From the Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599-7260
Received for publication, March 27, 2001
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ABSTRACT |
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Fusion proteins and full-length mutants were
generated to identify the Ca2+-free (apoCaM) and
Ca2+-bound (CaCaM) calmodulin binding sites of the skeletal
muscle Ca2+ release channel/ryanodine receptor (RyR1).
[35S]Calmodulin (CaM) overlays of fusion proteins
revealed one potential Ca2+-dependent (aa
3553-3662) and one Ca2+-independent (aa 4302-4430) CaM
binding domain. W3620A or L3624D substitutions almost abolished
completely, whereas V3619A or L3624A substitutions reduced
[35S]CaM binding to fusion protein (aa 3553-3662). Three
full-length RyR1 single-site mutants (V3619A,W3620A,L3624D) and one
deletion mutant ( Calcium release channels, also known as ryanodine receptors
(RyRs),1 control the release
of Ca2+ from endoplasmic and sarcoplasmic reticulum in a
wide range of tissues (1-3). Mammalian tissues express three
structurally and functionally related RyR isoforms referred to as the
skeletal muscle (RyR1), cardiac muscle (RyR2), and brain (RyR3)
ryanodine receptors. All three isoforms have been purified as 30 S
protein complexes composed of four 565-kDa RyR polypeptides in tight
association with four 12-kDa FK506-binding proteins. They are
cation-selective channels capable of multiple interactions with other
molecules. These include small diffusible molecules, such as
Ca2+, Mg2+, and ATP, and proteins, such as
triadin and calmodulin (CaM) (1-4).
CaM is a ubiquitous cytosolic protein that has a critical role in
regulating cellular functions by altering the activity of a large
number of proteins. CaM regulates all three RyR isoforms. RyR1 and RyR3
are activated by Ca2+-free CaM (apoCaM) and are inhibited
by Ca2+-bound CaM (CaCaM) (5-8), whereas RyR2 is not
activated by apoCaM but is inhibited by CaCaM (8-10). Determination of
the number of CaM binding sites and their location has been the focus
of several studies. Early studies using [125I]CaM (6, 11)
or fluorescence-labeled CaM (12) showed a stoichiometry of 1 CaCaM and
2-6 apoCaM binding sites/RyR1 subunit. More recent studies using
metabolically 35S-labeled CaM showed one binding
site/RyR1 monomer for both of apoCaM and CaCaM (8, 10, 13, 14). Binding
site localization studies with fusion proteins and synthetic peptides
revealed up to seven candidate CaM binding sites in RyR1 (15-18),
clearly exceeding the number of 1 [35S]apoCaM and 1 [35S]CaCaM binding site/RyR polypeptide. To resolve this
discrepancy, full-length RyR1 mutants were generated focusing on two
CaM binding domains identified in [35S]CaM overlays of
fusion proteins spanning the full-length RyR1 (10). The RyR1 mutants
were expressed in HEK293 cells, and their [35S]CaM
binding properties and regulation by CaM were determined. We found that
two amino acid substitutions (W3620A,L3624D) resulted in a loss of high
affinity CaCaM binding and inhibition of RyR1 by CaCaM (nanomolars).
The L3624D substitution also resulted in a loss of apoCaM binding and
activation of RyR1 by apoCaM. Portions of this study have been
published previously in abstract form (19).
Materials--
[3H]ryanodine was obtained from
PerkinElmer Life Sciences, Tran35S-label was from
ICN Radiochemicals (Costa Mesa, CA), unlabeled ryanodine was from
Calbiochem (La Jolla, CA), unlabeled CaM was from Sigma, and
complete protease inhibitors were from Roche Molecular Biochemicals.
Construction of Wild Type and Mutant cDNA
Plasmids--
cDNAs for RyR1 fusion proteins tagged with trpE and
GST were constructed using pATH and pGEX-5X vectors, respectively. The plasmids were transformed into BL21 Escherichia coli cells,
and protein expression was induced by manufacturer's protocol (for GST) and as described previously (for trpE) (20). FPI
(3225-3662), FPI-2 (3352-3392), FPI-3 (3391-3554), and FPI-4
(3553-3662) were expressed as trpE fusion proteins, and FPI-1
(3225-3353) and FPM (4302-4430) were expressed as GST fusion proteins
(amino acid sequences are shown in parentheses). The full-length rabbit
RyR1 cDNA (ClaI/XbaI) was constructed and
cloned into expression vector pCMV5 as described previously (21).
