EDITORIAL FOCUS
Matrix free Mg2+ and the
regulation of mitochondrial volume
Dennis W.
Jung and
Gerald P.
Brierley
Department of Medical Biochemistry, College of Medicine, The
Ohio State University, Columbus, Ohio 43210
 |
ABSTRACT |
Mitochondria must maintain volume homeostasis in
order to carry out oxidative phosphorylation. It has been postulated
that the concentration of free
Mg2+
([Mg2+]) serves as the
sensor of matrix volume and regulates a
K+-extruding
K+/H+
antiport (K. D. Garlid. J. Biol. Chem.
255: 11273-11279, 1980). To test this hypothesis, the fluorescent
probe furaptra was used to monitor
[Mg2+] and free
Ca2+ concentration ([Ca2+]) in the matrix of
isolated beef heart mitochondria, and
K+/H+
antiport activity was measured by passive swelling in potassium acetate. Concentrations that result in 50% inhibition of maximum activity of 92 µM matrix [Mg2+] and 2.2 µM
[Ca2+] were determined for the
K+/H+ antiport. Untreated mitochondria average
670 µM matrix [Mg2+], a value that would permit <1%
of maximum
K+/H+
antiport activity. Hypotonic swelling results in large decreases in
matrix [Mg2+], but
swelling due to accumulation of acetate salts does not alter
[Mg2+]. Swelling in
phosphate salts decreases matrix
[Mg2+], but not to
levels that permit appreciable antiport activity. We conclude that
1) it is unlikely that matrix
[Mg2+] serves as the
mitochondrial volume sensor, 2) if
K+/H+
antiport functions as a volume control transporter, it is probably regulated by factors other than
[Mg2+], and
3) alternative mechanisms for
mitochondrial volume control should be considered.
potassium-hydrogen antiport; heart mitochondria; mitochondrial free
magnesium concentration; mitochondrial volume control; furaptra
 |
INTRODUCTION |
MITOCHONDRIA IN SITU maintain a high negative membrane
potential (
) in an intracellular milieu containing >140 mM
K+. Mitchell (35) recognized that
the electrophoretic inward movement of
K+ would constitute a threat to
the osmotic integrity of the mitochondrion and that a mechanism for
volume control was necessary. He proposed that an electroneutral
K+/H+
antiporter must be present to extrude excess entering
K+. The presence of such a
transporter initially proved difficult to demonstrate, but it is now
clear that
K+/H+
antiport activity can be unmasked when mitochondrial
Mg2+ is depleted and the pH
elevated in hypotonic media (see Refs. 9 and 15 for reviews). In
addition, a
K+/H+
antiporter has been isolated and reconstituted (33), and a brief report
of its cloning and partial sequencing has appeared (45).
The permeability of mitochondria to
K+ increases with increasing

in vitro (12), and an ATP-sensitive electrophoretic
K+ channel is also present (see
Ref. 16 for review). It is now thought that electrophoretic
K+ influx and electroneutral
efflux must be regulated to maintain mitochondrial volume homeostasis
(16). An Mg2+-sensitive
electrophoretic anion channel (4) is also present and is believed to
participate in net salt extrusion and osmotic contraction.
Garlid (14, 15) has proposed that the mitochondrial
K+/H+
antiporter is regulated by the matrix free
Mg2+ concentration
([Mg2+]). In this
"Mg2+ carrier-brake" model,
matrix [Mg2+] is
postulated to serve as the volume sensor. Mitochondrial swelling due to
the uptake of K+ salts of
Mg2+-liganding anions, such as
citrate and phosphate, would decrease matrix free
[Mg2+] and promote the
disassociation of Mg2+ bound to
negative regulatory sites on the
K+/H+
antiport and the anion channel. The resulting activation of these two
transporters would bring about net salt efflux and osmotic contraction.
This system is seen as adjusting the steady-state rate of
K+/H+
antiport to equal the rate of electrophoretic
K+ uniport so that matrix volume
remains unchanged (15).
Although this elegant model was proposed several years ago, evidence
that matrix [Mg2+] has
properties consistent with those of a volume sensor is inconclusive (9,
21, 27), and a precise relationship between matrix [Mg2+] and
K+/H+
antiport has yet to be established. It is well known that mitochondria can take up and extrude Mg2+ by
respiration-dependent processes and that matrix
[Mg2+] can vary with
metabolic conditions (see Ref. 27 for review). It is also clear that
the antiport is reversibly inhibited by [Mg2+] (15). However,
to confirm that the antiport is regulated by matrix
[Mg2+], it must be
shown that matrix
[Mg2+] varies in the
concentration range that controls antiport activity (18). To confirm
that matrix [Mg2+]
acts as a mitochondrial volume sensor, its concentration should be
shown to vary in an appropriate way with volume changes.
