From the Department of Pharmacology and Physiology, University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642
Received for publication, February 16, 2001, and in revised form, April 9, 2001
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ABSTRACT |
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Recent studies have shown that, in a wide
variety of cells, mitochondria respond dynamically to physiological
changes in cytosolic Ca2+ concentrations
([Ca2+]c). Mitochondrial Ca2+
uptake occurs via a ruthenium red-sensitive calcium uniporter and a
rapid mode of Ca2+ uptake. Surprisingly, the molecular
identity of these Ca2+ transport proteins is still unknown.
Using electron microscopy and Western blotting, we identified a
ryanodine receptor in the inner mitochondrial membrane with a molecular
mass of approximately 600 kDa in mitochondria isolated from the rat
heart. [3H]Ryanodine binds to this mitochondrial
ryanodine receptor with high affinity. This binding is modulated by
Ca2+ but not caffeine and is inhibited by Mg2+
and ruthenium red in the assay medium. In the presence of ryanodine, Ca2+ uptake into isolated heart mitochondria is suppressed.
In addition, ryanodine inhibited mitochondrial swelling induced by
Ca2+ overload. This swelling effect was not observed when
Ca2+ was applied to the cytosolic fraction containing
sarcoplasmic reticulum. These results are the first to identify a
mitochondrial Ca2+ transport protein that has
characteristics similar to the ryanodine receptor. This mitochondrial
ryanodine receptor is likely to play an essential role in the dynamic
uptake of Ca2+ into mitochondria during Ca2+ oscillations.
Mitochondria play a central role in numerous fundamental cellular
processes ranging from the generation of ATP to the regulation of
cytosolic Ca2+ homeostasis and apoptosis (1-3). Impairment
of intracellular Ca2+ homeostasis and mitochondrial
function has been implicated in the development of neurodegenerative
diseases, diabetes, and cardiomyopathy (4-6). Measuring changes in
mitochondrial Ca2+ concentrations
([Ca2+]m)1
in living cells with highly sensitive fluorescence dyes and targeted luminescence probes has drawn new attention on the regulation of
mitochondrial Ca2+ homeostasis and its biological
implications (7-9). Several studies show that, in many cell types,
mitochondria respond dynamically to physiological oscillations of free
[Ca2+]c (10-14).
In cardiac muscle cells, mitochondria also respond to
physiological changes in [Ca2+]c (15).
However, controversy remains whether the mitochondrial Ca2+
uptake mechanisms can sequester Ca2+ rapidly on a
beat-to-beat basis (15). Established Ca2+ uptake mechanisms
are the CaUP and rapid mode of Ca2+ uptake (1, 16). The
mitochondrial CaUP is activated by Ca2+ concentrations
greater than 10 µM, which are usually only achieved within cytosolic microdomains (9). In isolated liver mitochondria, rapid mode of Ca2+ uptake responds with mitochondrial
Ca2+ uptake in response to Ca2+ pulses of less
than 300 nM (16). Although these Ca2+ uptake
mechanisms are kinetically and pharmacologically well characterized,
their molecular identity has yet to be determined.
An intriguing observation is the considerable similarity in biochemical
and pharmacological properties between the mitochondrial CaUP and the
SR-RyR (1, 17). Both, the CaUP and the SR-RyR are activated by changes
in [Ca2+]c and inhibited by adenine
nucleotides, Mg2+, and RR. The strikingly similar
properties of these proteins led to our hypothesis that mitochondria
contain a RyR. Here we show with immunological, biochemical,
pharmacological, and physiological techniques that heart mitochondria
contain a functional RyR within the IMM. This mRyR underlies fast
mitochondrial Ca2+ uptake and, therefore, is uniquely
positioned to regulate dynamically Ca2+-mediated cellular
processes such as ATP production during heartbeat (18).
Materials--
Ryanodine was purchased from Calbiochem and
[3H]ryanodine from Amersham Pharmacia Biotech. Antibodies
against RyR (clone 34C), developed by J. Airey and J. Sutko, were
obtained from the Developmental Studies Hybridoma Bank maintained by
the Department of Biological Sciences of the University of Iowa (Iowa
City, IA) and from RBI/Sigma. The antibodies against the
voltage-dependent anion channel (VDAC) and the sarco- and
endoplasmic reticulum Ca2+-ATPase (SERCA) were obtained
from Calbiochem and Santa Cruz, respectively. All other chemicals were
purchased from Sigma unless noted.
Isolation of Rat Heart Mitochondria--
Heart mitochondria were
isolated in isotonic ice-cold mannitol/sucrose buffer (M/S buffer; in
mM: 225 mannitol, 10 sucrose, 0.5 EGTA, 1 glutathione; 10 HEPES, pH 7.4) by differential centrifugation and subsequent
purification on a Percoll gradient (19). The final two washes were done
in EGTA-free buffer. The isolated mitochondria were stored on ice and
used for experiments for up to 4 h after finishing the isolation
procedure. Experiments were done with mitochondria having intact RR-
and carbonyl cyanide 3-chlorophenylhydrazone-sensitive Ca2+
uptake mechanisms, as measured with a Ca2+-sensitive microelectrode.