Single and multiple base changes were introduced by pfu
polymerase-based chain reaction using mutagenic oligonucleotides and
the QuickChangeTM site-directed mutagenesis kit
(Stratagene, La Jolla, CA). For the fusion proteins, FPI-4 was used as
the template of mutagenesis. The construction of the full-length RyR1
mutants made use of mutated FPI-4 fragments,
EclXI/SspBI(10872-11054). Alternatively,
a partial fragment, PvuI/NdeI(8600-11304), was
used as the template of mutagenesis. Deletion of sequences encoding
amino acids 4274-4535 was performed using two NarI
restriction enzyme sites (22). Mutated and deleted sequences were
confirmed by sequencing. Mutated and deleted full-length expression
plasmids were prepared by ligation of two fragments (ClaI/PvuI and PvuI/XbaI
containing the mutated or deleted sequence) and expression vector pCMV5
(ClaI/XbaI).
Expression of Full-length RyR1 in HEK293 Cells--
RyR1
cDNAs were transiently expressed in HEK293 cells with the
LipofectAMINE Plus (Life Technologies, Inc.) or Fugene6 (Roche Molecular Biochemicals) methods according to the manufacturers' instructions. Cells were maintained at 37 °C and 5% CO2
in high glucose Dulbecco's modified Eagle's medium containing 10%
fetal bovine serum and plated the day before transfection. For each 10-cm tissue culture dish, 3-6 µg cDNA was used. Cells were
harvested 42-48 h after transfection. Cells were washed twice with
3-ml ice-cold phosphate-buffered saline containing 1 mM
EDTA and Complete protease inhibitors and harvested in the same
solution by removal from the plates by scraping. Cells were collected
by centrifugation, washed in the same buffer without EDTA, and stored
at [35S]Calmodulin Overlay--
CaM binding to RyR1
fusion proteins was assayed by [35S]CaM overlays using
whole cell preparations or inclusion bodies. [35S]CaM was
prepared using Tran35S label as described previously (10).
Proteins were separated by SDS-polyacrylamide gel electrophoresis and
transferred to nitrocellulose membranes. Nonspecific binding sites were
blocked by treating membranes with a solution, 150 mM KCl,
20 mM KPipes, pH 7.0, containing 1 mg/ml bovine
serum albumin (BSA) and 100 µM Ca2+ (blocking
buffer) for 1 h. CaCaM binding was analyzed by incubating membranes with 100 nM [35S]CaM in 150 mM KCl, 20 mM KPipes, pH 7.0, 0.04% Tween 20, and 100 µM Ca2+ for 1 h and washing with
blocking buffer 4 times. Dried membranes were exposed to x-ray film,
and radioactivity was determined by autoradiography. ApoCaM binding was
analyzed in buffer solutions containing 5 mM EGTA instead
of 100 µM Ca2+.
[35S]Calmodulin Binding--
Crude membrane
fractions prepared as described below were incubated for 2 h at
room temperature with solutions, 5 or 15 nM [35S]CaM in 10 mM KPipes, 10 mM
imidazole, pH 7.0, containing 0.15 M sucrose, 150 mM KCl, 0.125 mg/ml BSA, 5 mM glutathione
(reduced form), 20 µM leupeptin, 200 µM
Pefabloc, and either 5 mM EGTA (apoCaM binding) or 200 µM Ca2+ (CaCaM binding). Aliquots were taken
for determination of total radioactivity and centrifuged for 45 min at
30 p.s.i. in a Beckman Airfuge to obtain bound
[35S]CaM. Radioactivities were determined by
scintillation counting. Nonspecific binding of [35S]CaM
was determined by incubating equal protein amounts of
vector-transfected or non-transfected HEK293 cells. In parallel
experiments, Bmax values of
[3H]ryanodine binding were determined. Membranes were
incubated for 5 h at room temperature with a saturating
concentration of [3H]ryanodine (30 nM) in 20 mM imidazole, pH 7.0, 0.6 M KCl, 0.15 M sucrose, 20 µM leupeptin, 200 µM Pefabloc, and 200 µM Ca2+.