The availability of the fluorescent probe furaptra as an indicator of
free [Mg2+] (39) makes
a direct test of these issues feasible. This laboratory has defined the
properties of furaptra when it is sequestered in the mitochondrial
matrix (25, 28) and, more recently, has used this probe to examine the
relationship between total mitochondrial Mg2+ and matrix
[Mg2+] (29). The
present study evaluates matrix
[Mg2+] changes with
mitochondrial swelling under three different conditions and compares
the rate of
K+/H+
antiport, as measured by mitochondrial swelling in a hypotonic potassium acetate medium, with matrix
[Mg2+], as reported by
the fluorescence of furaptra. The results indicate that matrix
[Mg2+] does not change
in a way that is consistent with the role of a volume sensor or that of
a regulator of
K+/H+
antiport. It is concluded that alternative mechanisms for sensing mitochondrial volume and regulating osmotic contraction should be considered.
 |
EXPERIMENTAL PROCEDURES |
Furaptra loading and divalent cation depletion of mitochondria.
Beef heart mitochondria were prepared as previously described (10) and
suspended in 0.25 M sucrose-5 mM TES, pH 7.4, at 25 mg of protein per
milliliter. Furaptra (also called mag-fura 2) was loaded into the
mitochondrial matrix by incubating 25 mg of mitochondria in 2 ml of
sucrose-TES containing 0.1 mM EGTA, 20 mM NaCl, 1 mM ATP sodium salt,
and 8 µM furaptra-AM (Molecular Probes) at 24°C. After 18 min, 6 ml of a KCl medium (100 mM KCl, 15 mM HEPES, 1 mM EGTA, 1 mM EDTA, pH
7.4) were added, then 2 µM BrA-23187 was added to deplete the
mitochondria of divalent cations. After an additional 5-min incubation,
the mitochondria were diluted with 8 ml of ice-cold KCl medium and
collected by centrifugation (11,000 g
for 10 min). The pellets were resuspended in 10 ml of a KCl medium
containing less EGTA (100 µM) and no EDTA, reisolated (11,000 g for 10 min), and resuspended at 25 mg/ml in KCl-HEPES containing 15 µM EGTA. During the depletion procedure, endogenous Mg2+ is
reduced from 30-40 nmol/mg protein to ~2 nmol/mg, as estimated by atomic absorption analysis (8). Furaptra loading of mitochondria without cation depletion was carried out as previously described (25,
26). Ionophores were obtained from Calbiochem.
Swelling measurements.
Passive osmotic swelling was followed as the light-scattering change at
520 nm with a Brinkman PC801 colorimeter, as previously described (11).
The light-scattering variable
was calculated from reciprocal
absorbance, as described by Beavis et al. (5). The swelling records
shown in Fig. 5A were made using an
SLM DW-2C spectrophotometer in the split-beam mode.
Fluorescence measurements.
The fluorescence of furaptra was measured as emission at 510 nm with
excitation ratios taken at 340/380 nm with a Perkin-Elmer LS-5B
fluorometer (25). The fluorescence can be calibrated in terms of matrix
[Mg2+] or
Ca2+ concentration
([Ca2+]) by the ratios
method (19), since Ca2+ binding to
furaptra leads to changes in the fluorescence spectra that are quite
similar to those due to Mg2+ (39).
Because the background fluorescence of mitochondria is significant
relative to that of furaptra, each experiment was repeated using
mitochondria that did not contain furaptra, and net fluorescence
intensities (furaptra-loaded
nonloaded) were used to form
ratios before calculating
[Mg2+] or
[Ca2+], as previously
described (25, 26).
The dissociation constant
(Kd)
for Mg2+-furaptra is very
dependent on ionic strength. The values determined by Jung et al. (28) of 1.29 mM in 55 mM and 2.1 mM in 100 mM
K+ salts, respectively, were used
to calculate matrix
[Mg2+]. The
Kd of
Ca2+-furaptra in 55 mM
K+ salts did not appear to be
available. A value of 13.7 µM was determined by recording excitation
spectra at pH 7.8 and 25°C in a 55 mM KCl medium containing 5 mM
TES, 2 mM nitrilotriacetic acid, 227 nM
K+-furaptra, and variable
CaCl2 (0-2.52 mM) to give
[Ca2+] from 0 to 0.6 mM. The [Ca2+] was
calculated using the program of Schoenmakers et al. (42) and taking the
apparent log stability constant for
Ca2+-nitrilotriacetic
acid to be 6.80 for the conditions specified.