Preparation of Mitochondrial Subfractions--
Mitochondrial
subfractions were prepared as described previously (20). Briefly,
isolated mitochondria from 2 rat hearts were osmotically shocked first
in 10 mM
Na2HPO4/NaH2PO4 for
20 min on ice followed by the addition of sucrose (20% final
concentration). Mitochondrial membranes were disrupted by sonication
(two times for 30 s), and eventually intact mitochondria were
removed by centrifugation at 7,000 × g. The
supernatant was transferred as a final layer onto a continuous sucrose
gradient (from 60% to 30% in 10 mM HEPES, pH 7.4, plus
protease inhibitor mixture (Roche Molecular Biochemicals) and
centrifuged for at least 8 h at 70,000 × g to
separate mitochondrial membrane vesicles. The mitochondrial subfractions were tested for specific marker proteins (succinate dehydrogenase (SDH) for IMM, creatine kinase for contact sites between
IMM and outer mitochondrial membrane (OMM), and the VDAC for OMM). The
characterized fractions were diluted 2-fold with M/S buffer,
supplemented with protease inhibitor mixture (Roche), and centrifuged
for 90 min at 300,000 × g to sediment the mRyR. The
pellet was resuspended in a small volume of M/S buffer and stored in
liquid nitrogen until needed.
Immunogold Labeling Methods--
For immunogold labeling of RyR,
isolated heart mitochondria were embedded in Lowicryl mixture at
Denaturing SDS-Gel Electrophoresis--
For the immunological
detection of the RyR and SERCA, 100 µg of protein was loaded on a 5%
SDS-polyacrylamide gel. The separated proteins were transferred onto a
nitrocellulose membrane for 90 min at 100 V. For VDAC, 50 µg of
protein was loaded onto a 12% SDS-polyacrylamide gel and transferred
for 45 min at 100 V onto a nitrocellulose membrane. Western blots were
performed using the Aurora chemiluminescence assay (ICN) with an
alkaline phosphatase-linked secondary antibody.
[3H]Ryanodine Binding--
For binding assays, 100 µg of mitochondrial protein were incubated with different
concentrations of [3H]ryanodine in 0.5 ml of binding
buffer (in mM: 170 KCl, 0.02 CaCl2, 10 MOPS, pH
7.0) for 16 h at 25 °C. For nonspecific binding, Ca2+ was replaced by 6 mM EGTA. At the end of
the incubation time, the reaction mixture was filtered under reduced
pressure through glass fiber filters (Whatman) and washed with ice-cold
buffer (170 mM KCl, 10 mM MOPS).
Measurements of Mitochondrial Net Calcium Uptake in Isolated Rat
Heart Mitochondria with a Ca2+-selective
Electrode--
Mitochondria (1 mg) were diluted in 1 ml of M/S buffer
containing 10 µM EGTA, and the free Ca2+
concentration was calculated to be 60 µM. Net
Ca2+ uptake was measured by monitoring changes in external
Ca2+ concentration in the reaction medium using a
Ca2+-selective electrode (Microelectrode Inc., Bedford, NH).
Rhod-2 Loading of Isolated Mitochondria--
Mitochondria were
suspended in M/S buffer containing 10 µM EGTA (to chelate
Ca2+ impurities in distilled water) to a final
concentration of ~10 mg of protein/ml. After incubation in 5 µM rhod-2 AM (Molecular Probes) for 10 min at room
temperature, mitochondria were washed three times to remove external
rhod-2. Mitochondria were used for experiments 30 min after finishing
the loading protocol to ensure cleavage of the rhod-2 AM into the
membrane-impermeable rhod-2 free acid.
Flash Photolysis of Caged Ca2+ and Measurement of
[Ca2+]m Dynamics--
For
[Ca2+]m measurement, isolated, rhod-2-loaded
mitochondria in a 0.5-µl droplet were adhered to a glass coverslip
and mounted on the stage of an Eclipse TE200 microscope equipped with a
SuperFluor 40X (1.3 numeric aperture oil immersion objective, Nikon).
This solution was mixed with 5 µl of "caged-Ca2+"
solution containing (in mM: 130 KCl, 10 HEPES, 10 succinate, 10 o-nitrophenyl EGTA, 5 CaCl2, 2 Mg-ATP, 1.2 MgCl2, pH 7.2). For the pulsatile photolytic
release of Ca2+ in the external media, a 1-ms flash of
~250 J of UV light (340-360 nm) was produced at 0.2 Hz.
Determination of relative changes in [Ca2+]m
was performed using digital imaging microscopy with a
monochrometer-based system and high speed CCD camera
(T.I.L.L.-Photonics). Rhod-2 labeled mitochondria were excited at
530 ± 15 nm and fluorescence emission collected using a 565 nm
long pass filter (Chroma). Images were acquired at a rate of 2-4 Hz
without binning and displayed as
Mitochondrial Swelling--
Mitochondrial swelling was induced
as described previously (23). Briefly, isolated heart mitochondria (1 mg of protein) were diluted in 1 ml of modified M/S buffer (in
mM: 120 KCl, 65 mannitol, 30 sucrose, 10 succinate, 5 Na2HPO4/NaH2PO4, 10 HEPES, pH 7.2). The absorbance was recorded with a spectrophotometer (Spectronic) at 540 nm for 2-3 min to obtain a stable base line, followed by the addition of Ca2+ to induce mitochondrial swelling.
Immunological Detection of RyR in Isolated Heart
Mitochondria--
Electron microscopic analysis revealed that ~70%
of isolated adult rat heart mitochondria, treated with gold-labeled
antibodies against SR-RyR, were labeled with 1-4 gold particles (Fig.
1A). The majority of the gold
particles were found in the cristae membrane of IMM (73.3% of total),
although some labeling was detected in the peripheral IMM. In contrast,
no significant labeling was found in the OMM and extramitochondrial
membranes (3.5% of total counted gold particles). In absence of the
RyR antibody, only 5% of the mitochondria showed labeling with a
maximum of 1 gold particle (Fig. 1B).
