 0.001), but had no
effect on the RyR1. Conversely, calmodulin, which is known to activate
the RyR1 at a very low [Ca2+] (15), produced a
significant activation of the RyR1 (p = 0.001), but had
no effect on the RyR2. Thus, it appears that these domain peptides
produce a calmodulin-like function for the RyR2.
One of the important unsolved questions in the E-C coupling is how
various stimuli received in the cytoplasmic domains of the RyR are
transmitted to the trans-membrane Ca2+ channel domain to
regulate the channel functions. Several pieces of indirect evidence in
the literature suggest the hypothesis that the NH2-terminal
~700-amino acid region of the RyR may represent one of the putative
regulatory domains of channel function. This is the region with
frequent occurrence of human malignant hyperthermia mutations and in
the porcine animal model of this disease (19-26). These mutations
consistently produce an abnormal regulation of the RyR (27-31, 42). A
monoclonal antibody raised against the region containing
Gly342 in fact produced a significant activation of the
RyR1 and a Ca2+ hypersensitization of the RyR1 (32). Thiol
alkylation of the 170 kDa NH2-terminal region affected
intramolecular S-S formation, suggesting that this region may be
involved in intramolecular domain-domain interactions (33, 34).
Furthermore, the NH2-terminal region of the RyR is highly
homologous to the corresponding region of the IP3 receptor,
which contains the IP3 binding site and which plays an
important role in the regulation of the IP3 receptor channel function (35-38).
We initiated this study to identify the putative regulatory domain(s)
located in the NH2-terminal region of the RyR and to characterize its (their) function utilizing synthetic peptides as
probes. This strategy is based upon the following principle. If any
subdomain of this region interacts with the target domain of functional
importance, the extrinsically added peptide corresponding to that
NH2 domain would bind to its target domain. Then, this peptide-to-target domain interaction would produce some effects on the
RyR function that are similar to those obtained by the in
vivo NH2 domain-to-target domain interaction. This would
not only permit us to identify the putative regulatory domain, but would also allow us to characterize its mode of regulation.
One of the most important findings of the present study is that the
synthetic peptides (peptide 1-2s and 1-2c), which correspond to the
domain 1-2 encompassing the Leu590-Gly628
(RyR1) and Leu601-Gly639 (RyR2) regions,
respectively (see Table I), produced significant activation of the RyR2
and induced Ca2+ release from cardiac SR. Domain 1-2 of
the RyR1 and its counterpart of the RyR2 share the same sequence in the
region of subdomain 1. The corresponding peptide 1 produced about the
same effects as peptide 1-2c, indicating that the essential activating
function is localized in subdomain 1. Interestingly, these peptides
were virtually incapable of inducing Ca2+ release from
skeletal SR. Generally, they produced no effect on ryanodine binding to
the RyR1 either, as long as the assay temperature was kept below
36 °C. This indicates that the activation is specific to the cardiac
isoform of the RyR under such limited conditions. At 36 °C, however,
the domain peptide 1-2 became capable of activating the RyR1. The
extents of activation varied over a broad range, from no activation to
a full activation comparable with that of the RyR2. This would indicate
that there is a common mechanism between both RyR isoforms by which the
RyR channel is regulated by mediation of an intramolecular
domain-domain interaction, but in the RyR1 the peptide probe revealed
such a mechanism only at a high temperature. A large variation in the
extent of activation seen at 36 °C suggests that in the vicinity of
this temperature there is a conformational transition from a
"tighter" domain-domain interaction to a somewhat "loose"
interaction, permitting the added peptides to exert an additional or
complimentary activation. Another important aspect of this study is the
finding that peptide 3 corresponding to the other domain encompassing
the Asp324-Val351 segment affected neither the
RyR1 nor the RyR2. Thus, it appears that the ability to activate the
RyR2 is specifically localized in the domain 1, although further
screening studies might reveal more regulatory regions/domains with
similar or different functions.