The basic reaction medium contained 55 mM potassium acetate, 5 mM TES
(pH 7.8, 25°C), 30 µM EGTA, 2 µg/ml oligomycin, 2 µg/ml rotenone, and 0.5 mg/ml furaptra-loaded, divalent cation-depleted mitochondria. This corresponds closely to the 110 mosM medium of
Nakashima and Garlid (37). Other additions or modifications are noted
in the figure legends.
Matrix pH was determined using the fluorescence of
carboxyseminaphthorhodofluor-1 (SNARF-1), as described by Baysal et al. (3), or 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein
(BCECF), as described by Brierley et al. (11).
 |
RESULTS |
Inhibition of
K+/H+
antiport by
[Mg2+].
It has been established that passive swelling in potassium acetate is a
convenient and reliable measure of
K+/H+
antiport activity (17, 37). Swelling is due to the rapid uptake of
acetic acid, its ionization, and the exchange of matrix H+ for external
K+ on the activated
K+/H+
antiport to give a net accumulation of potassium acetate (17). Beef
heart mitochondria depleted of divalent cations swell passively when
suspended in this medium (10), which indicates that the K+/H+
antiport has been activated. There is essentially no swelling in the
absence of divalent cation depletion (10).
The swelling is inhibited in a concentration-dependent manner by
MgCl2 added to the medium (Fig.
1A). The
matrix [Mg2+] as
determined from furaptra fluorescence in a parallel incubation is shown
in Fig. 1B. Exogenous
Mg2+ enters the divalent
cation-depleted mitochondria on residual BrA-23187 in exchange for 2 H+ and requires 150-200 s to
reach a steady state (Fig. 1B).
Matrix [Mg2+] does not
equilibrate with external
[Mg2+] under these
conditions, since lysis of the mitochondria with detergent results in a
large fluorescence signal increase (not shown) (28). With 1 mM
MgCl2 added to the medium, matrix
[Mg2+] is only ~250
µM after 200 s (Fig. 1B). Because
furaptra measures matrix
[Mg2+] directly, the
failure of the BrA-23187 to equilibrate
[Mg2+] across the
inner membrane is not relevant to the present measurements or
conclusions.

View larger version (42K):
[in this window]
[in a new window]
|
Fig. 1.
Inhibition of
K+/H+
antiport activity by matrix free
Mg2+ concentration
([Mg2+]). Beef heart
mitochondria were depleted of divalent cations and loaded with
furaptra. Depleted mitochondria were suspended at 0.5 mg/ml in basic
reaction medium (pH 7.8) containing indicated concentrations of
MgCl2.
A:
K+/H+
antiport activity measured by passive swelling in hypotonic
K+ acetate. Light scattering was
recorded, and light-scattering variable ( ) was calculated.
B: matrix
[Mg2+] estimated in a
parallel incubation by means of furaptra fluorescence and calculated
using a dissociation constant
(Kd) of 1.29 mM. C: matrix pH determined under
identical conditions with carboxyseminaphthorhodofluor-1
(SNARF-1)-loaded mitochondria and calculated as described in Ref. 3.
There was no change in pH record in presence of 0.6 mM
MgCl2.
|
|
The swelling shows a lag of 30-40 s, a maximum rate that is
maintained for
50 s, and then a decline as the incubation continues (Fig. 1A) (11). It is known that
the
K+/H+
antiport is strongly affected by pH (6, 11, 37). The rate of swelling
in potassium acetate increases without apparent limit as external pH is
increased (37). This may be due in part to decreased competition
between K+ and
H+ for an external catalytic site
(11), but the
K+/H+
antiport has also been shown to be inhibited allosterically by matrix
protons (6, 11). It is also well established that depletion of matrix
divalent cations with A-23187 results in decreased matrix pH (6, 11).
However, the mitochondria used in this study were depleted of divalent
cations in a KCl medium at pH 7.4 and washed in KCl, conditions that
minimize matrix acidification. A parallel incubation with mitochondria
loaded with SNARF-1 (Fig. 1C)
indicates that when the depleted mitochondria are suspended in
potassium acetate medium at pH 7.8, matrix pH is initially 7.55 but
increases to pH 7.7 at ~50 s and 7.75 at 100 s (Fig. 1C; see also Fig. 5 in Ref. 11 for
BCECF records of a quite similar protocol). This establishes that the
pH is
0.10 to
0.05 when the swelling reaches its
maximum rate (Fig. 1A) and that the inhibition due to added
[Mg2+] is not
complicated by changing H+
concentration. Also, the
Kd for
Mg2+-furaptra is relatively
insensitive to pH in this range (32).