Using Western blot analysis, we confirmed the specific detection of
RyR-like protein in the IMM from osmotically shocked rat heart
mitochondria (Fig. 1C). Western blots performed on the
cytosolic and mitochondrial subfractions demonstrated immunoreactivity
against RyR in all fractions except the OMM (Fig. 1C,
anti-RyR). A positive signal against RyR protein in the IMM
was obtained in all preparations (n = 6). In all tested
fractions, the RyR antibody labeled a protein of ~600 kDa. The purity
of the IMM fraction was verified by the presence of SDH activity, an
enzyme localized exclusively in the IMM. Furthermore, this fraction was
free of SERCA and VDAC (Fig. 1C, anti-SERCA,
anti-VDAC) in all preparations, indicating that the IMM
fraction is devoid of any significant contamination from SR and OMM
fragments. In addition, SERCA pump proteins were not detected in four
independently isolated mitochondrial preparations (Fig. 1D),
indicating that even the intact mitochondria had minimal SR contamination.
An extensive effort to exclude the possibility that the mRyR was not
caused by contamination with SR-RyR was deemed essential, because
mitochondria are located in close proximity to the SR in cardiac muscle
cells (14, 24). The chosen marker proteins, such as SERCA, VDAC, and
SDH, are specific for their intracellular location. Labeling of the RyR
in the purified IMM with a specific antibody is consistent with the
result obtained by electron microscopy, showing that the mRyR is
localized within the IMM (Fig. 1A). The molecular weight of
the detected RyR proteins in the IMM and the cross-reactivity with the
used SR-RyR specific antibody suggests that the mRyR is structurally
homologous to the SR-RyR.
[3H]Ryanodine Binding to Isolated Heart
Mitochondria--
Various physiological and pharmacological effectors
including Ca2+, caffeine, Mg2+, and RR modulate
ryanodine binding to the SR-RyR (25, 26). To characterize the
pharmacological properties of mRyR, we studied [3H]ryanodine binding to heart mitochondria in presence
of these modulators. [3H]Ryanodine bound to isolated
heart mitochondria in presence of 20 µM Ca2+
with an apparent affinity (Kd) of 9.8 ± 2.1 nM (n = 3, in triplicate for all binding
experiments; Fig. 2A).
Depending on the experimental conditions employed, it has been reported that SR-RyR exhibited an apparent Kd between 2 and
200 nM for ryanodine (27, 28). The maximal density of mRyR
binding sites (Bmax) was 398.4 ± 12 fmol/mg of protein (n = 3; Scatchard plot in Fig.
2A), which is ~10 times less than that described for
[3H]ryanodine binding to purified SR membranes under
similar experimental conditions (28, 29). To confirm that
[3H]ryanodine binding was not due to contamination by SR,
binding was performed with equal amounts of proteins from intact
isolated mitochondria and mitochondrial subfractions. The results
showed that ~90 ± 5% (n = 3) of the total
[3H]ryanodine bound to mitochondria was due to binding to
the IMM and not to other fractions.
The amount of [3H]ryanodine binding to mitochondria as a
function of free Ca2+ concentration in the assay media was
biphasic. Binding increased at pCa between 5 and 7 and
decreased at pCa between 3 and 4 with maximal binding at
pCa 5.3 (Fig. 2B). Surprisingly, unlike the cardiac SR-RyR, caffeine did not enhance mitochondrial ryanodine binding (n = 3). Caffeine-insensitive RyR, however,
have been described in canine salivary glands (30) and in Jurkat cells (31).
Mg2+ is known to decrease the Bmax
for [3H]ryanodine binding and to inhibit SR
Ca2+ efflux in skeletal and cardiac muscle (26, 32, 33).
This is consistent with single-channel studies, where millimolar
concentrations of Mg2+ reduced the open probability of the
RyR and maintained the channel in a closed state (25, 26, 32).
Accordingly, we studied the effects of Mg2+ on
[3H]ryanodine binding in isolated heart mitochondria and
observed a 50% inhibition in presence of 0.33 mM
Mg2+ (n = 3, Fig. 2C). Under
comparable experimental conditions, it has been shown that up to 1 mM Mg2+ did not have any inhibitory effects on
ryanodine binding in cardiac SR-RyR (34-36). This suggests that, in
cardiac muscle cells, the mRyR is more sensitive to Mg2+
inhibition of [3H]ryanodine binding than the SR-RyR.
RR inhibits the release of Ca2+ from the SR in skeletal and
cardiac muscles by decreasing the open probability of the RyR (26, 37).
After RR treatment, [3H]ryanodine binding to isolated rat
heart mitochondria was strongly inhibited with IC50 = 105 nM (n = 3, Fig. 2D). This
suppression of mitochondrial [3H]ryanodine binding by RR
is much more potent than that observed in cardiac SR-RyR, which had
IC50 values between 290 and 1,000 nM (34, 35).
Consistent with the binding data, we have shown that RR (1-5
µM) blocks mitochondrial Ca2+ uptake without
much effect on SR Ca2+ release in chemically skinned
cardiac myocytes (14). These results indicate that significant
differences exist in the potency of RR in inhibiting mRyR and
SR-RyR.
The binding data provide pharmacological evidence of the existence of
mRyR in the IMM. They also show that there are distinct differences
between mRyR and SR-RyR with respect to their abundance and their
sensitivities to caffeine, Mg2+, and RR. Therefore, the
mRyR and SR-RyR would operate at different capacities under similar
conditions and could be regulated and modulated differentially.