As shown in the present ryanodine binding assays, these peptides are
capable of activating the RyR2 at zero or nearly zero [Ca2+]. It is particularly interesting to note that the
mode of activation by these domain peptides at nominal zero
Ca2+ is similar to that of the effect of calmodulin on the
RyR1. Since calmodulin has no effect on the RyR2 and domain peptides
have no effects on the RyR1, it is tempting to speculate that the
domain peptides (hence, the corresponding in situ domain,
domain 1-2) have a calmodulin-like function on the RyR2 at very low
Ca2+ concentrations. As shown in this study, at a threshold
level of [Ca2+] for the activation of cardiac muscle,
i.e. 0.2 µM (43, 44), these peptides produced
a significant activation of the RyR2 and induced a rapid
Ca2+ release from cardiac SR. This suggests that the
corresponding domain (i.e. domain 1-2) may be involved in
the enhancement of cardiac contractility in the vicinity of the
threshold [Ca2+] required for activation. At maximally
activating or even higher [Ca2+] values, these peptides
still produced a significant enhancement of the
Ca2+-dependent activation of the RyR2. Thus,
the domain corresponding to these peptides (i.e. domain
1-2) seems to play several important functions, e.g.
(a) activation of the RyR2 at nearly zero Ca2+,
and (b) amplification of the
Ca2+-dependent activation of the RyR2 and
opening of the Ca2+ channel at near threshold or
subthreshold [Ca2+] values.
The amino acid sequence of peptide 1-2s represents the common sequence
of domain 1-2s of the RyR1 of several animal species. Similarly,
peptide 1-2c represents the common sequence of the RyR2 of several
animal species. Thus, the difference in four amino acid residues
between the two peptides (cf. underlined residues in Table
I) represents definitive characteristics distinguishing between the
RyR1 and the RyR2. As shown in the present studies, the
AC50 of peptide 1-2c was significantly lower than that of peptide 1-2s. This indicates that the affinity of binding to the putative regulatory domain described above is higher for the cardiac sequence than the skeletal sequence. The extent of activation by
peptide 1-2c appears to be significantly larger than that of peptide
1. These results suggest that the skeletal-type sequence that is
present in subdomain 2 may function as a suppressor of the activating
function localized in subdomain 1, whereas the cardiac-type sequence of
subdomain 2 may function as an enhancer. Interestingly, the differences
in the apparent affinity among these peptides became evident only at
lower [Ca2+] values (0 or 0.2 µM), and such
differences were abolished at higher [Ca2+] values (10 µM), suggesting that the affinity of these peptides increases with [Ca2+]. Thus, the cardiac sequence of
domain 1-2 may be important for the regulation at low
[Ca2+] values, but the common sequence in domain 1 may be
sufficient at higher [Ca2+] values.
The remaining important questions concern the general mechanism by
which the RyR is regulated in a Ca2+-dependent
manner, and more specifically the mechanism by which the
Ca2+-dependent activation of the RyR2 is
amplified by domain peptide 1-2. Since this domain peptide is a copy
of domain 1-2 and it has discrete effects on the RyR, it is
conceivable that within the RyR molecule this domain is interacting
with its target domain to regulate the Ca2+ channel. We
tentatively locate the target domain in the postulated modulatory
domain containing the putative high affinity Ca2+ binding
site (45). Regarding the mode of channel regulation, there are at least
two alternative possibilities as follows. First, the interdomain
interaction may facilitate the Ca2+-dependent
channel opening. Conversely, it is also possible that the interdomain
interaction serves as a channel blocking mechanism. Then, why did the
added domain peptide activate the RyR channel in a RyR2-specific
manner? In the case of the first possibility (namely the interdomain
interaction produces channel activation), the interaction between
domain 1-2 and the Ca2+-modulatory domain (see above)
would be much tighter in the RyR1 than in the RyR2. In other words, the
domain-mediated regulatory mechanism is fully functional in the RyR1,
but its function is only partial in the RyR2. Therefore, the
interaction of the domain peptide with the target domain will exert
additional or supplementary effects in the RyR2. At higher
temperatures, the interdomain interactions would become less tight, and
the activating effects of peptides would become pronounced even in the
RyR1. In the case of the second possibility (i.e. the
interdomain interaction produces channel closing), the
isoform-dependent differences in the tightness of interdomain interaction and its temperature dependence would be entirely opposite to the above. For instance, the interdomain interaction would be much tighter in the RyR2 than in the RyR1, producing more prominent de-blocking effects of the added peptides in
the RyR2. Clearly more work is required to elucidate the detailed mechanism(s) of the channel regulation mediated by domain-domain and
peptide-domain interactions.