Specific [3H]ryanodine binding was determined as
described above.
The time courses of [35S]CaM binding to and dissociation
from skeletal SR vesicles were determined by a filtration assay. To minimize nonspecific binding of [35S]CaM, Whatman GF/B
filters were blocked in buffer, 0.15 M KCl, 20 mM KPipes, pH 7.0, containing 10 mg/ml BSA. Vesicles on the filters were washed with three 5 ml of ice-cold buffer, 0.15 M KCl, 20 mM KPipes, pH 7.0, containing 0.1 mg/ml BSA and 100 µM Ca2+
([35S]CaCaM binding and [35S]CaCaM
dissociation) or 100 µM EGTA ([35S]apoCaM dissociation).
[3H]Ryanodine
Binding--
[3H]Ryanodine binding experiments were
performed with crude membrane fractions. HEK293 cell pellets were
resuspended in 20 mM imidazole, pH 7.0, 0.3 M
sucrose, 150 mM KCl, 1 mM glutathione (oxidized
form), Complete protease inhibitors, and 0.1 mM EGTA and
homogenized with a Tekmar Tissumizer for 5 s at a setting of
13,500 rpm. Homogenates were centrifuged for 45 min at 40,000 rpm in a
Beckman Ti75 rotor, and pellets were resuspended in the above buffer
without EGTA and glutathione. Unless otherwise indicated, membranes
were incubated with 2.5 nM [3H]ryanodine in
20 mM imidazole, pH 7.0, 0.3 M sucrose, 250 mM KCl, 0.5 mM glutathione (oxidized), 0.25 mg/ml BSA, protease inhibitors, and indicated Ca2+
concentrations. Nonspecific binding was determined using a
1000-2000-fold excess of unlabeled ryanodine. After 20 h,
aliquots of the samples were diluted with 8.5 volumes of ice-cold water
and placed on Whatman GF/B filters preincubated with 2%
polyethyleneimine in water. Filters were washed with three 5 ml of
ice-cold 100 mM KCl, 1 mM KPipes, pH 7.0, solution. The radioactivity remaining with the filters was determined
by liquid scintillation counting to obtain bound
[3H]ryanodine.
[35S]Calmodulin Overlays of Wild Type and Mutant RyR1
Fusion Proteins--
In a previous study, we used 15 fusion proteins
spanning the full coding sequence of the RyR1 polypeptide to identify
candidate CaM binding domains (10). We found that two fusion proteins including amino acids 3225-3662 of RyR1 (FPI) and amino acids 4302-4430 (FPM) specifically bound [35S]CaM in a
Ca2+-dependent and independent manner,
respectively (Fig. 1) (10). In this
study, we further subdivided the larger of the two fusion proteins
(FPI) into four fragments (FPI-(1-4)) using specific restriction
enzyme sites. The fragments were expressed as trpE fusion proteins.
FPI-1 (3225-3353) was also expressed as a GST fusion protein because
the expression level of the trpE fusion protein was very low. Since all
fusion proteins were insoluble, [35S]CaM overlays were
done with whole cell fractions in Fig. 1. The amounts of proteins on
the gels were adjusted to show similar Coomassie Blue staining for the
fusion proteins (Fig. 1A). Fig. 1B shows that in
the presence of 100 nM [35S]CaM and 100 µM Ca2+, three of the fusion proteins clearly
showed detectable [35S]CaM binding. The strongest binding
was observed for FPI-4 followed by FPI and FPM. We also performed
[35S]CaM overlays in the presence of 5 mM
EGTA instead of 100 µM Ca2+. As previously
found (10), FPM bound [35S]apoCaM at a level comparable
with CaCaM. FPI did not show apoCaM binding and, as expected, neither
did the FPI-derived fragments. These results show that
[35S]CaM could bind to two fusion proteins derived from
RyR1; binding to one fusion protein was
Ca2+-dependent, whereas binding to the other
was Ca2+-independent.