Ryanodine Inhibition of Mitochondrial Ca2+
Uptake--
We next investigated functional aspects of mRyR in the
sequestration of Ca2+ using two different methods. Adding
mitochondria to a buffer containing 60 µM free
Ca2+ caused a significant mitochondrial Ca2+
uptake, as measured by a decrease in the extramitochondrial
Ca2+ concentration with a Ca2+-selective
microelectrode (Fig. 3A,
control). In the presence of 100 or 10 µM
ryanodine, mitochondrial Ca2+ uptake was suppressed by
60 ± 2.7% or 41.2 ± 1.9%, respectively (Fig.
3A, n = 15). Removal of the OMM with
digitonin (25 µg/mg of mitochondrial protein for 30 s) to
minimize possible SR-contamination had no significant effect on
mitochondrial Ca2+ uptake in the presence or absence of
ryanodine (Fig. 3D).
The same inhibitory effect in mitochondrial Ca2+ uptake was
observed with dantrolene, a compound that has been shown to inhibit the
skeletal muscle SR-RyR and therefore Ca2+ release (17). The
effect of dantrolene on the cardiac SR-RyR is still controversial
(38-40). However, incubation of isolated mitochondria with 10 µM dantrolene decreased mitochondrial Ca2+
uptake by 55.9 ± 7.8% (Fig. 3, B and D;
n = 5). Finally, the presence of 30 µM
cyclopiazonic acid (CPA), an inhibitor of SERCA, did not alter
mitochondrial Ca2+ uptake compared with untreated
mitochondria (96 ± 2.4%, n = 3), indicating
little contamination of the SR-RyR (Fig. 3, C and
D).
The relatively slow response time of Ca2+ microelectrodes
made them unable to follow the rapid mitochondrial Ca2+
uptake during Ca2+ pulses. To investigate whether the mRyR
contributes to rapid Ca2+ uptake, we measured the
Ca2+ responses of single or small clusters of isolated,
rhod-2-loaded mitochondria using high speed digital imaging in
combination with pulsatile flash photolysis of caged Ca2+.
As shown in Fig. 4 (A and
C), Ca2+ uptake was stimulated in rhod-2-loaded
isolated heart mitochondria after flash photolysis of caged
Ca2+ in the external solution. The slow decay in the
fluorescence may result from absence of Na+ in the solution
that inhibited Na+-dependent Ca2+
efflux. In Fig. 4B, a low affinity Ca2+
indicator Oregon Green Bapta-5N was used in separate experiments to
confirm that pulsatile flash photolysis resulted in elevations in
extramitochondrial Ca2+ that reached to their peak value
within sampling time of one frame (250-500 ms). After pre-incubating
mitochondria with 100 µM ryanodine, a significant
component of the evoked change in mitochondrial fluorescence was
suppressed (Fig. 4C). The cumulative change in fluorescence
(
The ability of mitochondria to sequester significant amount of
Ca2+ through a ryanodine inhibitory pathway suggests that
the mRyR could play an important role in buffering high concentrations of [Ca2+]c. Consistent with our experiments,
a decrease in [Ca2+]m in the presence of
ryanodine has been observed in A10 cells when perfused with more then 1 µM Ca2+ (42). Moreover, the ability of
mitochondria to respond instantaneously to fast Ca2+ pulses
through a ryanodine inhibitable pathway suggests that the mRyR may be
responsible for Ca2+ sequestration during heartbeats.
Inhibition of Mitochondrial Swelling by Ryanodine--
Excessive
accumulation of Ca2+ in the mitochondrial matrix
depolarizes the mitochondrial membrane potential and disrupts
fundamental mitochondrial functions like oxidative phosphorylation and
ATP production, which results in opening of the mitochondrial
permeability transition pore in isolated heart and liver mitochondria
(23, 43). This is accompanied by mitochondrial swelling and can be measured by the decrease of absorbance at 540 nm. Inducing
mitochondrial swelling in isolated intact heart mitochondria with 100 µM Ca2+ led to a decrease in absorbance of
19.5 ± 3.4% (n = 8). Upon preincubation with
2-20 µM ryanodine, we observed a
concentration-dependent inhibition of
Ca2+-induced mitochondrial swelling (Fig.
5A). Interestingly, less then
2 µM ryanodine significantly enhanced mitochondrial
swelling in all experiments (Fig. 5, A and B;
n = 6). CPA had no effect on the inhibition of
mitochondrial swelling after treatment with 20 µM
ryanodine (Fig. 6). In addition, the
concentration dependence in ryanodine-mediated inhibition of
mitochondrial swelling by ryanodine was also not altered by CPA
treatment (data not shown). An accelerated mitochondrial swelling in
the presence of less than 2 µM ryanodine or the
inhibition of swelling by higher ryanodine concentrations is in
agreement with the literature, where low ryanodine concentrations
switch the SR-RyR into an open state and higher concentrations keep the
Ca2+ channel in the RyR closed (17, 44).
Dantrolene (10 µM) was as effective as 20 µM ryanodine in blocking mitochondrial swelling (Fig. 6).
Finally, the SR-containing cytosolic fraction itself revealed no
significant changes in absorbance after the addition of
Ca2+ (Fig. 6). In these experiments the same amount of
cytosolic protein and experimental protocol was used as that described
for mitochondrial swelling. Therefore, the possibility that the effect
of mitochondrial swelling could be mimicked by cytosolic components
like the SR was excluded.
Mitochondrial swelling due to the opening of the mitochondrial
permeability transition pore has been implicated in triggering apoptosis and necrosis of several cell types (45). The ability of
ryanodine to prevent such mitochondrial swelling by blocking influx of
Ca2+ may provide some insights into the development of
novel therapeutic agents for mitochondria-mediated cell injury and death.