Our recent study on polylysine-induced Ca2+ release from
the skeletal muscle SR (46) suggested the presence of a tight
coordination between Ca2+ release and the subsequent
re-uptake of the released Ca2+. As shown in the present
study, there appears to be a similar release/uptake coordination
mechanism also in the cardiac SR. Assuming that the domain peptides
used here are simulating the exact features of the in vivo
channel regulation mechanism, we would propose that the mechanism
discussed above is involved not only in the potentiation of channel
opening, but also in the acceleration of Ca2+
re-uptake.
In conclusion, the synthetic peptides corresponding to one of the
conserved NH2-terminal domains of the RyR1 and the RyR2 (designated as domain 1-2) activated the RyR2. These peptides activated the RyR2 at nearly zero [Ca2+], and also
amplified Ca2+-dependent activation of the
RyR2. They also induced a rapid Ca2+ release from the
cardiac SR. Thus, it appears that the domain corresponding to these
peptides, namely domain 1-2, is capable of performing these functions
in situ, by interacting with the putative regulatory domain(s).
We thank Dr. Renne C. Lu, Dr. Paul Leavis,
Gina Pagani, and Elizabeth Gowell for help in the synthesis and
purification of the peptides.
*
This work was supported by National Institutes of Health
Grant AR16922 and a grant from the Muscular Dystrophy Association.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Boston Biomedical
Research Inst., 20 Staniford St., Boston, MA 02114. Tel.: 617-912-0384; Fax: 617-912-0308; E-mail: ikemoto@bbri.org.
The abbreviations used are:
RyR, ryanodine
receptor;
MES, 2-(N-morpholino)ethanesulfonic acid;
MOPS, 3-(N-morpholino)propanesulfonic acid;
SR, sarcoplasmic
reticulum;
IP3, inositol 1,4,5-trisphosphate.
A Postulated Role of the Near Amino-terminal Domain of the
Ryanodine Receptor in the Regulation of the Sarcoplasmic Reticulum
Ca2+ Channel*
,,, and§¶
Boston Biomedical Research Institute,
Boston, Massachusetts 02114 and the § Department of
Neurology, Harvard Medical School, Boston, Massachusetts 02115
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
78 °C.
78 °C.
-amino protecting group. The peptides were cleaved and deprotected with 95% trifluoroacetic acid and purified by
reversed-phase high pressure liquid chromatography.
0.05 was accepted as statistically significant.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Subdomains of RyR selected and the amino acid sequence of the synthetic
peptides corresponding to these domains
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Fig. 1.
Peptide 1-2s enhances
[3H]ryanodine binding to cardiac SR vesicles (RyR2).
Dog cardiac muscle microsomes (1.5 mg/ml) were incubated with 10 nM [3H]ryanodine in a solution containing 0.3 M KCl, 10 µM CaCl2, 20 mM MOPS, pH 7.2, and various concentrations of peptide
1-2s as indicated. Ryanodine binding in the absence of peptides
(control) was 0.81 ± 0.13 pmol/mg. Each datum point
represents the mean ± S.D. of four or more experiments carried
out in duplicate.
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Fig. 2.
Effects of peptide 1-2s on
[3H]ryanodine binding to the RyR2 and the RyR1 at two
assay temperatures: A, 36 °C; B,
22 °C. Histograms show the frequency of occurrences of
different extents of enhancement of ryanodine binding to the RyR2 and
the RyR1 induced by 200 µM peptide 1-2s. The assay
solutions consisted of 0.3 M KCl, 10 µM
CaCl2, 20 mM MOPS, pH 7.2, with 10 nM or 8 nM [3H]ryanodine for dog
cardiac muscle microsomes and rabbit skeletal muscle microsomes,
respectively.
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Fig. 3.
Lack of effect of peptide 3s on
[3H]ryanodine binding to cardiac SR vesicles (RyR2) and
skeletal triads (RyR1). Ryanodine binding assays were carried out
at 36 °C in the presence of various concentrations of peptide 3s as
described under "Experimental Procedures." Each datum
point represents the mean ± S.D. of at least three
experiments carried out in duplicate.
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Fig. 4.
Concentration dependence of activation of
RyR2 by peptides 1-2s, 1-2c, and 1 at 10 µM Ca2+. Ryanodine
binding assays were performed at 36 °C (see "Experimental
Procedures"). Each datum point represents the mean ± S.D. of at least three experiments carried out in duplicate.
1 > 1-2s.
The concentrations for half-maximal activation (AC50) of
ryanodine binding to the RyR2 by three isoforms of domain peptide 1-2
0.05 (1-2c
versus 1-2s). **, p 0.05 (1-2s
versus 1).