Primary sequence predictions suggest the presence of several CaM
binding sites in RyR1 (23, 24). One of these sites was predicted to be
present in FPI-4 (3614-3637). Using nnPredict (University of
California, San Francisco, CA), we identified a stretch of amino acids
(aa 3617-3628) predicted to form an amphipathic [35S]Calmodulin Binding to Wild Type and Mutant
RyR1s--
We introduced three site-specific mutations in the
full-length RyR1 that led to nearly a complete loss (W3620A,L3624D) or a reduction (V3619A) of [35S]CaM binding to FPI-4 (Fig.
2). We also generated a deletion mutant (RyR1 [3H]Ryanodine Binding to Wild Type and Mutant RyR1s--
We next examined the functional effects of CaCaM on the four RyR1
mutants shown in Fig. 3 by determining their
[3H]ryanodine binding properties in the absence and
presence of exogenously added CaM. The highly specific plant alkaloid
ryanodine is widely used as a probe of channel activity because of its
preferential binding to the open RyR channel states (1-3). The four
mutants exhibited a specific [3H]ryanodine binding
affinity (determined by Scatchard analysis) and Ca2+
activation/inactivation profile comparable with wt-RyR1 with the
exception of RyR1 [35S]Calmodulin Binding to Native
RyR1--
Dissociation and chase experiments were performed to
determine whether CaCaM and apoCaM share a common binding domain in
native RyR1s using a filtration assay. As shown in Fig.
5A, the dissociation of
[35S]CaM from skeletal muscle SR vesicles enriched in
RyR1 is not largely dependent of whether CaM is bound in the presence
or absence of Ca2+ but rather on whether Ca2+
is present in the dissociation buffer with apoCaM dissociating at a
significantly greater rate than CaCaM. In a similar set of experiments, SR vesicles were preincubated with or without
non-radioactive CaM in either the presence or absence of
Ca2+ followed by the binding of radioactive CaCaM. The
resulting rates of [35S]CaM binding were dramatically
slower to vesicles pretreated with non-radioactive CaM (Fig.
5B, open symbols) than the rates of binding to
vesicles not pretreated with CaM (Fig. 5B, closed symbols). Furthermore, the rates were relatively independent of whether the preincubation had been performed in the absence or presence
of Ca2+. These experiments strongly support the mutant
results that CaCaM and apoCaM bind to a common region of RyR1.
Calmodulin has a dual effect on skeletal muscle Ca2+
release channel activity. CaM activates the channel at Ca2+
concentrations below 1 µM, whereas at Ca2+
concentrations above 1 µM, the channel activity is
inhibited by CaM. The data we have presented here indicate that these
effects are mediated through a single CaM binding domain that is shared by apoCaM and CaCaM.
Several studies have reported the stoichiometry of CaM binding to RyR1
using SR vesicles (6, 8, 10-14) and purified RyR1 preparations (6,
10). The initial studies using either 125I (6, 11)
or fluorescently (12) labeled CaM revealed that the native
RyR1 binds with nanomolar affinity 1 CaM/subunit in the presence of
Ca2+, and that there are as many as six high affinity
binding sites for apoCaM on each of the four RyR1 subunits that
comprise the functional channel. More recent studies using
35S metabolically labeled CaM indicate that the tetrameric
skeletal muscle channel complex binds 4 CaM molecules both in the
absence and presence of Ca2+ or 1 CaM/subunit (8, 10, 13).
These results imply that chemical modification of CaM increases the
number of CaM binding sites of RyR1.
Previous studies performed to localize the CaM binding sites relied on
the use of fusion proteins and synthetic peptides (Fig. 6). CaM overlays of RyR1 fusion proteins
using 125I (15) or digoxigenin-labeled (16) CaM revealed up
to seven regions that bound CaM. With the exception of one site, CaM
binding was abolished in the presence of EGTA, indicating that it was Ca2+-dependent. Our protein overlays using
[35S]CaM identified potential binding domains in two
fusion proteins; one of which, FPM (aa 4302-4430), bound CaM both in
the absence and presence of Ca2+. The other fusion protein,
FPI-4 (aa 3553-3662), bound CaM only in the presence of
Ca2+ in agreement with previous studies using fusion
proteins (15, 16).