The fastest rates of swelling (
/min) after the lag phase were
determined from Fig. 1A and plotted
as the percentage of maximum K+/H+
activity vs. the matrix
[Mg2+] at the time
these rates were achieved (Fig. 2). The
data were fit to a sigmoidal curve, and the concentration that results
in 50% inhibition of maximum activity
(IC50) of
K+/H+
activity is 92 µM Mg2+. The
average value of matrix
[Mg2+] (670 µM) that
is found in our preparations of furaptra-loaded heart mitochondria (29)
is indicated in Fig. 2.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 2.
K+/H+
antiport activity as a function of matrix
[Mg2+]. Fastest
swelling rates after initial lag period were determined at ~100 s
from traces in Fig. 1A by linear
regression analysis.
[Mg2+] values were
taken from Fig. 1B at time fastest
rate was measured. Data were curve fit to a sigmoidal plot with
Graphpad and then normalized, with maximum and minimum set to 100 and
0%, respectively. Concentration that results in 50% inhibition of
maximum activity (IC50) of
K+/H+
activity by matrix
[Mg2+] is 92 µM.
Average matrix [Mg2+]
of furaptra-loaded heart mitochondria is 670 µM (arrow) (29).
|
|
Inhibition of
K+/H+
antiport by
[Ca2+].
The procedure used for Mg2+ (Fig.
1) was repeated to evaluate inhibition due to
Ca2+ by adding 0-1.0 mM
CaCl2 to the medium (records not
shown). The fastest rates of swelling and the matrix
[Ca2+] corresponding
to the time of the fastest swelling rates were determined and plotted
as percent
K+/H+
activity vs. matrix
[Ca2+] (Fig.
3). These data were curve fit to a
hyperbola, and a linear Hanes plot indicates an
IC50 of 2.2 µM for inhibition of
K+/H+
activity by [Ca2+].

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 3.
K+/H+
antiport activity as a function of matrix free
Ca2+ concentration
([Ca2+]). Incubation
conditions and calculations were identical to those described in Fig. 1
legend, except CaCl2 (0-1 mM)
replaced MgCl2. Fastest swelling
rates were determined from light-scattering records, and corresponding
matrix [Ca2+] were
calculated from furaptra fluorescence with a
Kd of 13.7 µM.
Data were curve fit using Graphpad and then normalized, with maximum
and minimum set to 100 and 0%, respectively.
Inset: Hanes plot.
IC50 for inhibition of
K+/H+
activity by matrix
[Ca2+] is estimated to
be 2.2 µM.
|
|
The experiment in Fig. 4 shows that when
Ca2+ and
Mg2+ are present in the medium,
the effect of the two cations on
K+/H+
activity is additive. When added individually,
MgCl2 (0.2 and 0.4 mM) or
CaCl2 (40 µM) inhibits the
swelling of beef heart mitochondria in a potassium acetate medium
significantly. Addition of Ca2+
with either concentration of Mg2+
results in an even greater inhibition of swelling, and added Mg2+ increases the inhibition seen
with Ca2+ alone (Fig. 4).

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 4.
Mg2+ and
Ca2+ show additive inhibition of
K+/H+
antiport activity. Conditions and calculations were the same as those
described in Fig. 1A with indicated
concentrations of MgCl2 or 40 µM
CaCl2 added to medium.
|
|
Changes in matrix
[Mg2+]
with swelling due to Pi accumulation.
If matrix [Mg2+] is to
function as a volume sensor, its concentration should change with
mitochondrial swelling. Heart mitochondria that have not been depleted
of divalent cations show only a minimal change in volume (Fig.
5A), but
matrix [Mg2+] declines
from an initial value of ~1 mM to ~0.7 mM (Fig.
5B) during respiration with
succinate and rotenone in a KCl medium. This is apparently due to an
increase in Mg2+ ligands, such as
citrate, and is linked to a reduction of matrix pyridine nucleotides
under these conditions (25, 26).

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of external
[Mg2+] on
Pi-induced swelling and matrix
[Mg2+].
A: furaptra-loaded beef heart
mitochondria suspended at 0.5 mg/ml in KCl (0.1 M) containing HEPES (10 mM, pH 7.4), rotenone (3 µg/ml), EGTA (2 mM), and
MgCl2 to buffer
[Mg2+] at 0, 0.5, 1.0, and 2.0 mM after addition of succinate (3.3 mM) and
Pi (3.3 mM). Swelling was measured
in a DW-2C spectrophotometer and was nearly identical at all 3 external
Mg2+ concentrations.