The present study demonstrates that mitochondria contain a RyR
within the IMM, which shares several, similar biochemical, pharmacological, and physiological properties with both the SR-RyR and
the CaUP. Based on the results presented here, a contamination of the
purified mitochondria by SR-RyR can be excluded for several reasons. 1)
Preparations of isolated mitochondria were free of detectable amounts
of SERCA protein. 2) The separated OMM fraction tested positive for
VDAC but negative for SDH activity. 3) The separated IMM tested
positive for SDH but negative for the OMM protein VDAC and the SR
protein SERCA. 4) CPA had no effect on mitochondrial Ca2+
uptake and mitochondrial swelling. 5) [3H]Ryanodine
binding was more sensitive to Mg2+ and RR inhibition. 6)
Caffeine had no effect on ryanodine binding.
The localization of mRyR in the IMM may raise the question of whether
the RyR, like other intracellular membrane proteins, could have
multiple intracellular locations. It has been shown that the inositol
1,4,5-triphosphate receptor is localized in the nucleus (46, 47) and
the plasma membrane (48) in addition to the ER and SR. Likewise,
proteins of the cell death-regulating Bcl-2 family have been shown to
be localized in the OMM, the nucleus membrane, and the ER membranes
(49). A Na+/Ca2+ exchanger is localized in the
IMM but also in the plasma membrane (1).
An increase in [Ca2+]c activates the RyR in
the SR or ER to release Ca2+ from intracellular stores due
to an outwardly directed Ca2+ electrochemical gradient.
Conversely, the inwardly directed Ca2+ electrochemical
gradient in mitochondria could result in Ca2+-induced
Ca2+ uptake following the activation of mRyR. Finally,
because of the pharmacological similarities to the CaUP, it is tempting
to speculate that the mRyR is the CaUP.
The proximity between the mitochondria and other Ca2+
transport proteins, such as SR- or ER-RyR and L-type Ca2+
channels, would allow mitochondria to sense microdomains with Ca2+ concentrations sufficient to open the mRyR (Fig.
7). Ca2+ released by SR-RyR
has been shown to activate mitochondrial Ca2+ uptake and
subsequently causes an increase in electron transport chain activity
and NAD(P)H fluorescence (50). The rapid influx of Ca2+
into the mitochondria would also contribute to local cytosolic Ca2+ buffering, thus regulating biological processes such
as ATP production and intracellular Ca2+ signaling.
Conversely, dysregulation in a mRyR may play an important role in the
development of disease states such as heart failure and
neurodegeneration.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C. Gold labeling of ultrathin sections of embedded
mitochondria against RyR was performed as described previously (21,
22).
F/F0, where
F/F0 = 100[(F
F0)/F0], and
F is the recorded fluorescence and F0
was obtained from the average of 15 sequential frames prior to
uncaging. Estimation of the evoked changes in external Ca2+
([Ca2+]ext) was performed using the low
affinity Ca2+ indicator, Oregon Green Bapta 5N (OGB-5N;
Molecular Probes). Using a range of standard solutions buffered at set
Ca2+ concentrations (Molecular Probes) and the equation
[Ca2+]ext = Kd
[(F
Fmin)/(Fmax
F)], the dissociation constant (Kd),
Fmin, and Fmax were
determined to be 21 µM, 57, and 1050, respectively. For
measurement of [Ca2+]ext, a 5-µl droplet
containing 50 µM OGB-5N was excited at 488 ± 15 nm
and fluorescence emission collected through a 525 ± 25 nm band
pass filter (Chroma).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Immunogold labeling and Western blot analysis
of a RyR in the IMM. A, immunogold labeling of RyR
(arrows) in the IMM of isolated adult rat heart mitochondria
(original magnification, ×50,000). Mitochondria were isolated as
described under "Experimental Procedures." The final sediment
containing the purified mitochondria was fixed in paraform aldehyde and
embedded in low temperature resin prior to immunogold labeling.
B, as a negative control the gold-labeled secondary antibody
was applied in absence of the RyR antibody, yielding no significance
detection of RyR protein. C, Western blot analysis against
RyR of mitochondrial subfractions from isolated and osmotically shocked
heart mitochondria. Characterized subfractions were centrifuged for 90 min at 300,000 × g to sediment the RyR. C,
SR containing cytosol; M, purified intact mitochondria;
CS, contact sites; anti-RyR, labeling against
RyR; anti-SERCA and anti-VDAC, Western blots
against SERCA and VDAC as negative controls for contamination of the
IMM with cytosolic membrane fragments and OMM. Arrows
indicate the position and the molecular weight of the marker molecules.
D, cytosol and mitochondria of four independent performed
preparations were probed against SERCA. Lanes
1C-4C represent the cytosolic fractions obtained after the
first centrifugation of the homogenate at 12,000 × g.
Lanes 1M-4M represent the corresponding
mitochondria after Percoll purification. C, cytosol;
M, mitochondria.
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Fig. 2.
[3H]Ryanodine binding to
isolated mitochondria in the presence or absence of modulators of the
RyR. All assays were done in triplicate, and all figures represent
a mean of at least three independent experiments. A,
ryanodine binding to isolated heart mitochondria. Data were fit for a
single class of binding sites by Scatchard plot (inset).