View larger version (22K):
[in a new window]
Fig. 5.
Time courses of calcium release from dog
cardiac SR microsomes induced by various concentrations of peptides
1-2s, 1-2c, and 1. The SR moiety was first loaded with
Ca2+ by ATP-dependent uptake and then mixed
with a solution containing indicated concentrations of peptides to
induce Ca2+ release. The time course of Ca2+
release was monitored in a stopped-flow apparatus using fluo-3 as a
Ca2+ probe (for details, see "Experimental
Procedures"). Experiments were performed at 22 °C. Each
trace was obtained by signal averaging a total of 15-20
traces.
View larger version (12K):
[in a new window]
Fig. 6.
The initial rate of SR Ca2+
release induced by various concentrations of peptides 1-2s, 1-2c, and
1. The Ca2+ release traces shown in Fig. 5 were fitted
by a model {y = y0 + A1(1 e
k1t) A2(1 ek2t)}:
A1, the amount of Ca2+
release; k1, the rate constant of
Ca2+ release; A2, the amount of
Ca2+ uptake; k2, the rate
constant of Ca2+ uptake. The initial rate of
Ca2+ release
{(dy/dt)t
0} was
calculated as the product of A1 and
k1.
40 µM) is followed by
a decrease of the fluo-3 signal. Both the magnitude and the rate of
this descending phase increased with the increase in peptide
concentration. As seen from the comparison among the three peptides at
the equivalent concentration (e.g. see 160 µM), the order of the ability of producing the descending
phase is 1-2c > 1 > 1-2s. Interestingly, this is the same
as the order of the ability of inducing Ca2+ release (see
above). The descending phase presumably represents re-uptake of the
released Ca2+ via the SR Ca2+ pump. Thus, it
appears that a faster and larger Ca2+ release results in a
faster and larger uptake of the released Ca2+ in a
coordinated manner. As seen in the release/uptake curve produced by 160 µM peptide 1-2c (Fig. 5), the Ca2+ level
often became even lower than the starting level. This indicates that
the Ca2+ pumping rate became considerably larger than the
steady-state pumping rate (the rate prior to the induction of
Ca2+ release) after peptide-induced Ca2+ release.
View larger version (14K):
[in a new window]
Fig. 7.
The Ca2+ dependence of
[3H]ryanodine binding to the RyR2 in the absence and
presence of peptide 1. [3H]Ryanodine binding
experiments were performed at 36 °C in the presence or absence of
100 µM peptide 1 at various [Ca2+] values.
Various free Ca2+ concentrations in the assay solution were
generated by mixing various concentrations of CaCl2 with 1 mM EGTA based upon computer calculation (47). The assay
conditions are described under "Experimental Procedures." Each
datum point represents the mean ± S.D. of at least
three experiments carried out in duplicate.
View larger version (39K):
[in a new window]
Fig. 8.
RyR2-specific activation by peptide 1 occurs
even at nominal zero [Ca2+].
[3H]Ryanodine binding experiments were carried out at
36 °C with 200 µM peptide 1 and 5 µM
calmodulin. The assay solution is as described under "Experimental
Procedures." Each bar represents the mean ± S.D. of
three experiments carried out in duplicate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Inui, M.,
Saito, A.,
and Fleischer, S.
(1987)
J. Biol. Chem.
262,
1740-1747 2.
Imagawa, T.,
Smith, J. S.,
Coronado, R.,
and Campbell, K. P.
(1987)
J. Biol. Chem.
262,
16636-16643 3.
Inui, M.,
Saito, A.,
and Fleischer, S.
(1987)
J. Biol. Chem.
262,
15637-15642 4.
Lai, F. A.,
Erickson, H. P.,
Rousseau, E.,
Liu, Q. L.,
and Meissner, G.
(1988)
Nature
331,
315-319[CrossRef][Medline]
[Order article via Infotrieve]
5.
Takeshima, H.,
Nishimura, S.,
Matsumoto, T.,
Ishida, H.,
Kangawa, K.,
Minamino, N.,
Matsuo, H.,
Ueda, M.,
Hanaoka, M.,
Hirose, T.,
and Numa, S.
(1989)
Nature
339,
439-445[CrossRef][Medline]
[Order article via Infotrieve]
6.
Otsu, K.,
Willard, H. F.,
Khanna, V. K.,
Zorzato, F.,
Green, N. M.,
and MacLennan, D. H.