A540, absorbance at 540 nm.
B: matrix
[Mg2+] was estimated
in a parallel incubation and calculated from furaptra fluorescence with
a Kd of 2.1 mM.
Total mitochondrial Mg2+ after 600 s was estimated by atomic absorption and showed a decrease of 1 nmol/mg
protein for zero external
[Mg2+] and no change
for external [Mg2+] of
0.5 and 1.0 mM. Matrix pH (pHi)
was measured in a parallel incubation with use of BCECF-loaded
mitochondria (11).
|
|
When no exogenous Mg2+ is present,
addition of Pi under these
conditions causes the mitochondria to accumulate potassium phosphate and swell osmotically (Fig. 5A). As
the mitochondria swell, matrix [Mg2+] decreases to
~0.4 mM (Fig. 5B). There is some
loss of total Mg2+ from the
mitochondria under these conditions (8, 25, 29), so the decrease in
matrix [Mg2+] can be
due to a reduction in total Mg2+
as well as the increased concentration of phosphate. When
[Mg2+] is present in
the suspending medium at 0.5-2.0 mM, there is little effect on
swelling after Pi addition (Fig.
5A), but matrix [Mg2+] shows a
different pattern of change. Matrix
[Mg2+] decreases
initially from 0.7 to ~0.6 mM on
Pi addition in the presence of 1 mM external Mg2+, but then
rebounds and, after 100 s, attains a level comparable to that before
the addition of Pi (Fig.
5B). With continued incubation, matrix [Mg2+]
increases to ~0.9 mM. A similar response is seen with 0.5 mM external
[Mg2+] (Fig.
5B). These results demonstrate that
mitochondria can swell significantly due to the uptake of potassium
phosphate without a sustained decrease in matrix
[Mg2+].
The accumulation of phosphate under the conditions of Fig. 5 results
from symport of phosphate and H+
(or
Pi/OH
antiport) on the phosphate transporter. This converts a portion of the
pH to 
and favors the electrophoretic uptake of
K+ (12) and osmotic swelling. A
parallel incubation using mitochondria loaded with BCECF (records not
shown) shows that matrix pH increases to 7.8 with succinate
respiration, drops rapidly to pH 7.4 on addition of
Pi, and rebounds spontaneously to
pH 7.7 over the next 60 s (Fig. 5B)
as cation uptake restores the
pH component.
 |
DISCUSSION |
Inhibition of
K+/H+
antiport by matrix
[Mg2+].
The present studies show that the
K+/H+
antiport activity of divalent cation-depleted heart mitochondria is
inhibited by matrix [Mg2+] with an
IC50 of 92 µM (Fig. 2). This
value is significantly less than
[Mg2+] of 200-400
µM estimated by Garlid (15). The higher value was calculated from an
observed IC50 for external
[Mg2+] of 50-65
µM in the presence of A-23187 "sufficient to assure equilibrium" with respect to
Mg2+/2H+
exchange and the assumption that the
pH was
0.3 to
0.4
(matrix acid). However, direct measurement with fluorescent probes
shows that the
pH is dissipated rapidly under these conditions (Fig. 1C). The present
IC50 values would seem to be the
more reliable, because furaptra provides a direct measure of matrix
[Mg2+] that is
independent of assumptions regarding
pH and does not depend on
equilibration of Mg2+ by the
ionophore. When the measured
pH of
0.05 (Fig.
1C) is used in Garlid's
calculation, IC50 values of
60-80 µM are obtained. The substantial agreement between these
values and our estimate of 92 µM is reassuring in view of our
previous reservations regarding the calibration of furaptra
fluorescence in terms of absolute matrix
[Mg2+] (28).
Kakar et al. (30) reported an IC50
of 300-350 µM for
[Mg2+] inhibition of
Rb+ uptake by a reconstituted
K+/H+
antiport from rat liver mitochondria. However, this result was obtained
using proteolipid vesicles with high leak rates, a very low
Rb+ concentration (1 mM) relative
to the Michealis-Menten constant for transport of this cation (130 mM),
and assay conditions that are far from those found in intact mitochondria.
The matrix [Mg2+] of
isolated beef heart mitochondria when loaded with furaptra averages 670 µM and varies with total mitochondrial Mg2+ (29). This level of matrix
[Mg2+] would put
K+/H+
antiport activity at <1% of maximal on the basis of the plot shown
in Fig. 2. For the antiport to respond rapidly to small changes in
matrix [Mg2+], as
postulated by the carrier-brake model,
[Mg2+] must be reduced
to a level that is on the ascending portion of this activity plot (36).
