From the Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599-7260
Received for publication, November 29, 2000, and in revised form, March 26, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Metabolically 35S-labeled
calmodulin (CaM) was used to determine the CaM binding properties of
the cardiac ryanodine receptor (RyR2) and to identify potential channel
domains for CaM binding. In addition, regulation of RyR2 by CaM was
assessed in [3H]ryanodine binding and single-channel
measurements. Cardiac sarcoplasmic reticulum vesicles bound
approximately four CaM molecules per RyR2 tetramer in the
absence of Ca2+; in the presence of 100 µM
Ca2+, the vesicles bound 7.5 CaM molecules per tetramer.
Purified RyR2 bound approximately four [35S]CaM molecules
per RyR tetramer, both in the presence and absence of Ca2+.
At least four CaM binding domains were identified in
[35S]CaM overlays of fusion proteins spanning the
full-length RyR2. The affinity (but not the stoichiometry) of CaM
binding was altered by redox state as controlled by the presence of
either GSH or GSSG. Inhibition of RyR2 activity by CaM was influenced
by Ca2+ concentration, redox state, and other channel
modulators. Parallel experiments with the skeletal muscle isoform
showed major differences in the CaM binding properties and regulation
by CaM of the skeletal and cardiac ryanodine receptors.
The ryanodine receptors
(RyRs)1 are large, high
conductance Ca2+ release channels found in a specialized
subcompartment of the endoplasmic reticulum of many tissues (1). In
muscle cells, this subcompartment is referred to as the sarcoplasmic
reticulum (SR). There are three known mammalian RyR isoforms: RyR1,
which is the dominant isoform in skeletal muscle; RyR2, which is found in cardiac muscle; and RyR3, which is expressed in many tissues at low
levels but is mostly associated with diaphragm and brain. In both
skeletal and cardiac muscle, Ca2+ release through the RyR
in response to a signal received from the T-tubule membrane via the
dihydropyridine receptor is a crucial step in excitation-contraction
coupling (2). This event is highly regulated by small molecules such as
Ca2+, Mg2+ and adenine nucleotides (3, 4), and
through protein-protein interactions such as with triadin and
calmodulin (CaM) (5-8).
CaM is a small (16.7 kDa) cytosolic protein, the structure of which has
been determined by both x-ray crystallography and NMR (9, 10). The
protein resembles a dumbbell, with two globular heads linked by a
solvent-exposed CaM shows a biphasic regulation of RyR1, activating the channel at
submicromolar cytosolic Ca2+ while inhibiting the channel
at higher Ca2+ concentrations (7). RyR2, on the other hand,
does not show activation by apoCaM but is inhibited by CaCaM in a
manner similar to RyR1 (13, 14). Several studies have reported the
stoichiometry of CaM binding to the RyR1. Early studies using
125I-CaM (7) or fluorescently labeled (15) CaM indicated a
stoichiometry of 1 molecule of CaCaM and 4-6 molecules of apoCaM bound
per subunit. Binding site localization studies with fusion proteins and
synthetic peptides indicated three to six potential binding sites per
subunit with a variable Ca2+ dependence (16, 17). More
recent studies using metabolically 35S-labeled CaM
indicate a stoichiometry of one binding site per RyR1 subunit for both
apo- and CaCaM (8). A recent report suggests that cardiac SR vesicles
bind five CaCaM molecules but only 1 apoCaM molecule per RyR2 tetramer
(14).
This study presents a systematic analysis of the CaM binding properties
of RyR2 and compares them to RyR1 assayed under identical conditions.
Our data indicate that the purified RyR2 binds approximately one
[35S]CaM molecule per subunit in both 100 µM Ca2+ and 5 mM EGTA. In native
cardiac SR vesicles, [35S]CaM binds approximately two
sites per RyR2 subunit in the presence of Ca2+ and a single
site per subunit in the absence of Ca2+. Two possible
explanations for this discrepancy are that a second CaCaM-binding
protein in SR vesicles is lost on purification or that purification
induces a conformational change masking the second site. We have also
analyzed the effects of redox state on CaM binding to both RyR1 and
RyR2. RyR1 and RyR2 are sensitive to redox regulation, showing a
2-3-fold reduction in CaCaM affinity in the presence of GSSG, which is
accentuated in the absence of Ca2+ to a 4-9-fold reduction
in affinity. CaCaM inhibition of native RyR2, unlike that of RyR1,
is greatly diminished by the presence of two allosteric regulators
of the ryanodine receptor, caffeine and AMPPCP. Unlike RyR1,
apoCaM inhibited RyR2, as determined in [3H]ryanodine
binding and single-channel measurements.
SR Vesicle Preparations and RyR Purification--
Heavy SR
vesicles were isolated from rabbit hind limb and back muscle and canine
cardiac muscle as previously described (18). In selected experiments,
endogenous CaM was removed by incubating SR vesicles for 30 min at
24 °C with 1 µM myosin light chain kinase-derived calmodulin binding peptide (CaMBP) in the presence of 100 µM Ca2+ followed by centrifugation through a
layer of 15% sucrose to remove complexed CaM and CaMBP. Where
indicated, the centrifugation step was omitted. For purification, SR
vesicles were solubilized in CHAPS, purified by sucrose density
gradient centrifugation, and reconstituted into phosphatidylcholine
liposomes (19). The endogenous SR-associated concentration of CaM was
determined by the ability of CaM to stimulate phosphodiesterase
hydrolysis of cAMP as described previously (20).
[35S]Calmodulin Expression and
Purification--
Calmodulin was metabolically labeled with
35S according to a protocol generously provided by Drs.
