Calmodulin Oxidation and Methionine to Glutamine Substitutions
Reveal Methionine Residues Critical for Functional Interaction with
Ryanodine Receptor-1*
Edward M.
Balog
§,
Laura E.
Norton
,
Rachel A.
Bloomquist
,
Razvan L.
Cornea
,
D. J.
Black¶,
Charles F.
Louis
,
David D.
Thomas
, and
Bradley R.
Fruen
From the From
Department of Biochemistry, Molecular
Biology, and Biophysics, University of Minnesota, Minneapolis,
Minnesota 55455, the ¶ Department of Molecular and Cellular
Biochemistry, The Ohio State University, Columbus, Ohio 43210, and the
Department of Biology, Georgia State University,
Atlanta, Georgia 30303
Received for publication, September 6, 2002, and in revised form, January 30, 2003
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ABSTRACT |
Calmodulin (CaM) binds to the skeletal
muscle ryanodine receptor Ca2+ release channel (RyR1)
with high affinity, and it may act as a Ca2+-sensing
subunit of the channel. Apo-CaM increases RyR1 channel activity, but
Ca2+-CaM is inhibitory. Here we examine the functional
effects of CaM oxidation on RyR1 regulation by both apo-CaM and
Ca2+-CaM, as assessed via determinations of
[3H]ryanodine and [35S]CaM binding to
skeletal muscle sarcoplasmic reticulum vesicles. Oxidation of all nine
CaM Met residues abolished functional interactions of CaM with RyR1.
Incomplete CaM oxidation, affecting 5-8 Met residues, increased the
CaM concentration required to modulate RyR1, having a greater effect on
the apo-CaM species. Mutating individual CaM Met residues to Gln
demonstrated that Met-109 was required for apo-CaM activation of RyR1
but not for Ca2+-CaM inhibition of the channel.
Furthermore, substitution of Gln for Met-124 increased the apo-
and Ca2+-CaM concentrations required to regulate RyR1.
These results thus identify Met residues critical for the productive
association of CaM with RyR1 channels and suggest that oxidation of CaM
may contribute to altered regulation of sarcoplasmic reticulum
Ca2+ release during oxidative stress.
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INTRODUCTION |
Skeletal muscle contraction is initiated by Ca2+
efflux from the sarcoplasmic reticulum
(SR)1 via the SR
Ca2+ release channel/ryanodine receptor (RyR1). The
homotetrameric RyR1 is the largest known ion channel having a molecular
mass of more than 2,000 kDa (1). The N-terminal two-thirds of the channel forms a large cytoplasmic domain to which numerous signaling proteins are anchored, including the FK506 binding protein and calmodulin (CaM) (2, 3). In vitro, the channel is activated by Ca2+ in the nM to µM range and
inactivated by µM-mM Ca2+. CaM
binding to the channel enhances the sensitivity to both Ca2+ activation and inactivation.
The 148-amino acid Ca2+-binding protein, CaM, is composed
of N-terminal and C-terminal globular domains connected by a flexible, central tether. CaM has an unusually high Met content, indeed 9 of the
148 amino acids are Met residues, resulting in an ~6-fold higher Met
content than the average protein (4). In vertebrate CaM these Met
residues are clustered primarily in the N (residues 36, 51, 71, and 72)
and the C (residues 109, 124, 144, and 145) termini. A 9th Met is
located in the tether at residue 76. High affinity Ca2+
binding to EF-hand motifs in each of the globular domains induces a
structural rearrangement that reveals the Met-rich hydrophobic patches
(5). These hydrophobic patches mediate Ca2+-CaM interaction
with a large and diverse group of proteins that share little sequence
homology (6). A less appreciated aspect of CaM regulation is the
ability of Ca2+-free CaM (apo-CaM) to regulate certain
targets (7). However, the role of Met residues in apo-CaM interaction
with targets is not clear.
Although the high Met content of CaM contributes to effective target
binding, the Met residues in the Ca2+-bound form of CaM are
surface-exposed and susceptible to oxidation. Oxidation converts Met to
Met sulfoxide, a physiologically relevant product (8). Indeed, Met
sulfoxide-containing CaM has been isolated from the brains of aged
animals (9).
The present study examines the functional effects of CaM oxidation on
the regulation of RyR1. To identify which of the individual Met
residues are important for the functional interaction of CaM with RyR1,
we used site-directed mutagenesis to change each of the nine CaM Met
residues to Gln, introducing an oxygen atom at the same position in the
side chain as the sulfoxide. Our results define CaM Met residues that
are critical for the functional interaction between CaM and RyR1 and
suggest that CaM oxidation may contribute to altered regulation of SR
Ca2+ release during oxidative stress.