For example, a decrease in matrix [Mg2+] to 200 µM
would permit ~10% of maximum antiport activity (Fig. 2).
Is matrix
[Mg2+]
an adequate volume sensor?
For matrix [Mg2+] to
be the sensor of mitochondrial volume changes, its concentration must
change with swelling and contraction. We have shown that matrix
[Mg2+] decreases
markedly with hypotonic swelling when heart mitochondria are exposed to
a series of KCl buffers of decreasing tonicity (see Fig. 8 in Ref. 25).
A Kd of 1.5 mM
was used to calculate the matrix
[Mg2+] reported
previously (25). We have now recalculated the matrix [Mg2+] from these
experiments using values for the
Kd of
Mg2+-furaptra that are appropriate
for the changing ionic strength of the media and the assumption that
matrix K+ concentration is close
to the external concentration when the rapid hypotonic swelling is
complete. These calculations show that matrix
[Mg2+] decreases from
1.34 mM in hypertonic (150 mM) KCl to near 100 µM when the
mitochondria are extensively swollen (Fig.
6). When the mitochondria are then
contracted by addition of KCl to a final concentration of 150 mM,
matrix [Mg2+]
increases to within 80% of the original value (Fig. 6).

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 6.
Effects of hypotonic swelling on matrix
[Mg2+]. Values for
matrix [Mg2+] reported
in Fig. 8 of Ref. 25 were recalculated using
Kd of 0.17, 1.2, 2.1, and 3.35 mM for Mg2+-furaptra
in presence of 0, 50, 100, and 150 mM KCl, respectively (28).
Furaptra-loaded mitochondria were suspended at 0.5 mg/ml in HEPES
buffer (10 mM, pH 7.4) containing rotenone (3 µg/ml), EGTA (2 mM),
and indicated concentration of KCl. Change in matrix
[Mg2+] when
mitochondria are contracted by addition of KCl to a final concentration
of 150 mM is also shown.
|
|
This indicates that as the matrix increases in volume because of the
uptake of water alone, Mg2+ does
not dissociate from its binding sites in appreciable amounts and matrix
[Mg2+] decreases
dramatically. Approximately 97% of the total
Mg2+ in the matrix is bound (13,
29). It was the extrusion of K+
after hypotonic swelling that led to the proposal of the
Mg2+ carrier-brake model (14), and
the data of Fig. 6 are in line with this model. However, hypotonic
swelling would not be expected to occur in mitochondria in situ.
In contrast to hypotonic swelling, osmotic swelling caused by the
respiration-dependent accumulation of
K+ and acetate (which is not an
effective Mg2+ ligand) does not
result in decreased matrix
[Mg2+] (29). Furaptra
fluorescence shows that matrix
[Mg2+] increases
slightly from its original value of 0.57 mM to 0.65 mM during extensive
swelling (change in absorbance at 540 nm of 0.14) in acetate (see Fig.
6 in Ref. 29). This suggests that K+ can displace enough bound
Mg2+ under these conditions to
keep matrix [Mg2+]
effectively buffered. The lack of change in matrix
[Mg2+] with acetate
swelling is clearly not consistent with its proposed role as the
mitochondrial volume sensor (14, 15).
Garlid (14) showed that anions with a high affinity for
Mg2+ (mainly citrate and
phosphate) promote the loss of matrix
K+ from liver mitochondria,
presumably by activation of
K+/H+
antiport. The accumulation of citrate has been shown to decrease matrix
[Mg2+] in liver
mitochondria (13). However, heart mitochondria lack the dicarboxylate
(31) and the tricarboxylate (2) transporters. This prevents phosphate
from exiting the matrix of heart mitochondria in exchange for substrate
anions, as it does in liver, and makes significant uptake of citrate as
a K+ counterion during osmotic
swelling unlikely. It follows that phosphate would be the most likely
anion that could decrease matrix [Mg2+] by its
accumulation in heart mitochondria in situ.
Recent studies have confirmed that increasing matrix phosphate
decreases the proportion of total
Mg2+ that is free in the matrix
(29). When heart mitochondria swell as a result of phosphate uptake
(Fig. 5), matrix
[Mg2+] drops to ~400
µM if external Mg2+ is absent.
However, a change of this magnitude would increase K+/H+
antiport activity from <1% to only ~2% of maximum (Fig. 2). In addition, when physiological levels of external
Mg2+ are present, the decrease in
matrix [Mg2+] is
nowhere near this large, and even though the matrix has been expanded
to a considerable extent, there is no sustained decrease in matrix
[Mg2+] (Fig.
5B).