4274-4535) were generated and expressed in human
embryonic kidney 293 cells. L3624D exhibited greatly reduced
[35S]CaM binding affinity as indicated by a lack of
noticeable binding of apoCaM and CaCaM (nanomolar) and the requirement
of CaCaM (micromolar) for the inhibition of RyR1 activity. W3620A bound
CaM (nanomolar) only in the absence of Ca2+ and did not
show inhibition of RyR1 activity by 3 µM CaCaM. V3619A and the deletion mutant bound apoCaM and CaCaM at levels compared with
wild type. V3619A activity was inhibited by CaM with IC50 ~200 nM, as compared with IC50 ~50
nM for wild type and the deletion mutant.
[35S]CaM binding experiments with sarcoplasmic reticulum
vesicles suggested that apoCaM and CaCaM bind to the same region of the native RyR1 channel complex. These results indicate that the intact RyR1 has a single CaM binding domain that is shared by apoCaM and
CaCaM.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C. Sarcoplasmic reticulum (SR) vesicles were prepared from
rabbit skeletal muscle as described previously (6).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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Fig. 1.
[35S]CaM overlays to RyR1
fusion proteins. A, SDS-polyacrylamide gel of whole
cell fractions stained with Coomassie Brilliant Blue R250. The
asterisks indicate fusion proteins as detected with trpE
(FPI, FPI-2, FPI-3, and FPI-4) or GST (FPI-1 and FPM) antibodies.
[35S]CaM overlays in the presence of either 100 µM Ca2+ (B) or 5 mM
EGTA (C). RyR1 amino acids included in fusion protein are
3225-3353 (FPI-1), 3352-3392 (FPI-2), 3391-3554 (FPI-3), and
3553-3662 (FPI-4), 3225-3662 (FPI), and 4302-4430 (FPM). Standard
molecular masses are shown on the left side of
panels in kDa. The positions of trpE and GST are also
shown. Neither bound CaCaM nor ApoCaM (data not shown).
-helical structure
but not in perfect agreement with reported CaM binding motifs.
Therefore, we somewhat arbitrarily mutated three hydrophobic amino acid
residues (Val 3619 to Ala, Trp 3620 to Ala, and Leu 3624 to Ala and
Asp) lying on one face of the helix. We also substituted
cysteine 3635 with an alanine because CaM blockage of
N-ethylmaleimide alkylation of Cys 3635 suggested that this
residue may be important for CaM binding (25). All of the mutant fusion
proteins including wt were isolated as inclusion bodies and tested for
[35S]CaCaM binding using the overlay assay. Equivalent
amounts of wt and mutated FPI-4s were used based on Coomassie Blue
staining of SDS gels. The results of the overlay assay are shown in
Fig. 2. The strongest binding was
observed for wt and C3635A mutant proteins. Mutant proteins with V3619A
or L3624A substitutions showed reduced binding, whereas mutant proteins
with W3620A or L3624D substitutions barely showed detectable binding.
The results identify two amino acid residues (Trp 3620, Leu 3624) that
are critical for CaCaM binding to FPI-4. However, it was unclear
whether the results with the fusion protein were directly applicable to the full-length RyR1. The information gained was limited because FPI-4
did not bind apoCaM and, therefore, could not be used to locate the
apoCaM binding sites in RyR1. Also, [35S]CaM overlays
revealed two candidate CaCaM binding sites as opposed to one
site/subunit in the native RyR1. Therefore, we extended our mutant
studies to the intact RyR1.
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Fig. 2.
[35S]CaM overlay to wild type
and mutant FPI-4 fusion proteins. [35S]CaM overlays
in the presence of 100 µM Ca2+ are shown.
Standard molecular masses are shown on the left side of
panels in kDa.