Bmax was 398 ± 12 fmol/mg of protein, and
the Kd was 9.52 nM (r = 0.97 for linear regression). B, bound
[3H]ryanodine; F, free
[3H]ryanodine. B, Ca2+ dependence
of [3H]ryanodine binding. 100 µg of mitochondrial
protein was incubated with various concentration of Ca2+ in
presence of 9 nM [3H]ryanodine. C,
inhibition of [3H]ryanodine binding with different
concentrations of MgCl2. D, inhibition of
[3H]ryanodine binding by RR.
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Fig. 3.
Ryanodine inhibits mitochondrial
Ca2+ uptake. Mitochondria were incubated with
ryanodine, CPA, or dantrolene and added to M/S buffer containing 60 µM free Ca2+, and the external
Ca2+ concentration was measured with a
Ca2+-sensitive microelectrode. A, this panel
shows a representative experiment of ryanodine-inhibited mitochondrial
Ca2+ uptake. Mitochondria were preincubated with the
indicated concentration of ryanodine for 15 min at room temperature,
and the changes of the Ca2+ concentration in the M/S buffer
were measured. B, after incubation with 10 µM
dantrolene for 10 min at room temperature, mitochondrial
Ca2+ uptake was inhibited by 55%. C,
pretreatment of mitochondria with 30 µM CPA for 10 min at
room temperature did not affect mitochondrial Ca2+ uptake.
D, inhibition of mitochondrial Ca2+ uptake in
the presence or absence of modulators of the RyR. Maximal
Ca2+ uptake in untreated heart mitochondria was set to be
100%. Dig, removal of OMM with 25 µg of digitonin/mg of
mitochondrial protein prior measuring Ca2+ uptake;
CPA, 30 µM CPA; Rya, 100 µM ryanodine; Dig+Rya, digitonin treatment
plus incubation with 100 µM ryanodine; Dan, 10 µM dantrolene. Each bar represents the
averaged value of at least three independent experiments, and values
are given ± S.E.
F) evoked by each flash was 0.14 ± 0.02, 0.34 ± 0.05, 0.58 ± 0.08, 0.85 ± 0.04, and 1 for control; and 0.05 ± 0.02, 0.12 ± 0.04, 0.25 ± 0.07, 0.40 ±
0.09, and 0.60 ± 0.13 for ryanodine-treated mitochondria
(n = 6, Fig. 4D). The magnitude of
[Ca2+]m at higher number of flashes in the
control solution could be an underestimation, due to the saturation of
rhod-2 (Kd = 0.6-0.8 µM). This could
account for the decrease in the slope of control curve between flash
number 4 and 5 in Fig. 4D. Finally, this mitochondrial
Ca2+ uptake was not due to release of Ca2+ from
SR contamination, because repetitive UV flashes applied to
rhod-2-loaded mitochondria in a droplet containing 100 µM
D-myo-inositol 1,4,5-trisphosphate,
P4(5)-1-(2-nitrophenyl)ethyl ester, a concentration of
caged inositol 1,4,5-trisphosphate shown to evoke robust
Ca2+ release from reticular stores (41), induced no
measurable
F.
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Fig. 4.
Ca2+ dynamics of individual or
small clusters of isolated, rhod-2-loaded heart mitochondria induced by
photolytic release of caged Ca2+. A, raw
fluorescence image and the corresponding pseudocolored digital
images of isolated mitochondria evoked by repetitive flash photolysis
of 10 o-nitrophenyl EGTA, 50% bound with Ca2+,
in the external solution. Each image displayed is the first frame (320 ms) following the flash lamp artifact and F reflect rises
in [Ca2+]m. B, flash application
(dots) produced micromolar Ca2+ transients in
the extramitochondrial solution as estimated with OGB-5N
(Kd = 21 µM). C,
representative fluorescence recordings showing the flash-induced
changes in [Ca2+]m as shown in A
for both control (black trace) and 100 µM ryanodine-treated (red trace)
mitochondria. Breaks in records indicate removal of flash lamp
artifacts. D, averaged data comparing the cumulative
F produced by each flash for matched control and
ryanodine-treated mitochondria.
F is the difference of
measured fluorescence directly prior and after flash application. Data
from control and ryanodine-treated mitochondria were normalized to the
maximal cumulative
F for the control response of each
experiment. Data are expressed as mean ± S.E., and p
values <0.05 by paired t test were considered statistically
significant (asterisks). Data are normalized to the maximal
F for the control response of each experiment.
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Fig. 5.
Ryanodine inhibits Ca2+-induced
mitochondrial swelling. In all experiments, 1 mg of mitochondrial
protein were dissolved in modified M/S buffer and swelling was induced
by the addition of 100 µM Ca2+. A,
this figure shows a representative concentration-response curve of
ryanodine-inhibited mitochondrial swelling. Heart mitochondria were
preincubated for 15 min at room temperature with the indicated
concentration of ryanodine. Ca2+ was added at time 0 (arrow), and the change in absorbance at 540 nm was
followed. Data are expressed as the ratio of the absorbance at any
given time (A) divided by the base-line absorbance at time 0 (Ao). B, averaged changes of the inhibitory
effect of ryanodine (Rya) on mitochondrial swelling. Each
bar represents at least six independently performed
experiments. Data are expressed as mean values ± S.E., and
p values <0.01 by paired t test were considered
statistically significant (asterisks).
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Fig. 6.
Mitochondrial swelling in the presence or
absence of modulators of the RyR or SERCA. Each bar
represents the averaged decrease in absorbance of at least three
independently performed experiments. Experiments were performed as in
Fig. 5. Ca2+, 100 µM
Ca2+; CPA, incubation with 50 µM
CPA for 10 min at room temperature prior the addition of
Ca2+; Dan, preincubation with 10 µM dantrolene for 10 min; Rya, 20 µM ryanodine. C, SR containing cytosolic
fraction. Data are expressed as mean values ± S.E.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 7.