Cellular ATP is normally nearly saturated with
Mg2+ (13), but under pathological
conditions, ATP is hydrolyzed and
[Mg2+] increases. When
heart cells are subjected to hypoxia and depleted of ATP, cytosolic
[Mg2+] more than
doubles, increasing from 1.3 to 2.8 mM (44). Under pathological
conditions where
[Ca2+] and
[Mg2+] may be
elevated, inhibition, rather than activation, of
K+/H+
antiport activity would be expected. It has also been suggested that
[Mg2+] can serve as a
volume transducer in intact cells, but changes in
[Mg2+] with cell
volume have not been demonstrated (41).
These considerations make it seem unlikely that a decrease in matrix
[Mg2+] that would
permit significant activation of the
K+/H+
antiport can be attained in vivo by accumulation of
Mg2+ ligands, such as
Pi. Because matrix
[Mg2+] must fluctuate
more dramatically than these studies indicate to provide precise
regulation of
K+/H+
antiport activity, the concept that it can function as the sensor for
volume homeostasis must be called into question. Activation of the
electrophoretic anion channel would also seem unlikely, because the
IC50 for its inhibition by
[Mg2+] has been
reported to be 38 µM at pH 7.4 and 90 µM at pH 7.8 (4).
An additional argument against a role for matrix
[Mg2+] as the volume
sensor is that it can vary with metabolic conditions in the absence of
changes in matrix volume (25). For example, isolated heart mitochondria
respiring with glutamate and malate show a reversible increase in
matrix [Mg2+] of
0.1-0.2 mM while undergoing a state 4-3-4 cycle (see
Figs. 4 and 5 in Ref. 25). These changes are accompanied by changes in
the redox state of mitochondrial pyridine nucleotides and appear to
depend on alterations in matrix citrate content. The decrease in matrix
[Mg2+] shown in Fig. 5
before phosphate addition is also not dependent on swelling.
Inhibition of
K+/H+
antiport by matrix
[Ca2+].
K+/H+
antiport activity is also inhibited by
[Ca2+] with an
IC50 of 2.2 µM (Fig. 3). Garlid
(15) put this value at 50-90 µM (calculated from observed values
of 12-18 µM), but our lower estimate seems more appropriate for
the reasons discussed above for
[Mg2+]. Mitochondrial
K+/H+
antiport was reported to be inhibited by
Ca2+ in heart mitochondria treated
with A-23187 (43), but Nakashima et al. (36) concluded that
Ca2+ has no effect on this
activity unless Mg2+ is first
depleted. It has also been reported that
Ca2+ in the presence of
Mg2+ stimulates
K+/H+
antiport activity indirectly in rat liver mitochondria (36). The
present studies (Fig. 4) indicate that
Ca2+ can inhibit
K+/H+
antiport activity in the presence of
Mg2+ and
Mg2+ can increase the inhibition
with Ca2+.
It has been shown that Ca2+ can
increase net uptake of K+ and
swelling of mitochondria, and these
Ca2+-dependent volume increases
have been related to increased respiration rate and hormone signaling
to the mitochondria (see Ref. 21 for review). Matrix
[Ca2+] has been
reported to increase in mitochondria in situ and can reach levels of
4 µM (40). It would therefore not seem unreasonable for this cation
to have significant effects on
K+/H+
antiport activity and take part in mitochondrial volume homeostasis. However, it is not clear why changes in
[Ca2+] would affect
the antiport if it is already fully inhibited by matrix
[Mg2+] under normal
metabolic conditions.
Alternative models for mitochondrial volume regulation.
A volume control system consists of a sensor that detects volume
changes, a transducer that will convey a signal to a volume-regulatory transporter, and a change in the activity of the transporter (22, 23).
Because our study indicates that matrix
[Mg2+] is unlikely to
perform the role of volume sensor and transducer, other mechanisms for
mitochondrial volume sensing and control must be considered.
Volume-sensing elements may act by concentration or mechanical
mechanisms (22, 23). A change in the matrix protein concentration (relief of "macromolecular crowding") by itself may act as a
signal by affecting transducing elements, such as a kinase or
phosphatase, that would regulate a transporter (34). An obvious
mechanical signal could involve contact between the inner and outer
membranes functioning to activate a volume-regulatory transporter.
Alternatively, components analogous to cellular stretch receptors may
be present to sense swelling of the mitochondria (7). The fact that
N,N'-dicyclohexylcarbodiimide inhibition of
K+/H+
antiport activity in Mg2+-depleted
mitochondria occurs in hypotonic, but not in isotonic, media (17)
suggests that stretching of the membrane or dilution or dissociation of
screening matrix proteins may be involved in exposing the reactive sites.