Gerald Carlson and Kenneth Traxler (University of Missouri at Kansas
City). The cDNA encoding CaM was the generous gift of Dr. Claude
Klee (National Institutes of Health). Escherichia coli
transformed with the plasmid DNA were grown in M63 minimal media, and
expression was induced by heat shock at 42 °C followed by the
addition of 1 mCi/100 ml Tran35S-label (ICN Radiochemicals,
Costa Mesa, CA). Expression was allowed to continue for 3 h before
the bacteria were pelleted and resuspended in lysis buffer (50 mM MOPS, pH 7.5, 100 mM KCl, 1 mM
EDTA, 1 mM dithiothreitol, 100 µg/ml lysozyme) and
allowed to lyse overnight at 4 °C. After centrifugation at
30,000 × g for 30 min, CaM was purified from the
cleared lysate on phenyl-Sepharose in the presence of 4 mM
Ca2+ and eluted with 1 mM EDTA. Peak elution
fractions were dialyzed versus two changes of 0.15 M KCl, 20 mM K+-Pipes, 100 µM CaCl2, pH 7.0. CaM protein concentration
was determined by absorption spectroscopy by the equation [CaM] = (A277 [35S]Calmodulin Binding--
Unless otherwise
indicated, SR vesicles or purified RyR preparations were incubated with
1-300 nM [35S]CaM in 150 mM KCl,
20 mM K-Pipes, pH 7.0, 0.1 mg/ml bovine serum albumin (BSA,
Sigma A-0281), 0.2 mM Pefabloc, 20 µM
leupeptin with either 5 mM GSH or GSSG and 100 µM (200 µM CaCl2, 100 µM EGTA) or <10 nM (5 mM EGTA,
no added CaCl2) free Ca2+. Equilibrium
[35S]CaM binding was assayed after incubation at room
temperature for 2 h by centrifugation in a Beckman Airfuge for 30 min at 90,000 × g (SR vesicles) or for 180 min at
225,000 × g in a Beckman Type 75 Ti rotor (soluble and
reconstituted, purified RyR preparations). Centrifugation-based binding
assays are ideal in situations of rapid ligand dissociation and low
affinity since the receptor and ligand remain in equilibrium throughout
the separation period. Nonspecific binding, including the trapped
volume of [35S]CaM, was determined using a 100-1000-fold
excess of unlabeled calmodulin (SR vesicles) or by determining
[35S]CaM binding to CHAPS-solubilized phospholipid or
liposomes that lacked RyR2. Bound [35S]CaM was determined
by scintillation counting after solubilization of pellets in Tris-HCl
buffer, pH 8.5, containing 2% sodium dodecylsulfate. The time course
of [35S]CaM dissociation was determined at 23 °C with
the use of a filter assay. To minimize nonspecific binding of
[35S]CaM, Whatman GF/B filters were blocked for 1 h
in 0.15 M KCl, 10 mM K-Pipes, pH 7.0, buffer
containing 10 mg/ml BSA. Vesicles on the filters were washed with
3 × 5 ml of 0.15 KCl, 10 mM K-Pipes, pH 7.0, buffer
containing 0.1 mg/ml BSA.
[3H]Ryanodine Binding--
Specific
[3H]ryanodine binding was determined in the buffer system
used for [35S]CaM binding after incubation with 1-2
nM [3H]ryanodine for 20 h at 23 °C as
previously described (18). Bmax values of
[3H]ryanodine binding were determined by Scatchard
analysis or with 50 nM [3H]ryanodine in 0.6 M KCl buffer.
Fusion Protein Generation and Expression--
Fusion proteins
spanning the full-length coding sequences of rabbit RyR1 (fused to TrpE
or glutathione S-transferase) and RyR2 (fused to glutathione
S-transferase) were generated by using polymerase chain
reaction to add unique restriction sites to the 5' and 3' ends of the
region of interest (RyR2) or using existing restriction sites (RyR1)
followed by cloning in-frame into pATH or pGEX-5X (RyR1) or pGEX-5X
(Amersham Pharmacia Biotech, RyR2). The sequences expressed for each
fusion protein are as follows: for RyR2, FP1 (1), FP2 (263),
FP3 (561), FP4 (872-1207), FP5 (1157-1509), FP6 (1487-1817),
FP7 (1791-2112), FP8 (2084-2401), FP9 (2385-2754), FP10
(2724-3016), FP11 (3003-3182), FP12 (3160-3352), FP13 (3298-3595), FP13short (3298-3577), FP14 (3543-3961), FP15 (3931-4229), FP16 (4205-4478), FP17 (4404-4563), FP18 (4548-4748), FP19 (4726-4968); for RyR1, FPA (1), FPB (282), FPC (799-1209), FPD
(1209-1632), FPE (1632-2157), FPF (2156-2592), FPG (2502-2874), FPH
(2804-3224), FPI (3225-3662), FPJ (3622-3880), FPK (3879-4222), FPL
(4223-4302), FPM (4302-4430), FPN (4431-4771), FPO (4771-5037).
Fusion proteins were expressed in the BL21 Gold strain of E. coli (Stratagene) by induction (with 1 mM
isopropyl-1-thio- [35S]Calmodulin Overlays--
Equivalent amounts
of each fusion protein (as judged by Coomassie Brilliant Blue stain and
Western analysis) were loaded onto 10% SDS-polyacrylamide
electrophoresis gels. After electrophoresis, the proteins were
transferred to nitrocellulose (0.45-µm pore, Schleicher & Schuell) by
semidry techniques. The membranes were blocked (2 × 30 min) in
150 mM KCl, 20 mM K-Pipes, pH 7.0, 1 mg/ml BSA
with either 100 µM CaCl2 or 5 mM
EGTA. The membranes were then exposed to 100 nM
[35S]CaM in 150 mM KCl, 20 mM
K-Pipes, pH 7.0, 0.04% Tween 20 with the appropriate Ca2+
concentration for 1 h at room temperature followed by four
10-min washes in blocking buffer at 4 °C. The membranes were dried
overnight then exposed to Biomax MR x-ray film (Eastman Kodak Co.) for
1-10 days.
Single-channel Analysis--
Single-channel measurements using
purified RyR2 were carried out as previously described (18) in planar
lipid bilayers containing phosphatidylethanolamine, phosphatidylserine,
and phosphatidylcholine in the ratio of 5:3:2 (25 mg of total
phospholipid/ml of n-decane). The side of the bilayer to
which the proteoliposomes containing the purified RyR2 were added was
defined as the cis (cytoplasmic) side. The trans (SR lumenal) side of
the bilayer was defined as ground. Measurements were made with
symmetrical 0.25 M KCl, 20 mM K-Pipes, pH 7.0, with 50 µM free Ca2+ in the trans (lumenal)
chamber. The cis (cytosolic) solution was varied according to
experimental conditions. Data were acquired using test potentials of
±35 mV and were sampled at 10 kHz and filtered at 2 kHz. Channel open
probabilities (Po) were determined from at least 2 min of recordings for each condition.
Data Analysis--
Free Ca2+ concentrations were
determined as previously described (18). All data analyses were done
using SigmaPlot version 5.
[35S]Calmodulin Binding to RyR1 and RyR2--
Fig.
1A shows the results of
Scatchard analysis of CaM binding to cardiac SR vesicles in reducing (5 mM reduced glutathione, GSH, circles) and
oxidizing (5 mM oxidized glutathione, GSSG, triangles) conditions with 100 µM
(filled symbols) and <10 nM free (open
symbols) Ca2+, respectively. The data are also
summarized in Table I. In the presence of
100 µM Ca2+, cardiac SR vesicles bound 7.5 mol of [35S]CaM/mol of bound [3H]ryanodine.