4274-4535) to address
the significance of a Ca2+-independent CaM binding site
detected in the overlays in FPM (aa 4302-4430) (Fig. 1, B
and C). The mutant RyR1s were expressed in HEK293 cells, and
crude membrane fractions were prepared to determine their CaM binding
properties. In parallel experiments, the RyR1 expression levels were
quantified by a ligand binding assay using saturating
[3H]ryanodine concentrations as described under
"Experimental Procedures." Expression of full-length wt and mutant
RyR1s was confirmed by Western blot analysis using anti-RyR1 monoclonal
antibody D110 (26) (data not shown). In Fig.
3, we used 5 and 15 nM
[35S]CaM, which are near and beyond the dissociation
constants of apoCaM and CaCaM binding to the native and purified RyR1
under the assay conditions described under "Experimental
Procedures" (10). Cells expressing wt-RyR1 bound 3-4
[35S]apoCaM and 4-5
[35S]CaCaM/[3H]ryanodine binding site. As
there is only one high affinity [3H]ryanodine binding
site/RyR1 tetramer, these ratios corresponded to ~1 apoCaM and 1 CaCaM binding site/RyR1 subunit. RyR1 mutant with a V3619A substitution
and RyR1
4274-4535 bound [35S]apoCaM and
[35S]CaCaM not significantly different from wt-RyR1.
W3620A bound only [35S]apoCaM, whereas L3624D showed a
loss of high affinity [35S]CaM binding both in the
presence and absence of Ca2+. These studies provided
information beyond that obtained with the fusion proteins. The results
of Fig. 3 indicate that in the intact RyR1 amino acid residues
4274-4535 are not important for high affinity apoCaM and CaCaM
binding. Rather, they suggest that Leu 3624 constitutes a part of both
the apoCaM and the CaCaM binding site in the intact RyR1, whereas Trp
3620 appeared to be only a part of the CaCaM binding site, results that
could not be obtained with the mutant fusion proteins because FPI-4 did
not show apoCaM binding.
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Fig. 3.
[35S]CaM binding to wild type
and mutant RyR1s. Membrane fractions prepared from HEK293 cells
expressing wt or mutant RyR1s were incubated for 2 h at room
temperature with 5 or 15 nM [35S]CaM and
either 200 µM Ca2+ (A) or 5 mM EGTA (B). The ratios of
[35S]CaM binding values to maximal binding values of
[3H]ryanodine are shown. Maximal values of
[3H]ryanodine binding (in pmol/mg protein), determined as
described under "Experimental Procedures", were 0.22 ± 0.03 (wt), 0.21 ± 0.02 (V3619A), 0.21 ± 0.03 (W3620A), 0.18 ± 0.02 (L3624D), and 0.69 ± 0.08 ( 4274-4535). Data are the
means ± S.E. of 3-6 experiments. *, p < 0.05;
**, p < 0.01 as compared with wt at the same CaM
concentration.
4274-4535, which showed an ~10-fold increased sensitivity to activating Ca2+ in agreement with a previous
report (22) (data not shown). Fig.
4A shows that
[3H]ryanodine binding to wt-RyR1 was inhibited by CaCaM
in a concentration-dependent manner with an
IC50 ~50 nM. The maximal extent of inhibition
(60% by ~1 µM CaM) was comparable with that observed
for native RyR1s (6). The deletion mutant (RyR1
4274-4535) showed a
response to CaCaM essentially identical to wt-RyR1. V3619A required a
higher CaCaM concentration for the inhibition of
[3H]ryanodine binding (IC50 ~200
nM as compared with IC50 ~50 nM for wt-RyR1). L3624D exhibited a greatly reduced apparent affinity for
CaCaM as indicated by the requirement of 3 µM CaCaM for
partial inhibition of RyR1 activity, whereas W3620A did not show any
inhibition at 3 µM CaCaM. Fig. 4B shows that,
in agreement with the apoCaM binding data of Fig. 3B, 1 µM apoCaM significantly increased
[3H]ryanodine binding to wt and V3619A, W3620A, and
4274-4535 RyR1s but not L3624D. Taken together, the results of the
[35S]CaM (Fig. 3) and [3H]ryanodine binding
(Fig. 4) experiments suggest that Leu 3624 constitutes a part of the
CaCaM inhibiting and apoCaM activating sites of RyR1, whereas Trp 3620 appears to be only essential for CaCaM inhibition.