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EXPERIMENTAL PROCEDURES |
Materials
Pigs were obtained from the University of Minnesota Experimental
Farm. Tran35S-labeled Met and Cys were obtained from ICN
Radiochemicals (Costa Mesa, CA). [3H]Ryanodine was
purchased from PerkinElmer Life Sciences. Unlabeled ryanodine was obtained from Calbiochem. High performance liquid chromatography grade acetonitrile was purchased from Fisher Scientific. C4 ZipTips were from Millipore (Burlington, MA). RPMI 1640 medium was
from ICN. Spectrophotometric grade trifluoroacetic acid, AMPPCP, and
other reagents were from Sigma.
[3H]Ryanodine Binding to Skeletal Muscle Heavy
Sarcoplasmic Reticulum
Isolation of SR Vesicles--
Skeletal muscle SR vesicles were
prepared from porcine longissimus dorsi muscle (10). Muscle was
homogenized in 0.1 M NaCl, 5 mM Tris maleate
buffer, pH 6.8, and centrifuged for 30 min at 2,600 × g. The supernatant was filtered through gauze and
centrifuged for 30 min at 15,000 × g. Pelleted
membranes were extracted in 1.1 M KCl, 5 mM
Tris, pH 6.8, centrifuged at 130,000 × g for 45 min,
and then resuspended in 0.3 M sucrose, 0.4 M
KCl, 5 mM Tris, pH 6.8, buffer. SR was then centrifuged
through a discontinuous sucrose gradient (22, 36, and 45% sucrose) at
130,000 × g for 5 h, and the heavy SR fraction
was collected from the 36 and 45% sucrose interface. Heavy SR vesicles
were resuspended in 0.3 M sucrose, 0.1 M KCl, 5 mM Tris, pH 6.8, flash frozen in liquid nitrogen, and
stored at
70 °C. All buffers contained a protease inhibitor
mixture (100 nM aprotinin, 1 µM leupeptin, 1 µM pepstatin, 1 mM benzamidine, and 0.2 mM phenylmethylsulfonyl fluoride).
[3H]Ryanodine Binding--
Ryanodine selectively
binds to the open RyR and therefore provides a useful indicator of
channel activity (11). SR vesicles (0.2 mg/ml) were incubated at
37 °C in medium containing 120 mM potassium propionate,
10 mM PIPES, pH 7.0, 3 mM AMPPCP, 100 nM [3H]ryanodine, and a Ca-EGTA buffer set to
give the desired free Ca2+ concentration (12). After 90 min, SR vesicles were collected on Whatman GF/B filters and washed with
8 ml of ice-cold 100 mM KCl buffer. Estimates of maximal
[3H]ryanodine binding capacity of each SR vesicle
preparation were determined in medium that in addition contained 500 mM KCl, 6 mM ATP, and 100 µM
Ca2+. Nonspecific binding was measured in the presence of
20 µM nonradioactive ryanodine.
[3H]Ryanodine binding is expressed as a percent of
maximal [3H]ryanodine binding.
[35S]Calmodulin Binding to Skeletal Muscle SR
Vesicles
RyR1 is the major CaM-binding protein in heavy SR (13,
14), thus SR vesicle [35S]CaM binding reflects primarily
RyR1 CaM binding. [35S]CaM binding to SR vesicles was
performed as described by Balshaw et al. (15). SR vesicles
were incubated in 150 mM KCl, 20 mM PIPES, pH
7.0, 5 mM GSH, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 50 nM [35S]CaM, and 0-5000 nM
unlabeled CaM for 2 h at 24 °C. Pellets were collected after
centrifugation at 40,000 rpm for 20 min in a Beckman TLA-55 rotor at
20 °C, solubilized by overnight incubation in 10% SDS, and
resuspended in 200 µl of double distilled H2O. Bound [35S]CaM was determined by scintillation counting.
Nonspecific binding was determined using 100-fold excess unlabeled CaM.
[35S]CaM binding is expressed as
B/B0, where B is the
[35S]CaM bound in the presence of unlabeled CaM, and
B0 is the [35S]CaM bound in the
absence of unlabeled CaM.
Oxidation of Calmodulin
Because the thioether group of Met is not protonated at low pH,
it can be oxidized selectively under acidic conditions (8). 60 µM calmodulin was incubated in 50 mM
Homopipes, pH 5.0, 0.1 M KCl, 2.0 mM
MgCl2, 50 mM H2O2 at
room temperature for 0.5-24 h. The reaction was stopped by overnight
dialysis (molecular mass cutoff = 3,500) at 4 °C in distilled
water (5 × 1 liter) buffered with 10 mM ammonium
bicarbonate, pH 7.7.