(1990)
J. Biol. Chem.
265,
13472-13483 7.
Franzini-Armstrong, C.
(1970)
J. Cell Biol.
47,
488-499 8.
Coronado, R.,
Morrissette, J.,
Sukhareva, M.,
and Vaughan, D. M.
(1994)
Am. J. Physiol.
266,
C1485-C1504 9.
McPherson, P. S.,
and Campbell, K. P.
(1993)
J. Biol. Chem.
268,
13765-13768 10.
Meissner, G.
(1994)
Annu. Rev. Physiol.
56,
485-508[CrossRef][Medline]
[Order article via Infotrieve]
11.
Ogawa, Y.
(1994)
Crit. Rev. Biochem. Mol. Biol.
29,
229-274[Medline]
[Order article via Infotrieve]
12.
Sorrentino, V.,
and Volpe, P.
(1993)
Trends Pharmacol. Sci.
14,
98-103[CrossRef][Medline]
[Order article via Infotrieve]
13.
Wagenknecht, T.,
Grassucci, R.,
Berkowitz, J.,
Wiederrecht, G. J.,
Xin, H.-B.,
and Fleischer, S.
(1996)
Biophys. J.
70,
1709-1715 14.
Seiler, S.,
Wegener, A.,
Wang, D.,
Hathaway, D.,
and Jones, L.
(1984)
J. Biol. Chem.
259,
8550-8557 15.
Tripathy, A.,
Xu, L.,
Mann, G.,
and Meissner, G.
(1995)
Biophys. J.
69,
106-119 16.
Chen, S. R. W.,
and MacLennan, D. H.
(1994)
J. Biol. Chem.
269,
22698-22704 17.
Jayaraman, T.,
Brillantes, A. M.,
Timerman, A. P.,
Fleischer, S.,
Erdjument-Bromage, H.,
Tempst, P,
and Marks, A. R.
(1992)
J. Biol. Chem.
267,
9474-9477 18.
Timerman, A. P.,
Ogunbumni, E.,
Freund, E.,
Wiederecht, G.,
Marks, A. R.,
and Fleischer, S.
(1993)
J. Biol. Chem.
268,
22992-22999 19.
Quane, K. A.,
Healy, J. M. S.,
Keating, K. E.,
Manning, B. M.,
Couch, F. J.,
Palmucci, L. M.,
Doriguzzi, C.,
Fagerlund, T. H.,
Berg, K.,
Ording, H.,
Bendixen, D.,
Mortier, W.,
Linz, U.,
Muller, C. R.,
and McCarthy, T. V.
(1993)
Nat. Genet.
5,
51-55[CrossRef][Medline]
[Order article via Infotrieve]
20.
Quane, K. A.,
Keating, K. E.,
Manning, B. M.,
Healy, J. M. S.,
Monsieurs, K.,
Hefron, J. J. A.,
Lehane, M.,
Heytens, L.,
Krivosic-Hober, R.,
Adnet, P.,
Ellis, F. R.,
Monnier, N.,
Lunardi, J.,
and McCarthy, T. V.
(1994)
Hum. Mol. Genet.
3,
471-476 21.
Fujii, J.,
Otsu, K.,
Zorzato, F.,
De Leon, S.,
Khanna, V. K.,
Weiler, J. E.,
O'Brien, P. J.,
and MacLennan, D. H.
(1991)
Science
253,
448-451 22.
Phillips, M.,
Khanna, V. K.,
DeLeon, S.,
Frodis, W.,
Britt, B. A.,
and MacLennan, D. H.
(1994)
Hum. Mol. Genet.
3,
2181-2186 23.
Zhang, Y.,
Chen, H. S.,
Khanna, V. K.,
DeLeon, S.,
Phillips, M. S.,
Scahpper, K.,
Britt, B. A.,
Bronwell, A. K. W.,
and MacLennan, D. H.
(1993)
Nat. Genet.
5,
46-50[CrossRef][Medline]
[Order article via Infotrieve]
24.
Gillard, E. F.,
Otsu, K.,
Fujii, J.,
Khanna, V. K.,
De Leon, S.,
Dermedezi, J.,
Britt, B. A.,
Duff, C. L.,
Worton, R. G.,
and MacLennan, D. H.
(1991)
Genomics
11,
751-755[CrossRef][Medline]
[Order article via Infotrieve]
25.