Since there is only one high affinity [3H]ryanodine
binding site per tetramer (3, 4), this suggests that in the presence of
Ca2+, RyR2 binds 2 CaM molecules per subunit or that other
CaM-binding proteins are present in cardiac SR vesicles. One of the
binding sites appeared to be CaCaM-specific since, in the absence of
free Ca2+, the stoichiometry approximates one CaM molecule
per RyR2 subunit. Skeletal SR vesicles bound ~1 molecule of
[35S]CaM per RyR1 subunit both in the presence and
absence of Ca2+, as has been previously reported (8). The
results from these experiments have been corrected for the presence of
endogenous CaM in the vesicle preparations (1.0 ± 0.6 (n = 4) and 0.13 ± 0.05 (n = 5)
CaM per subunit for cardiac and skeletal SR vesicles, respectively), as
determined by phosphodiesterase activation assay.
After purification of RyR2 from cardiac SR vesicles (shown in Fig.
1B) in the presence of 5 mM GSH and after
reconstitution into proteoliposomes, the
CaCaM:[3H]ryanodine binding stoichiometry dropped from
~2 to 1 molecule per subunit in the presence of 100 µM
Ca2+, suggesting that other CaM-binding proteins have been
removed. Alternatively, one of the two CaCaM binding sites in RyR2 may have been conformationally destroyed or buried during purification. We
also considered the possibility that endogenous CaM remains associated
with the purified RyR. However, if there is such a population of CaM,
it would have to be very tightly bound to the receptor and, therefore,
without effect on CaCaM binding measured in this study, which has a
high rate of dissociation (see Fig. 2).
Purification did not significantly alter the
CaM:[3H]ryanodine binding stoichiometry for RyR2 in the
absence of Ca2+ or for RyR1 both in the presence and
absence of Ca2+ (Table I).
Decrease in free Ca2+ concentration from 100 µM to <10 nM lowered the affinity for CaM in
cardiac SR vesicles 20-fold in the presence of reduced glutathione
(GSH) (Fig. 1, A and B, circles) and
~40-fold in the presence of oxidized glutathione (GSSG)
(triangles). In either the presence or absence of
Ca2+, oxidized glutathione decreased the affinity for CaM
relative to reduced glutathione without changing
Bmax. Likewise, the CaM binding affinity of RyR1
was reduced 2.5-fold by GSSG in the presence of Ca2+ and
9-fold in the absence, again without changes in
Bmax (Table I).
The Ca2+-dependent changes in CaM binding to
cardiac SR vesicles are shown in Fig. 1C. Increase in free
Ca2+ from <10 to 100 nM was without
significant effect on CaM binding affinity or
CaM:[3H]ryanodine binding stoichiometry, implying that
there is apoCaM binding at resting cytosolic Ca2+ levels.
The binding stoichiometry nearly doubled as Ca2+
concentration was raised to 600 nM, to a value close to the
one at 100 µM Ca2+. Conversely, the increase
in CaM binding affinity occurred over a broad Ca2+
concentration range, requiring Ca2+ concentrations between
1 and 10 µM for maximal affinity.
Dissociation experiments were performed to determine the effects of
redox state on the stability of the apoCaM and CaCaM RyR complexes. As
shown in Fig. 2, the rate of dissociation from RyR2 is largely
independent of whether CaM is bound in the presence of 5 mM
GSH or 5 mM GSSG. The rate of dissociation in EGTA
containing media occurred with a Identification of Potential Calmodulin Binding Sites--
To
identify potential calmodulin binding sites within the linear sequence
of RyR1 and RyR2, we have generated fusion proteins spanning the full
coding sequence of the subunit. The design for the fusion proteins is
indicated under "Experimental Procedures." RyR1 sequences were
fused to TrpE (with the exception of RyR1 FP E, L, M, and N, which are
fused to GST to improve expression), whereas the RyR2 fusion proteins
were fused to GST. Since the vast majority of the fusion proteins were
insoluble, [35S]CaM overlays were performed with whole
cell fractions. Fig. 3A shows
that, in the presence of 100 µM CaCl2 and 100 nM [35S]CaM, five of the RyR2 fusion proteins
showed pronounced [35S]CaM binding. CaM binding was
detected for RyR2 fusion proteins 2 (aa 263-615), 10 (2724-3016), 13 (3298-3595), 14 (3543-3961), and 18 (4548-4748), whereas
inconsistent binding was observed for fusion proteins 1 (1), 3 (561), 6 (1487-1817), and 16 (4205-4478). Fig. 3B
shows that in 5 mM EGTA, the binding to FP10 is only
slightly decreased, whereas the binding to the remaining fusion
proteins is greatly reduced, indicative of a Ca2+
dependence of binding to FPs 2, 13, 14, and 18.
The CaM binding to FP13 is localized to the C-terminal portion of the
fusion protein since a truncated form, FP13short (3298-3577), does not
bind either CaCaM or apoCaM in overlay experiments (not shown). Hence,
it is likely that FPs 13 and 14 contain the same CaM binding domain.
RyR1 fusion proteins I (aa 3225-3662) and M (4302-4430) bound CaCaM;
in the presence of 5 mM EGTA, binding to FPM was not
significantly altered, and binding to FPI was abolished (not shown).
These results suggest that there are multiple potential CaM binding
sites within the linear RyR sequences, particularly RyR2. Most appear
to be buried in the large intact RyR2 channel protein because their
number exceeds the number of CaM binding sites in the intact receptor
(Table I).
Functional Implications of Calmodulin Binding--
It has been
previously reported that CaCaM inhibits Ca2+ efflux from
both skeletal and cardiac SR vesicles (13, 22). In addition, CaCaM
inhibits [3H]ryanodine binding to RyR1 in skeletal SR
vesicles (7, 14, 23) with little effect on [3H]ryanodine
binding to RyR2 in cardiac SR vesicles (14). We have used a CaM binding
peptide derived from the myosin light chain kinase (CaMBP) to determine
and correct for the presence of endogenous calmodulin (see
above) in studies of [3H]ryanodine binding, which
has not been done in previous studies. Relatively low CaMBP
concentrations (0.1 µM) were sufficient to reduce
[35S]CaM binding in the presence of 100 µM
free Ca2+ to skeletal SR vesicles to 4% of the control
value (Fig. 4A). For cardiac
SR vesicles, a higher CaMBP concentration (1 µM) was required to reduce [35S]CaM binding to comparable, low
levels. It is crucial that relatively low concentrations of CaMBP be
used if the experiments are done in the presence of the peptide since,
as shown in Fig. 4B, vesicles assayed in the presence of
high concentrations of CaMBP show a marked stimulation of
[3H]ryanodine binding to skeletal SR in either GSH or
GSSG by CaMBP at concentrations greater than 1 µM. A
lower degree of stimulation was observed with cardiac SR. CaMBP was
less effective in reducing [35S]CaM binding to SR
vesicles at [Ca2+] < 1 µM. Where
indicated, experiments were therefore done with SR vesicles pretreated
with CaMBP, as described under "Experimental Procedures."