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Fig. 4.
CaM inhibition and activation of
[3H]ryanodine binding to wild type and mutant RyR1s.
Specific [3H]ryanodine binding to wt, V3619A, W3620A,
L3624D, and 4274-4535 RyR1s was determined as described under
"Experimental Procedures" in the presence of 100 µM
Ca2+ (A) or 0.1 µM
Ca2+ and 1 mM AMPPCP (a nonhydrolyzable ATP
analog) (B) and the indicated concentrations of CaM.
Normalized [3H]ryanodine binding data are the means ± S.E. of four or more experiments. *, p < 0.05; **
p < 0.01 as compared with wt at the same CaM
concentration (A) and wt and mutants in the absence of CaM
(B).
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Fig. 5.
Time course of [35S]CaM binding
and dissociation. A, skeletal SR vesicles were prebound
with 100 nM [35S]CaM in 150 mM
KCl, 20 mM KPipes, pH 7.0, 5 mM glutathione
(reduced form), 0.1 mg/ml BSA, 0.2 mM Pefabloc, 20 µM leupeptin with 5 mM EGTA
(circle) or 100 µM Ca2+
(triangle) and then diluted 60-fold into media containing 50 nM non-radioactive CaM. Final EGTA-Ca2+
concentration was either 1 mM EGTA (filled
symbols) or 100 µM Ca2+ (open
symbols). Filter assays were performed as described under
"Experimental Procedures." B, skeletal SR vesicles were
preincubated for 1 h at room temperature in 0.15 M
KCl, 20 mM KPipes, pH 7.0, buffer containing 5 mM glutathione (reduced form), 0.2 mM Pefabloc,
20 µM leupeptin, and 0.1 mg/ml BSA with (open
symbols) or without (filled symbols) 100 nM
non-radioactive CaM. Free Ca2+ concentration was <0.01
µM (circle) or 50 µM
(triangle). Aliquots were then diluted 20-fold into media
containing 2.5 nM [35S]CaM and 0 (open
symbols) or 5 nM (closed symbols) unlabeled
CaM. Final CaM concentration was 7.5 nM, and final free
Ca2+ concentration was 50 µM. The
representative traces of three similar experiments are shown.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 6.
Diagram of calmodulin binding sites.
Sequence domains suggested by previous work and this study are shown.
Potential CaCaM binding domains are filled boxes, whereas
domains binding both apoCaM and CaCaM are light gray with a
black border. Arrows indicate amino acids that
were identified in this study to constitute part of the CaCaM (Trp
3620) and apoCaM and CaCaM (Leu 3624) binding sites.
Studies with fusion proteins show that the fragmentation of the 565 kDa
of RyR peptide into smaller pieces unmasks CaM binding sites not
detected in the large channel complex. It is therefore necessary that
full-length RyR1 mutants lacking putative CaM binding sites are
constructed and that the functional consequences of these mutations are
examined. Deletion of one of the potential CaM binding sites identified
in the [35S]CaM overlays, RyR14274-4535, was without
effect on high affinity CaM binding and the inhibition and activation
of [3H]ryanodine binding by CaCaM and apoCaM,
respectively (Figs. 3 and 4). In this study, we therefore focused on
amino acid residues covered by FPI-4 (aa 3553-3662), which contained a
CaM binding site implicated in all previous studies (Fig. 6). Three
amino acid substitutions (V3619A,W3620A,L3624D) in FPI-4, leading
to a reduction or nearly a complete loss of [35S]CaCaM
binding, were introduced in the full-length RyR1. One of the mutants
(L3624D) showed a loss of both high affinity apoCaM and CaCaM binding,
whereas a second mutant (W3620A) showed a specific loss of CaCaM
binding as it maintained the ability to bind [35S]CaM
(nanomolar) in the absence of Ca2+. These results suggest
that Leu 3624 is critical for conferring both apoCaM and CaCaM binding,
whereas Trp 3620 is critical only for CaCaM. The physiological
relevance of these findings was supported by [35S]CaM
dissociation and chase experiments, which indicated that the native
RyR1 has a site that interacts with both apoCaM and CaCaM. Using
cryoelectron microscopy and three-dimensional reconstruction, Samso
et al. (27) showed that apoCaM and CaCaM bind to two near but distinct cytoplasmic locations on each of the four subunits of the
RyR1. This observation suggests that apoCaM and/or CaCaM binding induce
major RyR1 protein conformational changes given that it is unlikely
that a shift of CaM by several amino acids can be detected at the
resolution achievable by electron microscopy.