Gillard, E. F.,
Otsu, K.,
Fujii, J.,
Duff, C. L.,
De Leon, S.,
Khanna, V. K.,
Britt, B. A.,
Worton, R. G.,
and MacLennan, D. H.
(1992)
Genomics
13,
1247-1254[CrossRef][Medline]
[Order article via Infotrieve]
26.
MacLennan, D. H.,
and Philips, M. S.
(1995)
in
Ryanodine Receptors
(Sorrentino, V., ed)
, pp. 155-180, CRC Press, Boca Raton, FL
27.
Mickelson, J. R.,
Gallant, E. M.,
Litterer, L. A.,
Johnson, K. M.,
Rempel, W. E.,
and Louis, C. F.
(1988)
J. Biol. Chem.
263,
9310-9315 28.
Fill, M.,
Coronado, R.,
Mickelson, J. R.,
Vilven, J.,
Ma, J.,
Jacobson, B. A.,
and Louis, C. F.
(1990)
Biophys. J.
50,
471-475 29.
El-Hayek, R.,
Yano, M.,
Antoniu, B.,
Mickelson, J. R.,
Louis, C. F.,
and Ikemoto, N.
(1995)
Am. J. Physiol.
268,
C1381-C1386 30.
Mickelson, J. R.,
and Louis, C. F.
(1996)
Physiol. Rev.
76,
537-592 31.
Tong, J.,
Oyamada, H.,
Demaurex, N.,
Grinstein, S.,
McCarthy, T. V.,
and MacLennan, D. H.
(1997)
J. Biol. Chem.
272,
26332-26339 32.
Zorzato, F.,
Menegazzi, P.,
Treves, S.,
and Ronjat, M.
(1996)
J. Biol. Chem.
271,
22759-22763 33.
Aghdasi, B.,
Zhang, J.,
Wu, Y.,
Reid, M. B.,
and Hamilton, S. L.
(1997)
J. Biol. Chem.
272,
3739-3748 34.
Wu, Y.,
Aghdasi, B.,
Dou, S. J.,
Zhang, J. J.,
Liu, S. Q.,
and Hamilton, S. L.
(1997)
J. Biol. Chem.
272,
25051-25061 35.
Furuichi, T.,
Yoshikawa, S.,
Miyawaki, A.,
Wada, K.,
Maeda, N.,
and Mikoshiba, K.
(1989)
Nature
342,
32-38[CrossRef][Medline]
[Order article via Infotrieve]
36.
Yoshikawa, F.,
Iwasaki, H.,
Michikawa, T.,
Furuichi, T.,
and Mikoshiba, K.
(1999)
J. Biol. Chem.
274,
328-334 37.
MacLennan, D. H.,
and Chen, S. R. W.
(1994)
in
Calcium as Cell Signal
(Maruyama, K.
, Noromura, Y.
, and Kohana, K., eds)
, pp. 141-153, Igaku-Shoin, Tokyo, Japan
38.
Mignery, G. A.,
Newton, C. L.,
Archer, B. T., III,
and Sudhof, T. C.
(1990)
J. Biochem. (Tokyo) 2
65,
12679-12685
39.
Ikemoto, N.,
Kim, D. H.,
and Antoniu, B.
(1988)
Methods Enzymol.
157,
469-480[Medline]
[Order article via Infotrieve]
40.
El-Hayek, R.,
Lokuta, A. J.,
Arevalo, C.,
and Valdivia, H. H.
(1995)
J. Biol. Chem.
270,
28696-28704 41.
El-Hayek, R.,
Yano, M.,
and Ikemoto, N.
(1995)
J. Biol. Chem.
270,
15634-15638 42.
MacLennan, D. H.,
and Philips, M. S.
(1992)
Science
256,
789-794 43.
Stern, M. D.,
and Lakatta, E. G.
(1992)
FASEB J.
6,
3092-3100[Abstract]
44.
Fabiato, A.
(1983)
Am. J. Physiol.
245,
C1-C14 45.
Chen, S. R. W.,
Zhang, L.,
and MacLennan, D. H.
(1993)
J. Biol. Chem.
268,
13414-13421 46.
Saiki, Y.,
and Ikemoto, N.
(1999)
Biochemistry
38,
3112-3119[CrossRef][Medline]
[Order article via Infotrieve]
47.
Yamada, S.,
and Ikemoto, N.
(1980)
J. Biol. Chem.
255,
3108-3119
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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