To correlate the binding of CaCaM to the inhibition of
[3H]ryanodine binding, we measured
[3H]ryanodine binding at increasing concentrations of CaM
both in the presence of GSH and GSSG for cardiac (Fig.
5) and skeletal (not shown) SR vesicles.
For cardiac SR, a higher extent of inhibition was observed in GSH than
GSSG. The KHi for inhibition of ryanodine binding (0.6 ± 0.2 and 1.7 ± 0.7 nM for RyR2 in
GSH and GSSG, respectively) was considerably lower than the
KD for [35S]CaM binding (see Table I).
The Hill coefficients for CaM inhibition of ryanodine binding to RyR2
were 1.0 ± 0.3 both in GSH and GSSG. Skeletal SR also had
KHi values (legend of Fig. 5) that were lower than the KD values and had Hill coefficients near
unity. The results suggest that inhibition of ryanodine binding perhaps requires only a single CaCaM bound/tetramer.
CaM inhibition of [3H]ryanodine binding is also modulated
by various regulators of the RyRs as indicated in Fig.
6 and Table II. At [Ca2+] > 1 µM, CaM (1 µM) inhibition of both cardiac
and skeletal RyR is observed in both oxidizing and reducing conditions
in the absence of MgAMPPCP (AMPPCP is a nonhydrolyzable ATP analogue).
CaM inhibits [3H]ryanodine binding to RyR2 by both
rendering the receptor less sensitive to activation by
Ca2+ and more sensitive to inhibition at high
Ca2+ as well as by lowering the maximal level of
[3H]ryanodine binding (Fig. 6, upper left
panel). In the presence of MgAMPPCP and at [Ca2+] < 10 µM, CaM inhibits cardiac SR ryanodine binding in both
reducing and oxidizing conditions (Fig. 6, upper right
panel). However, at [Ca2+] > 10 µM,
CaM inhibits RyR2 ryanodine binding only in reducing conditions. The
lower left panel of Fig. 6 indicates that in agreement with
a previous report (24), skeletal SR [3H]ryanodine binding
is markedly inhibited in the presence of GSH. [3H]Ryanodine binding to skeletal SR is activated by CaM
at low Ca2+ concentrations both in the absence or presence
of MgAMPPCP and the presence of GSH (see Table II) or GSSG and
inhibited at higher Ca2+ concentrations in both oxidizing
and reducing conditions (Fig. 6, lower two panels). In the
presence of 5 mM MgAMPPCP, free Ca2+
concentrations in excess of 0.3 mM were not tested because
of difficulties in keeping Ca2+ in solution.
In Table II, the effects of CaM on [3H]ryanodine binding
to RyR2 and RyR1 are compared in 0.1 and 100 µM
Ca2+ media that contained 10 mM caffeine, 5 mM AMPPCP, or 1 mM Mg2+. ApoCaM
significantly inhibits RyR2 in reducing conditions in either the
presence or absence of AMPPCP. This apoCaM inhibition was attenuated in
the presence of caffeine or in oxidizing conditions. CaCaM inhibition
of RyR2 was significant in control (+GSH) and in the presence of 1 mM MgCl2, with no significant effects in the
presence of caffeine or AMPPCP. ApoCaM stimulation of RyR1 under both
oxidizing and reducing conditions was maintained in the presence of 10 mM caffeine or 5 mM AMPPCP, compounds that further sensitize the RyR1 ion channel to low concentrations of Ca2+. CaCaM inhibition of ryanodine binding to RyR1 was
observed under each condition, although the magnitude of the inhibition
was decreased in the presence of AMPPCP. The inhibition by CaCaM in
AMPPCP was lower in the presence of GSSG than GSH, whereas the
inhibition in the presence of caffeine was unaffected by the redox
state of glutathione.
Calmodulin Interaction with Purified Ryanodine
Receptors--
Purification of RyR1 by CHAPS solubilization and
reconstitution into proteoliposomes had little effect on the
equilibrium [35S]CaM binding properties, as indicated in
Table I; furthermore, CaCaM inhibition of [3H]ryanodine
binding to purified RyR1 was comparable with that of SR vesicles (not
shown). Purification of RyR2, however, decreased the stoichiometry of
CaCaM binding without affecting the KD. In addition,
purified RyR2 (either solubilized or reconstituted) failed to show any
inhibition of [3H]ryanodine binding by CaCaM in
equilibrium binding experiments.
Single-channel measurements using purified RyR2 indicate that in an
applied electrical field CaM inhibition of RyR2 is maintained after purification and reconstitution
into proteoliposomes (Fig. 7, Table III).
In Fig. 7A, a single channel was recorded in the presence of
100 µM cis (cytoplasmic) Ca2+. The addition
of 1 µM cis CaM reduced channel open probability (Po) from 0.62 to 0.27. The Hill inhibition constant and coefficient of 7 experiments were 42 ± 1 nM and
1.4 ± 0.1, respectively (Fig. 7B). In 100 µM free cis Ca2+ and symmetrical 0.25 M KCl, the addition of 1 µM CaM to the cis chamber reduced Po from 0.73 ± 0.09 to
0.32 ± 0.08 (n = 7) (Table III). The inhibition
of purified RyR2 was attributed to a decrease in mean open time from
9.83 ± 6.57 to 1.45 ± 0.47 ms (n = 5)
(statistically significant normalized decrease in open time of 44 ± 12%) without other significant effects on channel gating
parameters. The inhibitory effect was magnified in the presence of 2 mM Mg2+ and 100 µM free
Ca2+ with a decrease in Po from
0.51 ± 0.15 to 0.05 ± 0.03 (n = 4). As in
the [3H]ryanodine binding studies using SR membranes,
MgATP reversed the inhibition by CaM at 100 µM free
Ca2+. At 100-200 nM free Ca2+
concentrations, the addition of 1 µM CaM had a weak
inhibitory effect alone as well as in the presence of caffeine; this
inhibition was statistically significant only when the data were
normalized (Po(+CaM)/Po( It has been known for more than 10 years that the
Ca2+-binding protein calmodulin is capable of inhibiting
Ca2+ release from isolated SR membranes both from cardiac
and skeletal muscle (13, 22). Since that time, extensive work has
attempted to characterize the nature of CaM binding to the ryanodine
receptor. Initial studies using either 125I (7) or
fluorescently (15) labeled CaM found that there are as many as six
binding sites for apoCaM on each of the four RyR1 subunits that compose
the functional channel. This number was supported in part by studies
using fragments of the full-length subunit, which indicated that there
were three regions that strongly bound CaM as well as at least three
other regions that had weaker CaM binding (16, 17). With the exception
of one site, CaM binding was Ca2+-dependent,
which indicated differences in the Ca2+ dependence of CaM
binding to the intact receptor and isolated fragments. More recent
studies using SR membranes and 35S metabolically labeled
CaM indicate that the tetrameric skeletal muscle channel complex binds
a total of four CaM molecules both for apo- and CaCaM or an average of
one CaM per subunit (8). The implication of these studies is that
chemical modification induces changes in calmodulin leading to
nonphysiological binding. The data that we have presented here show
that, in agreement with previous studies using [35S]CaM,
the skeletal muscle isoform binds ~4 molecules of CaM per tetramer
both in the presence and absence of Ca2+. Furthermore, the
stoichiometry of CaM binding to RyR1 is not influenced by the
redox state, whereas previous studies using sulfhydryl-reacting agents
and [125I]CaM binding have indicated a decrease in the
number of apoCaM binding sites (23).