Calmodulin Site-directed Mutagenesis, Expression, and
Purification
Recombinant rat CaM was expressed in Escherichia coli
using the pET-7 vector (16), purified via phenyl-Sepharose
chromatography (17), and dialyzed overnight at 4 °C against 2 mM HEPES, pH 7.0. CaM concentration was determined with the
Micro BCA assay (Pierce) using wild-type CaM as a standard. The
concentration of the CaM standard was determined using the published
molar extinction coefficient,
277-320 nm = 3,029 M
1 cm
1 (18). Glutamine was
substituted for each of the Met residues (residues 36, 51, 71, 72, 76, 109, 124, 144, and 145) using QuikChange mutagenesis kits (Stratagene,
La Jolla, CA). DNA sequence analysis confirmed the correct generation
of each mutant.
[35S]Methionine Incorporation
The biosynthesis of 35S-labeled CaM was carried out
essentially as described previously for [35S]FKBP12 (19).
Briefly, bacterial growth was initiated in M9 medium containing
ampicillin. When the A600 value of the bacteria reached 0.6 the pelleted bacteria were resuspended in RPMI 1640 medium
containing ampicillin, 1/40th of the Met and Cys concentration compared
with the regular RPMI 1640 medium, and
isopropy-D-thiogalactopyranoside was added to a final
concentration of 1 mM. A 1.4-mCi aliquot of
[35S]methionine and [35S]cysteine was then
added to the medium and the bacteria cultured for 5-7 h at the same conditions.
Matrix-assisted Laser
Desorption/Ionization-Time-of-Flight (MALDI-TOF) Mass
Spectrometry
MALDI-TOF mass spectrometry was performed at the University of
Minnesota Mass Spectrometry Consortium for the Life Sciences using a
Bruker Biflex III mass spectrometer (Bruker, Boston, MA) equipped with
a N2 laser (337 nm, 3-ns pulse length) and a microchannel plate detector. Data were collected in the linear mode, positive polarity, with an accelerating potential of 19 kV. Each spectrum was
the accumulation of ~200 laser shots. External calibration was
performed using horse heart cytochrome c and horse skeletal muscle myoglobin. The matrix used for samples and standards was a
saturated solution of 3,5-dimethoxy-4-hydroxycinnamic acid in 50:50,
acetonitrile:nanopure water, 0.1% trifluoroacetic acid. Prior to
MALDI-TOF analysis samples were desalted using Millipore's C4 ZipTips
according to the manufacturer's protocol.
Steady-state Fluorescence
The Ca2+-induced increase in tyrosine fluorescence
intensity is thought to reflect a reduced quenching in
Ca2+-bound CaM and has been used to monitor
Ca2+ binding to the C terminus of CaM (20, 21). Spectra
were collected at 25 °C using an ISS K2 fluorometer in ratio mode.
The 3 µM CaM samples were excited at 275 nm using a xenon
lamp, and corrected emission spectra were acquired from 280 to 400 nm
in 1-nm increments. Excitation and emission bandwidths were 8 nm.
Ca2+ titrations were performed by the addition of small
aliquots of concentrated CaCl2 to the sample in the apo
buffer (120 mM KCl, 20 mM PIPES, 1.0 mM EGTA, pH 7.0). A matching buffer scan was subtracted
from each spectrum. The fluorescence readings, at 305 nm, for each
titration were normalized to the high and low end points before
nonlinear least squares analysis.
Circular Dichroism (CD)
CD spectra were recorded from 250 to 200 nM with a
JASCO J-710 spectrophotometer coupled with a data processor. Spectra
were recorded digitally and fed through the data processor for signal averaging and base line subtraction. Spectra were recorded at 25 °C
with a CaM concentration of 150 µM in a solution of 2 mM HEPES, pH 7.0, and either 500 µM
Na2 EGTA or CaCl2 using quartz cuvettes with a
path length of 1.0 mm. Spectra were recorded with a scan speed of 20 nm/min, signal-averaged six times, and an equally signal-averaged
solvent base line was subtracted.
PAGE
CaMs were analyzed under denaturing conditions using SDS-PAGE
(22). Samples were incubated for 30 min in sample buffer containing either 5 mM CaCl2 or 5 mM EGTA
before loading onto 15% gel. No Ca2+ or EGTA was added to
the gel or running buffer.