A model of Ca2+ dynamics in
cardiac myocytes. Mitochondria sense microdomains with high
Ca2+ following activation of voltage-gated Ca2+
channels (VGCC) and Ca2+ release from
intracellular stores. These raises could trigger fast mitochondrial
Ca2+ uptake via the mRyR and/or CaUP, which may ultimately
affect physiological and pathological processes.
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ACKNOWLEDGEMENTS |
---|
The antibody against RyR developed by J. Airey and J. Sutko was obtained from the Developmental Studies Hybridoma Bank maintained by the Department of Biological Sciences of the University of Iowa (Iowa City, IA). Electron microscopy was done by Karen de Mesy Jensen (Department of Pathology, University of Rochester, Rochester, NY). We thank Drs. R. T. Dirksen, P. M. Hinkle, C. Franzini-Armstrong, current and past members of the Sheu laboratory for helpful comments, critical reading, and discussions on the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant HL-33333, National Institutes of Health Grant DK-54568, American Heart Association Grant 9920244T, and American Heart Association Grant 0050839T.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.
These authors contributed equally to this work.
§ To whom correspondence should be addressed: Dept. of Pharmacology and Physiology, Box 711, University of Rochester, School of Medicine and Dentistry, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 716-275-3381; Fax: 716-273-2652; E-mail: sheyshing_sheu@urmc.rochester.edu.
Published, JBC Papers in Press, April 10, 2001, DOI 10.1074/jbc.M101486200
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ABBREVIATIONS |
---|
The abbreviations used are: [Ca2+]m, mitochondrial Ca2+ concentration; [Ca2+]c, cytosolic Ca2+ concentration; CaUP, mitochondrial calcium uniporter; RR, ruthenium red; SR, sarcoplasmic reticulum; RyR, ryanodine receptor, SR-RyR, sarcoplasmic ryanodine receptor; mRyR, mitochondrial ryanodine receptor; SERCA, sarco- and endoplasmic reticulum Ca2+-ATPase; CPA, cyclopiazonic acid; IMM, inner mitochondrial membrane; OMM, outer mitochondrial membrane; VDAC, voltage-gated anion channel; MOPS, 4-morpholinepropanesulfonic acid; SDH, succinate dehydrogenase; M/S, mannitol/sucrose; OGB-5N, Oregon Green Bapta 5N.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Gunter, T. E.,
Gunter, K. K.,
Sheu, S-S.,
and Gavin, C. E.
(1994)
Am. J. Physiol.
267,
C313-C339 |
2. |
Duchen, M. R.
(2000)
J. Physiol. (London)
529,
57-68 |
3. | Bernardi, P., Petronilli, V., Di Lisa, F., and Forte, M. (2001) Trends Biochem. Sci. 26, 112-117[CrossRef][Medline] [Order article via Infotrieve] |
4. | Nishikawa, T., Edelstein, D., Du, X. L., Yamagishi, S., Matsumura, T., Kaneda, Y., Yorek, M. A., Beebe, D., Oates, P. J., Hammes, H. P., Giardino, I., and Brownlee, M. (2000) Nature 404, 787-790[CrossRef][Medline] [Order article via Infotrieve] |
5. | Beal, M. F. (1996) Curr. Opin. Neurobiol. 6, 661-666[CrossRef][Medline] [Order article via Infotrieve] |
6. |
Wallace, D. C.
(1999)
Science
283,
1482-1488 |
7. |
Babcock, D. F.,
Herrington, J.,
Goodwin, P. C.,
Park, Y. B.,
and Hille, B.
(1997)
J. Cell Biol.
136,
833-844 |
8. | Sheu, S.-S., and Jou, M. J. (1994) J. Bioenerg. Biomembr. 26, 487-493[Medline] [Order article via Infotrieve] |
9. | Rizzuto, R., Brini, M., Murgia, M., and Pozzan, T. (1993) Science 262, 744-747[Medline] [Order article via Infotrieve] |
10. |
Simpson, P. B.,
and Russell, J. T.
(1996)
J. Biol. Chem.
271,
33493-33501 |
11. | Hajnoczky, G., Robb-Gaspers, L. D., Seitz, M. B., and Thomas, A. P. (1995) Cell 82, 415-424[Medline] [Order article via Infotrieve] |
12. | Chacon, E., Ohata, H., Harper, I. S., Trollinger, D. R., Herman, B., and Lemasters, J. J. (1996) FEBS Lett. 382, 31-36[CrossRef][Medline] [Order article via Infotrieve] |
13. |
Duchen, M. R.,
Leyssens, A.,
and Crompton, M.
(1998)
J. Cell Biol.
142,
975-988 |
14. | Sharma, V., Ramesh, V., Franzini-Armstrong, C., and Sheu, S.-S. (2000) J. Bioenerg. Biomembr. 32, 97-104[CrossRef][Medline] [Order article via Infotrieve] |
15. | Hueser, J., Blatter, L. A., and Sheu, S.-S. (2000) J. Bioenerg. Biomembr. 32, 27-33[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Sparagna, G. C.,
Gunter, K. K.,
Sheu, S.-S.,
and Gunter, T. E.
(1995)
J. Biol. Chem.
270,
27510-27515 |
17. |
Franzini-Armstrong, C.,
and Protasi, F.
(1997)
Physiol. Rev.