Very little is known about the CaM binding properties of the cardiac
isoform of the ryanodine receptor. A recent report by Fruen et
al. (14) suggests that cardiac SR membranes bind a single molecule
of CaCaM per subunit. Their results also indicate that the binding of
apoCaM is greatly reduced, with a stoichiometry of ~1 molecule per
tetramer. The most likely cause of the discrepancy between their report
and ours (7.5 molecules of CaCaM per tetramer and 4 molecules apoCaM
per tetramer) is due to their use of a filtration-based assay given the
rapid rate of dissociation of CaM from the cardiac receptor,
particularly in the presence of EGTA, where the Recent data suggest there is a single CaM binding domain that binds
both apo- and CaCaM at distinct but closely apposed sequences (8, 29).
Attempts to localize the CaM binding domain in RyR2 illustrated
additional differences between skeletal and cardiac ryanodine
receptors. Only two potential CaM binding sites were identified in
fusion proteins derived from RyR1; one of which, fusion protein I (aa
3225-3662), bound only CaCaM, whereas the other, fusion protein M (aa
4302-4430), bound CaM both in 100 µM Ca2+ as
well as 5 mM EGTA. The fusion proteins derived from the
RyR2 sequence, however, revealed many more potential CaM binding sites. Two overlapping sites (FP13, aa 3298-3595, and FP14, aa 3543-3961) displayed pronounced CaM binding. Fusion proteins I (RyR1) and 13 and
14 (RyR2) contain a sequence implicated in each study, localizing CaM
binding sites in the RyR1 sequence (8, 16, 17, 28). C-terminal
truncation of 18 amino acids from FP13 (FP13Short, aa 3298-3577)
removed both Ca2+- and apoCaM binding in overlay
experiments, suggesting that the sequence (RyR2 aa 3578-3595)
HPQRSKKAVWHKLLSKQR is crucial for conferring CaM binding. Furthermore,
our data imply that the affinity of CaM binding to this site is
Ca2+-dependent. A portion of this sequence with
additional C-terminal residues (fusion protein PC26 RyR1, aa
3552-3661, and peptide PM2 RyR1, aa 3617-3634 and RyR2 aa 3583-3601)
was found to bind calmodulin in 10 µM Ca2+
but not in 10 mM EGTA (17); the corresponding peptide
derived from RyR2 was also found to have similar Ca2+
dependence (28). Interestingly, this single domain has been implicated
in both CaCaM and apoCaM binding, as both forms are capable of
protecting RyR1 from trypsin cleavage at arginines 3630 and 3637 (8). A
recent publication has used peptides derived from this RyR1 CaM binding
domain to further refine the putative apo and CaCaM binding sites as an
N-terminal CaCaM-specific domain from 3614 to 3634 and an overlapping
domain also capable of binding apoCaM from 3625 to 3644 (29). In RyR2,
however, the results do not entirely agree, as FP13 bound both apo- and
CaCaM (albeit to different extents) but does not contain this apoCaM
domain. Furthermore, in the Rodney et al. (29) study, a
peptide (3614-KSKKAVWHKLLSKQ-3627), which agrees with the critical
sequence for CaM binding to RyR2 FP13, was unable to bind either
Ca2+ or apoCaM. The results from our localization of
potential CaM binding sites as well as those reported by others are
summarized in Fig. 8.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical stalk. Each of the N- and C-terminal
domains contains two EF-hand Ca2+ binding motifs.
Ca2+ binding domains I and II in the N domain have a lower
Ca2+ affinity (10
5 M)
and are less Ca2+-selective than the corresponding domains
III and IV (10
6 M) in the C
domain. CaM binds to and regulates a myriad of target proteins involved
in almost every biological function in three distinct manners: 1) as
CaCaM 2) as apoCaM (without Ca2+ bound), and 3)
Ca2+ independent or constituently bound (11, 12). Each of
these binding events occurs through one of several poorly defined CaM binding motifs, the most common of which are composed of an amphipathic helix of ~20 amino acid residues that bind CaCaM or an IQ
sequence motif that preferentially binds apoCaM.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
A320)/
. For
expressed CaM,
was assumed to be 0.20 ml/(mg cm) (21).
-D-galactopyranoside for GST and 10 µg/ml indoacrylic acid for TrpE fusion proteins) and a 2-h incubation
at 37 °C. Whole cell pellets were collected by centrifugation at
1500 × g for 15 min followed by resuspension in
phosphate-buffered saline containing Complete protease inhibitors (Roche Molecular Biochemicals) and lysis by sonication.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (13K):
[in a new window]
Fig. 1.
Analysis of 35S-calmodulin
binding to cardiac SR vesicles and purified RyR2. Cardiac SR
vesicles (A) and proteoliposomes containing the purified
RyR2 (B) were incubated with increasing concentrations of
[35S]CaM in the presence of either 100 µM
free Ca2+ (filled symbols) or 5 mM
EGTA (open symbols) and reduced (GSH, circles) or
oxidized (GSSG, triangles) glutathione. Averaged
[35S]CaM/[3H]ryanodine binding ratios and
KD values from a total of between 5 and 16 experiments are shown in Table I. C,
Bmax (left axis, filled
circles) and KD (right axis,
open circles) values for [35S]CaM binding were
determined as a function of free Ca2+ concentration. The
data are mean ± S.D. for 8 experiments at <10 nM
Ca2+, 3 experiments at all other
[Ca2+].