Analysis
The CaM concentration dependence of SR vesicle
[3H]ryanodine binding and the inhibition of
[35S]CaM binding by unlabeled CaM were fit with the Hill
equation. The Ca2+ dependence of ryanodine binding was fit
with Equation 1, which assumes a high affinity Ca2+ binding
site, which when bound will activate the RyR and a lower affinity
Ca2+ binding site which when bound will inhibit channel
opening,
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(Eq. 1)
|
where B is the ryanodine bound,
Bmax is the maximal ryanodine binding,
EC50 and IC50 are the half-activating and
half-inhibiting Ca2+ concentrations, respectively, and
na and ni are the Hill
coefficients for activation and inhibition, respectively.
Statistics
Data are presented as the means ± S.E.
[3H]Ryanodine and [3S]CaM binding curves in
the presence and absence of CaM and CaM mutants were studied using a
one-way analysis of variance with Dunnett's multiple comparison as a
post hoc test or by Student's paired and unpaired
t tests as appropriate. The level of significance was
<0.05.
 |
RESULTS |
Effects of CaM Oxidation on Regulation of RyR1--
As shown
previously (14), apo-CaM activated and Ca2+-CaM inhibited
RyR1. Thus, in a medium containing 100 nM
Ca2+, CaM enhanced skeletal muscle SR vesicle
[3H]ryanodine binding (EC50 = 54 ± 4 nM, nH = 1.3 ± 0.2), and
in a medium containing 700 µM Ca2+, CaM
decreased SR vesicle ryanodine binding (IC50 = 46 ± 15 nM, nH = 1.4 ± 0.1). The
oxidation of CaM by incubation in 50 mM
H2O2 for 24 h abolished apo- and
Ca2+-CaM regulation of SR vesicle ryanodine binding (Fig.
1, A and B). In
addition, after incubation with 50 mM
H2O2 for 24 h CaM was no longer able to
inhibit [35S]CaM binding to SR vesicles in a medium
containing either 100 nM or 700 µM
Ca2+ (Fig. 1, C and D). Thus, the
inability of oxidized CaM to modulate SR vesicle ryanodine binding was
caused by the loss of CaM binding to SR vesicles. By comparison,
partial oxidation of CaM by incubation in 50 mM
H2O2 for 30 min did not fully abolish CaM
modulation of ryanodine binding; however, both the half-activating
(EC50 > 1000 nM) and half-inhibiting
(IC50 = 104 ± 8, nH = 1.3 ± 0.2) CaM concentrations were increased (Fig. 1, A and
B), with a larger effect occurring at 100 nM
Ca2+. These results suggest that the oxidation of critical
CaM Met residues alters the productive association of CaM with the
RyR1.

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Fig. 1.
Effects of CaM oxidation on CaM concentration
dependence of [3H]ryanodine and [35S]CaM
binding to skeletal muscle SR vesicles. Ryanodine (A
and B) and CaM (C and D) binding was
carried out as described under "Experimental Procedures" in the
presence of 100 nM Ca2+ (A and
C) or 700 µM Ca2+ (B
and D) and either native CaM or CaM that had been incubated
in 50 mM H2O2 for 30 min or 24 h. [3H]Ryanodine binding is expressed as a percent of
maximal [3H]ryanodine binding. [35S]CaM
binding is expressed as B/B0, where
B is the [35S]CaM bound in the presence of
unlabeled CaM, and B0 is the
[35S]CaM bound in the absence of unlabeled CaM. The
maximal [3H]ryanodine binding capacity of the SR vesicles
used in this study was 7.3 ± 0.5 pmol/mg, the maximal
apo-[35S]CaM binding capacity was 32.4 ± 1.6 pmol/mg, and the maximal Ca2+-[35S]CaM
binding capacity was 45.7 ± 2.7 pmol/mg. Curves fit to the native
CaM and CaM incubated with 50 mM
H2O2 for 30 min were derived from the Hill
equation.
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Mass Spectrometry of Oxidatively Modified CaM--
MALDI-TOF mass
spectrometry was performed to determine the extent of oxidative
modification of CaM incubated with 50 mM
H2O2 for either 30 min or 24 h. As shown
in Fig. 2, the mass spectrum of native
CaM displayed a single peak corresponding to a mass of 16,711 Da, which
corresponds, within the measurement error, to the theoretical mass of
unmodified CaM (16706.39 Da). Thus, the native CaM used in these
experiments consisted of a single population of unmodified CaM. In
contrast, the mass spectrum of CaM after incubation in 50 mM H2O2 for 30 min displayed four
peaks corresponding to masses of 16,783, 16,798, 16,815, and 16,830 Da.