Our data are in good agreement with a recent report by Moore et al. (13) who suggested that the region of the RyR1 identified in this study binds both apoCaM and CaCaM as both CaM forms were capable of protecting RyR1 from trypsin cleavage at arginines 3630 and 3637. Furthermore, these investigators showed that a synthetic peptide (aa 3614-3643), which included the two trypsin cleavage sites, bound both apoCaM and CaCaM (18). A shorter peptide (aa 3614-3635) bound CaCaM but showed a loss of apoCaM binding, whereas another peptide including neither Trp 3620 nor Leu 3624 (aa 3625-3644) bound apoCaM and with a reduced affinity CaCaM (18). Therefore, the results obtained with synthetic peptides (18) and the intact RyR1 in this study do not agree entirely.
The functional consequences of our mutations were assessed by
determining their Ca2+ dependence and
[3H]ryanodine binding properties. The RyR1 mutants bound
[3H]ryanodine with an affinity and showed a
Ca2+ dependence comparable with wt-RyR1 with the exception
of RyR14274-4535, which showed an ~10-fold increased sensitivity
to activating Ca2+, as previously reported (22). Therefore,
the mutations did not introduce major global conformational changes,
but rather they appeared to be mostly limited to the CaM binding sites.
The functional studies also allowed tests of the effects of micromolar concentrations of CaM as opposed to the binding studies that are limited to nanomolar CaM concentrations due to experimental restraints. [3H]Ryanodine binding to W3620A was not inhibited by 3 µM CaCaM, which suggests a complete loss or at least a
very large reduction of CaCaM binding affinity. L3624D and V3619A were
inhibited by CaM with IC50 ~3 µM and ~200
nM, respectively, as compared with IC50 ~50
nM for wt in agreement with the binding studies, which showed nearly a complete loss of CaCaM binding for L3624D but not for V3619A.
In addition to regulating the Ca2+ release channel, CaM
probably also influences Ca2+ release through other
proteins that interact with the release channel. Potential targets of
CaM regulation are the transverse tubule Ca2+ channel,
which via a direct interaction controls the SR Ca2+
release channel, calmodulin-dependent protein kinase,
and calmodulin-stimulated protein phosphatase (calcineurin) (1-3, 28,
29). Our work provides information for future studies of distinguishing
CaM regulation of the RyR1 from that of other proteins. We show that two single amino acid substitutions distinctly change the regulation of
the skeletal muscle Ca2+ release channel by CaM; one of
which (L3624D) results in a loss of activation by apoCaM and an
inhibition by CaCaM, whereas the other (W3620A) specifically abolishes
CaCaM inhibition.
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ACKNOWLEDGEMENTS |
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We thank David Balshaw for suggestions and John Johnson and Daniel A. Pasek for technical assistance.
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FOOTNOTES |
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* The work was supported by National Institutes of Health Grant AR18687.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.
§ Postdoctoral Fellow of Japan Society for the Promotion of the Science.
To whom correspondence should be addressed. Tel.: 919-966-5021;
Fax: 919-966-2852; E-mail: meissner@med.unc.edu.
Published, JBC Papers in Press, April 16, 2001, DOI 10.1074/jbc.M102729200
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ABBREVIATIONS |
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The abbreviations used are: RyR, ryanodine receptor; RyR1, skeletal muscle RyR; CaM, calmodulin; apoCaM, Ca2+-free CaM; CaCaM, Ca2+-bound CaM; HEK, human embryonic kidney; GST, glutathione S-transferase; SR, sarcoplasmic reticulum; KPipes, potassium 1,4-piperazinediethanesulfonic acid; BSA, bovine serum albumin; wt, wild type.
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