[35S]Calmodulin binding to cardiac and skeletal SR vesicles
and purified RyR2 and RyR1
View larger version (14K):
[in a new window]
Fig. 2.
Dissociation of bound
35S-calmodulin. Cardiac SR vesicles were prebound with
100 or 200 nM [35S]CaM in 100 µM free Ca2+ (filled symbols) or 5 mM EGTA (open symbols), respectively, in the
presence of 5 mM GSH (circles) or 5 mM GSSG (triangles) and then were diluted
60-fold into media containing 50 nM nonradioactive CaM and
either 100 µM free Ca2+ (filled
symbols) or 5 mM EGTA (open symbols). The
averaged time constants (in min) of [35S]CaM dissociation
±S.D. of 3-5 experiments for RyR2 and RyR1, respectively, was
8.5 ± 2.9 and 12.4 ± 4.2 (Ca2+ + GSH), 9.0 ± 2.6 and 8.9 ± 3.4 (Ca2+ + GSSG), 0.7 ± 0.5 and 3.8 ± 1.0 (EGTA + GSH), and 0.7 ± 0.5 and 4.0 ± 0.9 (EGTA + GSSG).
1/2 of ~40 s. The
rate of dissociation was decreased by more than 10-fold in the presence
of Ca2+, occurring with
1/2 of ~9 min. The
results suggest that the rate of dissociation of CaM from RyR2 is
largely independent of redox state but rather on whether
Ca2+ is present in the dissociation buffer, with apoCaM
dissociating at a significantly greater rate than CaCaM. Similar
results were obtained for RyR1 (see the legend of Fig. 2), with less
pronounced differences between the rates of dissociation of CaCaM and apoCaM.
View larger version (64K):
[in a new window]
Fig. 3.
Identification of potential calmodulin
binding sites. Fusion proteins 1-19 derived from the full-length
RyR2 sequence (fused to GST) as well as GST without an insert were
expressed in E. coli and equivalent amounts of expressed
fusion protein as well as nontransformed (NT) E. coli, as judged by Coomassie-stained SDS-polyacrylamide
electrophoresis gels, were transferred to nitrocellulose. After
blocking with 1 mg/ml BSA, 100 nM [35S]CaM
was overlaid for 1 h in 0.15 M KCl, 20 mM
K-Pipes, pH 7, 0.04% Tween 20 with 100 µM
CaCl2 (A) or 5 mM EGTA
(B). After four washes in blocking buffer and overnight
drying, the membranes were exposed to x-ray film.
View larger version (18K):
[in a new window]
Fig. 4.
CaMBP effects on
[35S]calmodulin and [3H]ryanodine
binding. A, [35S]CaM binding in 10 nM [35S]CaM and 100 µM free
Ca2+ was determined in either 5 mM GSH
(circles) or GSSG (triangles) as a function of
increasing CaMBP concentration to SR vesicles from either cardiac
(filled symbols) or skeletal (open symbols)
muscle. B, cardiac or skeletal SR vesicles were preincubated
with 1 µM CaMBP and centrifuged to remove endogenous CaM
and added CaMBP, then [3H]ryanodine binding was
determined in 0.15 M KCl, 20 mM K-Pipes, pH
7.0, 100 µM Ca2+, 0.1 mg/ml BSA in either 5 mM GSH or GSSG with increasing concentrations of CaMBP.
Data are the mean ± S.D. of 3-4 experiments.
View larger version (10K):
[in a new window]
Fig. 5.
Calmodulin inhibition of
[3H]ryanodine binding to cardiac SR vesicles.
Cardiac SR vesicles were preincubated with 1 µM CaMBP and
centrifuged to remove endogenous CaM and added CaMBP.
[3H]Ryanodine binding was determined in 0.15 M KCl, 20 mM K-Pipes, pH 7.0, 100 µM Ca2+, 0.1 mg/ml BSA, 0-2500
nM CaM, and either 5 mM GSH
(circles) or GSSG (triangles). Free CaM
concentrations were measured on paired samples using
[35S]CaM. Solid lines were obtained according
to the equation B = Bo (1 + (([CaM]/KHi)nHi) 1,
where B and Bo are bound
[3H]ryanodine in the presence and absence of CaM. The
averaged Hill inhibition constants (KHi) ± S.D. of 3-4 experiments in the presence of GSH and GSSG were 0.6 ± 0.2 nM and 1.7 ± 0.7 nM, respectively.
Corresponding Hill coefficients (nHi) were
1.0 ± 0.3 and 1.0 ± 0.3. Corresponding
KHi for RyR1 were 1.6 ± 0.2 nM
and 4.7 ± 0.4 nM, and nHi were
1.2 ± 0.4 and 1.1 ± 0.3.
View larger version (36K):
[in a new window]
Fig. 6.
Ca2+ dependence of calmodulin
inhibition of [3H]ryanodine binding.
[3H]Ryanodine binding was determined as a function of
free Ca2+ concentration for both cardiac (RyR2) and
skeletal (RyR1) SR vesicles either in the absence ( MgA) or
presence (+MgA) of 5 mM MgAMPPCP and either in
the absence (circles) or presence (triangles) of
1 µM added CaM and either with 5 mM GSH
(open symbols) or 5 mM GSSG (filled
symbols). Endogenous CaM was removed by pretreating vesicles with
1 µM CaMBP (experiments at <1 µM
Ca2+) followed by centrifugation or dissociated from the
receptor by carrying out the binding assay in the presence of 0.5 µM CaMBP (experiments at > 1 µM
Ca2+). The averaged data ± S.D. of 3-4 experiments
are fit to a two-site (one activation, one inhibition) logistic
function (34).
Regulation of native RyR1 and RyR2 by calmodulin in the presence of
other ligands
CaM) to
complex endogenous CaM or in the presence of 1 µM CaM
(+CaM). Data are mean ± S.D. of 3-4 experiments.
CaM) = 0.22 ± 0.05 for control and 0.66 ± 0.10 in the presence
of caffeine). Thus, RyR2 was not activated by apoCaM, as is the case
for RyR1 (7). It therefore appears that purification of RyR2 alters the
conformation of the channel sufficiently to mask in
[3H]ryanodine binding measurements the CaCaM binding site
that is responsible for inhibition of the channel, but that this site, mediating inhibition by CaCaM and presumably apoCaM (Fig.
1C), can be recovered in single-channel measurements by the
application of an electrical field.
View larger version (27K):
[in a new window]
Fig. 7.