Oxidation of Met to Met sulfoxide increased the mass by 16 Da.
Consequently, this partially oxidized CaM was composed of multiple
populations of CaM oxiforms with 5, 6, 7, or 8 Met residues oxidized to
their corresponding sulfoxide. CaM incubated in 50 mM
H2O2 for 24 h displayed a single peak
corresponding to a mass of 16,861 Da. Therefore, incubation of CaM in
50 mM H2O2 for 24 h produced a
single population of CaM with all 9 Met residues oxidized to their
corresponding Met sulfoxides.

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Fig. 2.
Mass spectra of native and oxidized CaM.
The mass peak (16,711 Da) of native CaM (A) corresponds,
within the experimental error, to the theoretical CaM mass (16,706.39).
The mass peaks (16,783, 16,798, 16,815, 16,830 Da) of CaM incubated in
50 mM H2O2 for 30 min
(B) correspond to the theoretical CaM mass with 5-8 of the
Met residues oxidized to Met sulfoxide. The mass peak (16,861 Da) of
CaM incubated in 50 mM H2O2 for
24 h (C) corresponds to the theoretical CaM mass with
all 9 Met residues oxidized to Met sulfoxide.
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|
Regulation of RyR1 by Met
Gln CaM Mutants--
To determine
the role of specific CaM Met residues in regulating RyR1, we used
site-directed mutagenesis to change each CaM Met to Gln. This
substitution introduced an oxygen atom at the same position in the side
chain as the sulfoxide. The greater polarity of the Gln side chain
relative to Met was expected to decrease the hydrophobic interactions
that normally stabilize the association of CaM with its target.
However, substituting Gln for Met is unlikely to significantly disturb
the structure of CaM because both amino acids have a similar propensity
to form
-helices (23).
Fig. 3 and Tables I and II summarize the
effects of CaM Met
Gln mutants
on the CaM concentration dependence of
skeletal muscle SR vesicle
[3H]ryanodine binding in medium containing 100 nM Ca2+ or 700 µM
Ca2+. In a medium containing 100 nM
Ca2+, all of the N-terminal Met
Gln mutants and M76Q,
in the tether, enhanced SR vesicle [3H]ryanodine binding.
However, the maximal activation by the M71Q mutant exceeded wild-type
CaM activation by ~50%. Although the M72Q CaM mutant enhanced
ryanodine binding to an extent similar to that of wild-type CaM, the
EC50 was ~3-fold greater than wild-type. In contrast, the
extent and concentration dependence of Ca2+-CaM inhibition
by all the N-terminal Met
Gln mutants and M76Q were similar to
wild-type CaM.

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Fig. 3.
Effects of the CaM Met Gln mutant on the CaM concentration dependence of skeletal muscle
SR vesicle [3H]ryanodine binding. Ryanodine binding
was performed as described under "Experimental Procedures" in
medium containing either 100 nM Ca2+
(A and B) or 700 µM
Ca2+ (C and D).
[3H]Ryanodine binding is expressed as a percent of
maximal [3H]ryanodine binding. Solid lines,
except in the case of M109Q in B, represent fits to the Hill
equation.
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With the exception of the M109Q CaM mutant, in 100 nM
Ca2+, all of the C-terminal Met
Gln mutants enhanced
ryanodine binding to an extent similar to that of wild-type CaM.
Substitution of Gln for Met at position 109 completely abolished
apo-CaM activation of RyR1. Replacing Met-124 with Gln increased the
apo-CaM EC50 nearly 23-fold compared with wild-type CaM. In
a medium containing 700 µM Ca2+, all of the
C-terminal Met
Gln mutants, including surprisingly M109Q, inhibited
ryanodine binding to an extent similar to that of wild-type CaM.
Similar to the effect in 100 nM Ca2+, the M124Q
mutation increased the IC50 for Ca2+-CaM
inhibition of ryanodine binding. However, the 5-fold increase in the
Ca2+-CaM IC50 was much smaller than the 23-fold
increase in the apo-CaM EC50 caused by this mutation.