77,
699-729 |
18. | McCormack, J. G., and Denton, R. M. (1993) Biochem. Soc. Trans. 21, 793-799[Medline] [Order article via Infotrieve] |
19. | Rehncrona, S., Mela, L., and Siesjo, B. K. (1979) Stroke 10, 437-446[Medline] [Order article via Infotrieve] |
20. | Ohlendieck, K., Riesinger, I., Adams, V., Krause, J., and Brdiczka, D. (1986) Biochim. Biophys. Acta 860, 672-689[Medline] [Order article via Infotrieve] |
21. | Herrera, G. A. (1989) Ultrastruct. Pathol. 13, 485-499[Medline] [Order article via Infotrieve] |
22. | de Mesy Jensen, K. L., and di Sant'Agnese, P. A. (1992) Ultrastruct. Pathol. 16, 51-59[Medline] [Order article via Infotrieve] |
23. |
Petronilli, V.,
Cola, C.,
Massari, S.,
Colonna, R.,
and Bernardi, P.
(1993)
J. Biol. Chem.
268,
21939-21945 |
24. | Ogata, T., and Yamasaki, Y. (1997) Anat. Rec. 248, 214-223[CrossRef][Medline] [Order article via Infotrieve] |
25. | Pessah, I. N., Stambuk, R. A., and Casida, J. E. (1987) Mol. Pharmacol. 31, 232-238[Abstract] |
26. | Meissner, G., Darling, E., and Eveleth, J. (1986) Biochemistry 25, 236-244[Medline] [Order article via Infotrieve] |
27. |
Inui, M.,
Saito, A.,
and Fleischer, S.
(1987)
J. Biol. Chem.
262,
15637-15642 |
28. | Lindsay, A. R., and Williams, A. J. (1991) Biochim. Biophys. Acta 1064, 89-102[Medline] [Order article via Infotrieve] |
29. |
Kijima, Y.,
Saito, A.,
Jetton, T. L.,
Magnuson, M. A.,
and Fleischer, S.
(1993)
J. Biol. Chem.
268,
3499-3506 |
30. | Yamaki, H., Morita, K., Kitayama, S., Imai, Y., Itadani, K., Akagawa, Y., and Dohi, T. (1998) J. Dent. Res. 77, 1807-1816[Abstract] |
31. | Hakamata, Y., Nishimura, S., Nakai, J., Nakashima, Y., Kita, T., and Imoto, K. (1994) FEBS Lett. 352, 206-210[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Meissner, G.,
and Henderson, J. S.
(1987)
J. Biol. Chem.
262,
3065-3073 |
33. |
Liu, W.,
Pasek, D. A.,
and Meissner, G.
(1998)
Am. J. Physiol.
274,
C120-V128 |
34. | Pessah, I. N., Waterhouse, A. L., and Casida, J. E. (1985) Biochem. Biophys. Res. Commun. 128, 449-456[Medline] [Order article via Infotrieve] |
35. | Holmberg, S. R., and Williams, A. J. (1990) Biochim. Biophys. Acta 1022, 187-193[Medline] [Order article via Infotrieve] |
36. | Zimanyi, I., and Pessah, I. N. (1991) J. Pharmacol. Exp. Ther. 256, 938-946[Abstract] |
37. |
Xu, L.,
Tripathy, A.,
Pasek, D. A.,
and Meissner, G.
(1999)
J. Biol. Chem.
274,
32680-32691 |
38. |
Fruen, B. R.,
Mickelson, J. R.,
and Louis, C. F.
(1997)
J. Biol. Chem.
272,
26965-26971 |
39. | Meissner, A., Min, J. Y., Haake, N., Hirt, S., and Simon, R. (1999) Eur. J. Heart Fail. 1, 177-186[Medline] [Order article via Infotrieve] |
40. | Fratea, S., Langeron, O., Lecarpentier, Y., Coriat, P., and Riou, B. (1997) Anesthesiology 86, 205-215[Medline] [Order article via Infotrieve] |
41. |
Giovannucci, D. R.,
Groblewski, G. E.,
Sneyd, J.,
and Yule, D. I.
(2000)
J. Biol. Chem.
275,
33704-33711 |
42. |
Nassar, A.,
and Simpson, A. W.
(2000)
J. Biol. Chem.
275,
23661-23665 |
43. | Halestrap, A. P., and Davidson, A. M. (1990) Biochem. J. 268, 153-160[Medline] [Order article via Infotrieve] |
44. | McGrew, S. G., Wolleben, C., Siegl, P., Inui, M., and Fleischer, S. (1989) Biochemistry 28, 1686-1691[Medline] [Order article via Infotrieve] |
45. |
Bernardi, P.,
Scorrano, L.,
Colonna, R.,
Petronilli, V.,
and Di Lisa, F.
(1999)
Eur. J. Biochem.
264,
687-701 |
46. |
Mak, D. O.,
and Foskett, J. K.
(1994)
J. Biol. Chem.
269,
29375-29378 |
47. | Putney, J. W. (1997) Cell Calcium 21, 257-261[Medline] [Order article via Infotrieve] |
48. |
Munger, S. D.,
Gleeson, R. A.,
Aldrich, H. C.,
Rust, N. C.,
Ache, B. W.,
and Greenberg, R. M.
(2000)
J. Biol. Chem.
275,
20450-20457 |
49. | Kroemer, G. (1997) Nat. Med. 3, 614-620[Medline] [Order article via Infotrieve] |
50. |
Szalai, G.,
Csordas, G.,
Hantash, B. M.,
Thomas, A. P.,
and Hajnoczky, G.
(2000)
J. Biol. Chem.
275,
15305-15313 |