Effect of cytosolic calmodulin on single
purified RyR2 ion channels. Proteoliposomes containing purified
RyR2 were fused with lipid bilayers. A, single-channel
currents were recorded at 35 mV (upward deflection from closed levels,
c-) in symmetric 0.25 M KCl, 20 mM
K+-Pipes, pH 7.0, media containing 5.08 mM
CaCl2 and 5 mM EGTA (free Ca2+ of
100 µM). Top trace, control,
Po = 0.62. Bottom trace, after the
addition of 1 µM CaM, Po = 0.27. B, dependence of cardiac Ca2+ release channel
activity on CaM concentration. Relative channel open probability
(Po/Po,control) was
obtained from 7 single-channel recordings similar to those shown in
A. Solid lines were obtained according to the
equation Po = Po,control(1 + (([CaM]/KHi)nHi) 1
where Po and
Po,control are single-channel activities
in the presence and absence of CaM. The Hill inhibition constant and
coefficient ± S.D. of seven experiments were 42 ± 1 nM and 1.4 ± 0.1, respectively. * and ** indicate the
means of data were significantly different from control at
p < 0.05 and p < 0.001, respectively.
Calmodulin inhibition of purified RyR2 single channel activities
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1/2 for
dissociation was about 40 s. For both skeletal and cardiac SR
vesicles, the CaM binding affinity was decreased both by the removal of
Ca2+ and by the presence of oxidizing GSSG. This agrees
with previous reports suggesting that superoxide anion decreases
cardiac SR CaM content (25) and sulfhydryl-reacting reagents diminish
CaM binding and inhibition of RyR1 (23, 26). The
Ca2+-dependent change in stoichiometry appears
to be the result of Ca2+ binding to CaM given the highly
cooperative nature of the increase, whereas the increase in affinity
may be due to Ca2+ binding to the cardiac ryanodine
receptor, as previously suggested for the skeletal receptor (27).
View larger version (6K):
[in a new window]
Fig. 8.
Schematic summary of potential calmodulin
binding sites. Sequence domains suggested by previous works (16,
17, 28, 29) and this report are aligned with cardiac binding domains
above and skeletal binding domains below a linear representation
of the full-length amino acid sequence of the ryanodine receptor.
CaCaM-specific binding domains are dark gray, whereas
domains binding both apo- and CaCaM are light gray with a
black border.
Previous reports show that the activity of the skeletal muscle ryanodine receptor is stimulated by CaM at low free Ca2+ concentrations (7, 14). This effect appears to be lacking in the few studies performed to date using cardiac SR vesicles (14). Recently it was also shown that cardiac SR vesicle ryanodine binding was not inhibited by CaCaM in the presence of adenine nucleotides, although SR Ca2+ efflux was inhibited by CaM in a manner similar to previous Ca2+ efflux measurements (14). We show here that inhibition of RyR2 by CaM is dependent on the assay conditions. [3H]Ryanodine binding to cardiac SR vesicles was inhibited by CaCaM in the absence and presence of Mg2+ and adenine nucleotide, with the effect being more pronounced at [Ca2+] < 10 µM in the presence of GSH. CaCaM was without a significant effect at 100 µM Ca2+ in the presence of GSSG and adenine nucleotide or caffeine. ApoCaM inhibited [3H]ryanodine binding to cardiac SR in the absence and presence of AMPPCP. In single-channel measurements, apoCaM had a small inhibitory effect in the absence and presence of caffeine.
We have also found that, although the use of the CaMBP is necessary to eliminate the effects of endogenous SR calmodulin, which is associated with both the skeletal and cardiac SR vesicles, it is imperative that it be used at low concentrations since it had a pronounced stimulatory effect on skeletal [3H]ryanodine binding after preincubation of the vesicles to remove the endogenous CaM. Since CaMBP is derived from a CaCaM-specific binding protein, it is ineffective at removing endogenous CaM in the absence of Ca2+. To correct for the effect of endogenous apoCaM, we first removed endogenous CaM by incubation with CaMBP in the presence of Ca2+ followed by centrifugation.
Purification of RyR2 decreases the stoichiometry of CaCaM binding, eliminating one site for [35S]CaCaM per subunit without affecting apoCaM binding, an effect not observed with RyR1. In addition, purification of RyR2 also eliminated CaCaM-dependent inhibition of [3H]ryanodine binding. This effect appears to be due to a conformational change in the purified receptor rather than to the removal of a necessary cofactor since single-channel measurements using purified RyR2 show that upon application of a transmembrane potential, CaCaM inhibition of the channel is restored. However, the KHi for inhibition of channel open probability was considerably higher than the KD for [35S]CaM binding. Whether CaM binding affinity is voltage-dependent or whether restoration of function correlates with unmasking of a second CaM binding site in RyR2 is not known because CaM binding to single channels cannot be measured. To resolve these issues, RyR2 mutants lacking putative CaM binding sites need to be constructed and examined.
Our data suggest that CaM is a major regulator of Ca2+
release from intracellular stores during excitation-contraction
coupling. Under conditions of oxidative stress, such as
exercise-induced fatigue or ischemia in which levels of GSSG are
increased relative to GSH, the affinity for both apo- and CaCaM is
decreased. In skeletal muscle, the oxidizing effects can result both in
an increase (oxidation of RyR1) and decrease (decrease in apoCaM
affinity) of basal Ca2+ release. One possible effect
consistent with our data is that oxidation will result in a pronounced
increase in peak Ca2+ release, resulting from RyR1
oxidation and diminished CaCaM binding affinity. In cardiac muscle, in
the presence of Mg2+ and adenine nucleotide, a shift from
reducing to oxidizing conditions will attenuate the inhibition of RyR2
by saturating CaM at low Ca2+ while eliminating inhibition
by CaM at high Ca2+. In addition, the CaM binding affinity
is reduced in the presence of oxidizing conditions, further sensitizing
the channel to activation by cytosolic Ca2+. This could
lead to an increase in the sensitivity of RyR2 to trigger
Ca2+ provided by the dihydropyridine receptor. Furthermore,
CaM is a key mediator of Ca2+-dependent
inactivation and facilitation of cardiac L-type Ca2+
channels (30-33) and is, therefore, a regulator of two of the most
tightly regulated steps in excitation-contraction coupling.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants AR18687 and HL27430.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.