The Ca2+ dependence of SR vesicle
[3H]ryanodine binding was examined in the absence and
presence of wild-type CaM and selected Met
Gln CaM mutants in
medium containing 3 mM AMPPCP and 3 mM MgCl2 (Fig. 4A and
Table III). Similar to previous reports
(24, 25), wild-type CaM significantly decreased both the
Ca2+ EC50 and IC50. Similarly, the
M71Q and M124Q CaM also lowered the Ca2+ EC50
and IC50. In contrast, in the presence of the M109Q CaM mutant, the Ca2+ EC50 was significantly lower
than in the absence of CaM, but the Ca2+ IC50
was not significantly different. However, in the presence of M109Q CaM,
the extent of channel activation was less than half that in the absence
of CaM, and the shift in the EC50 was caused by the lower
extent of activation rather than an enhancement of channel opening at
low Ca2+. For example, at 0.1 µM
Ca2+, wild-type CaM and both the M71Q and M124Q CaM mutants
significantly increased SR vesicle ryanodine binding compared with the
absence of CaM. In contrast, ryanodine binding in the presence of the M109Q CaM was not significantly different from the binding in the
absence of CaM.

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Fig. 4.
Ca2+ dependence of skeletal
muscle SR vesicle [3H]ryanodine binding in the absence of
CaM and in the presence of 5 µM
wild-type or Met Gln CaM mutants.
Ryanodine binding was performed as described under "Experimental
Procedures" in medium containing no CaM, 5 µM wild-type
CaM, 5 µM M71Q CaM, 5 µM M109Q CaM, or 5 µM M124Q CaM and either 3 mM AMPPCP and 3 mM MgCl2 (A) or 3 mM
AMPPCP and no MgCl2 (B).
[3H]Ryanodine binding is expressed as a percent of
maximal [3H]ryanodine binding. The solid lines
are the fits to Equation 1.
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Table III
Ca2+ dependence of skeletal muscle SR vesicle
[3H]ryanodine binding in media containing no CaM or 1 µM CaM
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To resolve better the effects of the Met
Gln substitutions on
apo-CaM function, channel activation was enhanced by performing binding
experiments in medium containing 3 mM AMPPCP but no
MgCl2 (Fig. 4B and Table III). Under these
conditions, wild-type, M71Q and M124Q CaM clearly enhanced the
Ca2+ sensitivity of channel activation while the M109Q CaM
did not.
Inhibition of [35S]CaM Binding to RyR1 by Met
Gln
CaM Mutants--
To determine whether the Met
Gln mutations
affected CaM regulation of RyR1 via changes in the affinity of CaM
binding to the RyR or via alterations in CaM regulatory efficacy, we
compared inhibition of SR vesicle [35S]CaM binding by
wild-type and Met
Gln CaM mutants (Fig.
5 and Table
IV). Compared with wild-type CaM, greater
concentrations of the M124Q CaM mutant were required to inhibit SR
vesicle [35S]CaM binding in medium containing either 100 nM or 700 µM Ca2+. Thus, the
effects of the M124Q mutation on CaM affinity for RyR1 were similar to
the effects of the mutation on SR vesicle [3H]ryanodine
binding.

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Fig. 5.
Inhibition of skeletal muscle SR vesicle
[35S]CaM binding by wild-type CaM and Met
Gln CaM mutants. [35S]CaM
binding was performed as described under "Experimental Procedures"
in medium containing 1 µM wild-type
[35S]CaM, either 100 nM Ca2+
(A) or 700 µM Ca2+ (B),
and the indicated concentrations of unlabeled wild-type CaM, M71Q CaM,
M72Q CaM, M76Q CaM, M109Q CaM, or M124Q CaM. Data are expressed as
B/B0, where B is the
amount of [35S]CaM bound in the presence of unlabeled
CaM, and B0 is the amount of
[35S]CaM bound in the absence of unlabeled CaM.
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Table IV
Inhibition of [35S]CaM binding to skeletal muscle SR vesicles
by wild-type and met Gen CaM mutants in media containing either
100 nM or 700 µM Ca2+
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At high Ca2+ concentrations, the M109Q CaM mutant inhibited
SR vesicle [35S]CaM binding in a manner similar to
wild-type CaM. However, in contrast to the inability of the M109Q
apo-CaM to enhance SR vesicle ryanodine binding, this mutant inhibited
[35S]CaM binding to SR vesicles. This suggests that at
low Ca2+, the M109Q apo-CaM mutant does bind to the RyR1,
but that binding does not affect channel opening.
SDS-PAGE, CD, and Intrinsic Tyrosine Fluorescence--
When CaM is
denatured for SDS-PAGE in the presence of Ca2+, the
mobility of the protein on SDS-PAGE is increased relative to that seen
after denaturation in the presence of EGTA (26). Although the
mechanisms underlying the mobility shift are not understood, it is
thought to reflect some difference in the ability of SDS to bind and/or
denature apo- and Ca2+-CaM (27). Thus, mutation-induced
structural changes in the Met
Gln mutant CaMs might be reflected in
altered mobility on SDS-PAGE. As can be seen from Fig.