A Postdoctoral Fellow of the Japan Society for the Promotion of 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, March 27, 2001, DOI 10.1074/jbc.M010771200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
RyR, ryanodine
receptor;
RyR1, skeletal muscle RyR;
RyR2, cardiac muscle RyR;
SR, sarcoplasmic reticulum;
CaM, calmodulin;
GSH, reduced glutathione;
GSSG, oxidized glutathione;
CaMBP, myosin light chain kinase-derived
calmodulin binding peptide;
BSA, bovine serum albumin;
AMPPCP, adenosine 5'-(,
-methylene)triphosphate;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
MOPS, 4-morpholinepropanesulfonic acid;
Pipes, 1,4-piperazinediethanesulfonic
acid;
GST, glutathione S-transferase;
aa, amino acids.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Sorrentino, V., and Volpe, P. (1993) Trends Pharmacol. Sci. 14, 98-103[CrossRef][Medline] [Order article via Infotrieve] |
2. | Tanabe, T., Mikami, A., Numa, S., and Beam, K. G. (1990) Nature 344, 451-453[CrossRef][Medline] [Order article via Infotrieve] |
3. | Meissner, G. (1994) Annu. Rev. Physiol. 56, 485-508[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Franzini-Armstrong, C.,
and Protasi, F.
(1997)
Physiol. Rev.
77,
699-729 |
5. |
Zhang, L.,
Kelley, J.,
Schmeisser, G.,
Kobayashi, Y. M.,
and Jones, L. R.
(1997)
J. Biol. Chem.
272,
23389-23397 |
6. | Ohkura, M., Furukawa, K., Fujimori, H., Kuruma, A., Kawano, S., Hiraoka, M., Kuniyasu, A., Nakayama, H., and Ohizumi, Y. (1998) Biochemistry 37, 12987-12993[CrossRef][Medline] [Order article via Infotrieve] |
7. | Tripathy, A., Xu, L., Mann, G., and Meissner, G. (1995) Biophys. J. 69, 106-119[Abstract] |
8. | Moore, C. P., Rodney, G., Zhang, J. Z., Santacruz-Toloza, L., Strasburg, G., and Hamilton, S. L. (1999) Biochemistry 38, 8532-8537[CrossRef][Medline] [Order article via Infotrieve] |
9. | Babu, Y. S., Sack, J. S., Greenhough, T. J., Bugg, C. E., Means, A. R., and Cook, W. J. (1985) Nature 315, 37-40[Medline] [Order article via Infotrieve] |
10. | Zhang, M., Tanaka, T., and Ikura, M. (1995) Nat. Struct. Biol. 2, 758-767[Medline] [Order article via Infotrieve] |
11. | Celio, M. R., Pauls, T., and Schwaller, B. (1995) Guidebook to the Calcium-binding Proteins , pp. 34-40, Oxford University Press, New York |
12. |
Rhoads, A. R.,
and Friedberg, F.
(1997)
FASEB J.
11,
331-340 |
13. |
Meissner, G.,
and Henderson, J. S.
(1987)
J. Biol. Chem.
262,
3065-3073 |
14. | Fruen, B. R., Bardy, J. M., Byrem, T. M., Strasburg, G. M., and Louis, C. F. (2000) Am. J. Physiol. 279, C724-C733 |
15. | Yang, H. C., Reedy, M. M., Burke, C. L., and Strasburg, G. M. (1994) Biochemistry 33, 518-525[Medline] [Order article via Infotrieve] |
16. |
Chen, S. R.,
and MacLennan, D. H.
(1994)
J. Biol. Chem.
269,
22698-22704 |
17. | Menegazzi, P., Larini, F., Treves, S., Guerrini, R., Quadroni, M., and Zorzato, F. (1994) Biochemistry 33, 9078-9084[Medline] [Order article via Infotrieve] |
18. |
Xu, L.,
Tripathy, A.,
Pasek, D. A.,
and Meissner, G.
(1999)
J. Biol. Chem.
274,
32680-32691 |
19. |
Lee, H. B.,
Xu, L.,
and Meissner, G.
(1994)
J. Biol. Chem.
269,
13305-13312 |
20. | Eu, J. P., Sun, J., Xu, L., Stamler, J. S., and Meissner, G. (2000) Cell 102, 499-509[Medline] [Order article via Infotrieve] |
21. | Richman, P. G., and Klee, C. B. (1979) J. Biol. Chem. 254, 5372-5376[Abstract] |
22. | Meissner, G. (1986) Biochemistry 25, 244-251[Medline] [Order article via Infotrieve] |
23. |
Zhang, J. Z.,
Wu, Y.,
Williams, B. Y.,
Rodney, G.,
Mandel, F.,
Strasburg, G. M.,
and Hamilton, S. L.
(1999)
Am. J. Physiol.
276,
C46-C53 |
24. |
Zable, A. C.,
Favero, T. G.,
and Abramson, J. J.
(1997)
J. Biol. Chem.
272,
7069-7077 |
25. |
Kawakami, M.,
and Okabe, E.
(1998)
Mol. Pharmacol.
53,
497-503 |
26. | Suko, J., Hellmann, G., and Drobny, H. (2000) J. Memb. Biol. 174, 105-120[CrossRef][Medline] [Order article via Infotrieve] |
27. | Rodney, G. G., Williams, B. Y., Strasburg, G. M., Beckingham, K., and Hamilton, S. L. (2000) Biochemistry 39, 7807-7812[CrossRef][Medline] [Order article via Infotrieve] |
28. | Guerrini, R., Menegazzi, P., Anacardio, R., Marastoni, M., Tomatis, R., Zorzato, F., and Treves, S. (1995) Biochemistry 34, 5120-5129[Medline] [Order article via Infotrieve] |
29. |
Rodney, G. G.,
Moore, C. P.,
Williams, B. Y.,
Zhang, J. Z.,
Krol, J.,
Pedersen, S. E.,
and Hamilton, S. L.
(2001)
J. Biol. Chem.
276,
2069-2074 |
30. |
Soldatov, N. M.,
Oz, M.,
O'Brien, K. A.,
Abernethy, D. R.,
and Morad, M.
(1998)
J. Biol. Chem.
273,
957-963 |
31. |
Qin, N.,
Olcese, R.,
Bransby, M.,
Lin, T.,
and Birnbaumer, L.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
2435-2438 |
32. | Peterson, B. Z., DeMaria, C. D., Adelman, J. P., and Yue, D. T. (1999) Neuron 22, 549-558[Medline] [Order article via Infotrieve] |
33. | Zuhlke, R. D., Pitt, G. S., Deisseroth, K., Tsien, R. W., and Reuter, H. (1999) Nature 399, 159-162[CrossRef][Medline] [Order article via Infotrieve] |
34. |
Meissner, G.,
Rios, E.,
Tripathy, A.,
and Pasek, D. A.
(1997)
J. Biol. Chem.
272,
1628-1638 |