6, none of the Met
Gln mutations altered the mobility of CaM in the presence of EGTA. Furthermore, none
of the mutations prevented the mobility shift upon the addition of
Ca2+.

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Fig. 6.
Electrophoretic mobility of wild-type and
Met Gln mutant CaM. Wild-type,
N-terminal Met Gln mutant (A), and C-terminal Met Gln mutant (B) CaM were run on SDS-PAGE as described under
"Experimental Procedures" after incubation in sample buffer
containing either 5 mM Ca2+ (+) or 5 mM EGTA ( ).
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To assess further the potential structural alterations induced by the
M109Q and M124Q CaM mutations, the CD spectra arising from these two
mutants were compared with the spectra of wild-type CaM. The far UV
spectra of wild-type CaM and the M109Q and M124Q mutant CaMs were not
significantly different, either in the presence of 500 µM
EGTA (Fig. 7A) or in the
presence of 500 µM Ca2+ (Fig. 7B).
Thus the Met
Gln mutations did not cause major changes in the
secondary structure of CaM.

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Fig. 7.
CD spectra of wild-type (solid
line), M109Q (dotted line), and M124Q
(broken line) CaM. Spectra were acquired as
described under "Experimental Procedures" in solutions containing
either 500 µM EGTA (A) or 500 µM
CaCl2 (B).
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To determine whether the M109Q or M124Q mutation altered the CaM
Ca2+ affinity we determined the Ca2+ dependence
of the change in intrinsic tyrosine fluorescence (20, 21). The apparent
Ca2+ affinities of wild-type CaM, M109Q CaM, and M124Q CaM
did not significantly differ (KCa: wild-type
CaM, 2.5 ± 0.3 µM; M109Q CaM, 1.5 ± 0.4 µM; M124Q CaM, 2.0 ± 0.3 µM).
 |
DISCUSSION |
The unusually high CaM Met content (9 of 148 residues) is thought
to allow CaM to associate with and regulate a large number of
structurally diverse proteins. Here we defined the role of specific CaM
Met residues in the regulation of RyR1.
Oxidation of all 9 CaM Met residues to their corresponding sulfoxide
abolished both apo- and Ca2+-CaM binding and modulation of
RyR1. In comparison, incomplete oxidation, i.e. oxidation of
5-8 CaM Met residues, did not alter the extent of either apo-CaM
activation or Ca2+-CaM inhibition, but rather increased the
CaM concentration required for these effects. Thus it appears that the
presence of 4 unoxidized Met residues is sufficient for the full extent
of CaM regulation of RyR1 but not to provide CaM with the normal high
affinity for RyR1. It is not clear, however, whether the ability of the
incompletely oxidized CaM to regulate RyR1 fully was the result of the
preservation of specific, vital Met residues or a critical Met surface area.
Apo-CaM function appeared to be more sensitive to Met modification than
Ca2+-CaM function. Incomplete oxidation caused a larger
increase in apo-CaM EC50 than in Ca2+-CaM
IC50. The differential effect of oxidation on the function of apo- and Ca2+-CaM was also reflected in the differing
effects of some of the Met
Gln mutants. Thus, the M124Q mutation
increased the apo-CaM EC50 by more than 20-fold but
increased the Ca2+-CaM IC50 by only 5-fold.
Even more dramatic were the effects of the M109Q mutant on apo-CaM
versus Ca2+-CaM function. This single Met
Gln substitution completely abolished activation of RyR1 but did not
alter Ca2+-CaM inhibition of the channel.
Met-109 and Met-124 were necessary for the high affinity interaction of
CaM with RyR1. Yuan et al. (28) found that in solution, the
N-terminal Met residues in apo-CaM are largely buried, whereas the
C-terminal Met residues are more exposed. They suggest that these
C-terminal Met residues may play a role in the binding of apo-CaM to
targets. Thus, in apo-CaM, Met-109 and Met-124 may be available to
interact with RyR1. Consequently, a mutation in either of these
residues significantly affects the functional interaction of apo-CaM
with RyR1. Upon Ca2+ binding to CaM, there is an increased
Met exposure (5). Therefore, replacement of an individual Met is less
deleterious to Ca2+-CaM than to apo-CaM function.
The M109Q mutation could potentially abolish apo-CaM activation of RyR1
via three mechanisms. The mutation could disrupt the structure of CaM
to such an extent that the mutant apo-CaM could not functionally
interact with the channel. In agreement with Chin and Means (29), the
Ca2+-induced mobility shifts on an SDS gel by M109Q CaM and
wild-type CaM were similar. In addition the mutant could be purified
via phenyl-Sepharose chromatography. Thus M109Q CaM preserved
sufficient hydrophobic surface to be retained by the phenyl-Sepharose
column and underwent a Ca2+-induced structural
rearrangement similar to wild-type CaM. Finally, the CD spectra of the
M109Q and M124Q CaM mutants were indistinguishable from wild-type CaM
in the absence of Ca2+ and, also in agreement with Chin and
Means (29), in the presence of Ca2+. Thus we were unable to
detect any substantial structural modification in these mutants.
Alternatively, the M109Q mutation could increase the Ca2+
affinity of the CaM such that a substantial fraction of the mutant CaM
would exist as inhibitory Ca2+-CaM in medium containing 100 nM Ca2+. However, the Ca2+
dependence of the change in intrinsic fluorescence did not differ between wild-type CaM and either M109Q or M124Q CaM.
Finally, a Met residue might be required in position 109 to make
specific interactions with RyR1. All of the Met
Gln mutants interacted, although with varying affinity, with RyR1. The initial nonspecific association of CaM with targets is thought to be followed by more precise interactions between specific residues (30). In low
Ca2+, M109Q CaM associated with RyR1, albeit with a low
affinity, but did not activate the channel. Therefore, it is likely
apo-CaM activation of RyR1 requires a specific interaction between
Met-109 and the channel.
Met
Gln substitutions have been used previously to define the Met
residues required for Ca2+-CaM activation of a number of
CaM-dependent protein kinases (29, 31) and the
plasma membrane Ca2+ pump (32). Although there was
variability in the Met residues required for normal enzyme regulation,
substitution of Gln for Met-124 decreased the affinity of CaM for all
of these targets. The M124Q mutation also decreased the maximal
CaM-dependent kinase activation but not CaM activation of
the Ca2+ pump. Whereas apo- and Ca2+-bound
M124Q CaM fully regulated RyR1, the substitution decreased the affinity
of CaM for the channel. Thus, Met-124 appears to be an important
determinant of the CaM affinity for all of these targets; however, its
importance in determining CaM regulatory efficacy is
target-dependent.
CaM is functionally bifurcated (29, 33), thus the N and C termini of
CaM may serve different roles in both apo- and Ca2+-CaM
regulation of RyR1. The effects of Met
Gln mutations clearly demonstrate the importance of the C-terminal lobe Met-109 and Met-124
in apo-CaM activation of RyR1. In addition, M124Q was the only
substitution that significantly altered the interaction of
Ca2+-CaM with RyR1. Rodney et al. (34) proposed
a model of RyR1 regulation by CaM in which Ca2+ binding to
the CaM C-terminal pair of EF-hands mediates the conversion of CaM from
an activator of RyR1 to an inhibitor. The requirement of Met-109 for
apo-CaM activation of RyR1 but not for Ca2+-CaM inhibition
of the channel suggests that a critical component of the
Ca2+-induced structural change converting CaM from an
activator to an inhibitor entails altering the interaction between CaM
Met-109 and RyR1.
In summary, oxidation of all 9 CaM Met residues abolished the
functional interaction between CaM and RyR1. Incomplete oxidation decreased CaM affinity for RyR1 but not the extent of channel regulation. Site-specific substitution of Met with Gln at residue 109 abolished apo-CaM activation of RyR1 without altering
Ca2+-CaM inhibition of the channel. Substitution of Met-124
with Gln decreased the affinity of both apo- and Ca2+-CaM
for RyR1. Thus these results identify Met residues critical for the
productive interaction of CaM for RyR1 and suggest that oxidation of
CaM may contribute to RyR1 dysfunction during oxidative stress.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Deb Ferrington for helpful
discussions and LeeAnn Higgins for assistance with the mass spectrometry.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant GM 31382 and by the American Heart Association, Northland
Affiliate.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: 6-155 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455. Tel.: 612-625-3292; Fax: 612-625-2163; E-mail: balog004@tc.umn.edu.
Published, JBC Papers in Press, February 13, 2003, DOI 10.1074/jbc.M209180200
 |
ABBREVIATIONS |
The abbreviations used are:
SR, sarcoplasmic
reticulum;
AMPPCP, adenosine 5'-(
,
-methylenetriphosphate);
apo-CaM, Ca2+-free CaM;
CaM, calmodulin;
HOMOPIPES, homopiperazine-N,N'-bis-2-(ethansulfonic acid);
MALDI-TOF, matrix-assisted laser desorption/ionization-time of
flight;
PIPES, 1,4-piperazinediethanesulfonic acid;
RyR, ryanodine
receptor.
 |
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