Whereas there is considerable evidence that a
K+/H+
antiport functions as a volume control transporter (15), the
possibility that other mechanisms may be involved should be considered.
Mitochondrial K+/H+
antiport activity requires extensive depletion of matrix
[Mg2+], elevated pH,
and hypotonic conditions for its demonstration, and there have always
been questions as to whether it might be an artifact derived from
another transporter by the conditions imposed (see the discussion in
Ref. 9). When Mitchell (35) proposed that electroneutral exchangers
must exist to allow efflux of cations against the membrane potential,
he also suggested an alternative mechanism, namely, that an occasional
bursting and resealing of the inner mitochondrial membrane would allow
equilibration of matrix solutes with those in the cytosol. It seems
possible that matrix K+ and
Mg2+ might be equilibrated by
diffusion through a transient pore, such as the mitochondrial
permeability transition pore (20), especially since ion-conducting
substates of the pore appear to be available. If the permeability
transition pore or a substate indeed opens and closes, or flickers,
under normal metabolic conditions, as has been suggested by several
studies (1, 24, 38), it is possible that it could be
triggered at appropriate times in each individual mitochondrion to
allow equilibration of small solutes. Gunter and Pfeiffer (20)
considered this mechanism and found that it was not unreasonable from
an energetic standpoint.
It is obvious that further work is necessary to clarify exactly the
mechanism of mitochondrial volume regulation and the physiological role
of the
K+/H+ antiport.
 |
ACKNOWLEDGEMENTS |
These studies were supported in part by National Heart, Lung, and
Blood Institute Grant HL-09364 to G. P. Brierley and a grant-in-aid to
D. W. Jung from the American Heart Association Ohio Affiliate.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: D. W. Jung,
Dept. of Medical Biochemistry, The Ohio State University, 1645 Neil
Ave., Columbus, OH 43210 (E-mail: jung.7{at}osu.edu).
Received 11 May 1999; accepted in final form 17 August 1999.
 |
REFERENCES |
1.
Altschuld, R. A.,
C. M. Hohl,
L. C. Castillo,
A. A. Garleb,
R. C. Starling,
and
G. P. Brierley.
Cyclosporin inhibits mitochondrial calcium efflux in isolated adult rat ventricular cardiomyocytes.
Am. J. Physiol.
262 (Heart Circ. Physiol. 31):
H1699-H1704,
1992[Abstract/Free Full Text].
2.
Azzi, A.,
M. Glerum,
R. Koller,
W. Mertens,
and
S. Spycher.
The mitochondrial tricarboxylate carrier.
J. Bioenerg. Biomembr.
25:
515-524,
1993[Medline].
3.
Baysal, K.,
G. P. Brierley,
S. Novgorodov,
and
D. W. Jung.
Regulation of the mitochondrial Na+/Ca2+ antiport by matrix pH.
Arch. Biochem. Biophys.
291:
383-389,
1991[Medline].
4.
Beavis, A. D.
Properties of the inner membrane anion channel in intact mitochondria.
J. Bioenerg. Biomembr.
24:
77-90,
1992[Medline].
5.
Beavis, A. D.,
R. D. Brannan,
and
K. D. Garlid.
Swelling and contraction of the mitochondrial matrix. I. A structural interpretation of the relationship between light scattering and matrix volume.
J. Biol. Chem.
260:
13424-13433,
1985[Abstract/Free Full Text].
6.
Beavis, A. D.,
and
K. D. Garlid.
Evidence for the allosteric regulation of the mitochondrial K+/H+ antiporter by matrix protons.
J. Biol. Chem.
265:
2538-2545,
1990[Abstract/Free Full Text].
7.
Bernardi, P.,
and
G. F. Azzone.
Electroneutral H+-K+ exchange in liver mitochondria. Regulation by membrane potential.
Biochim. Biophys. Acta
724:
212-223,
1983[Medline].
8.
Brierley, G. P.,
M. H. Davis,
and
D. W. Jung.
Respiration-dependent uptake and extrusion of Mg2+ by isolated heart mitochondria.
Arch. Biochem. Biophys.
253:
322-332,
1987[Medline].
9.
Brierley, G. P.,
and
D. W. Jung.
K+/H+ antiport in mitochondria.
J. Bioenerg. Biomembr.
20:
193-209,
1988[Medline].
10.
Brierley, G. P.,
M. S. Jurkowitz,
T. Farooqui,
and
D. W. Jung.
K+/H+ antiport in heart mitochondria.
J. Biol. Chem.
259:
14672-14678,
1984[Abstract/Free Full Text].
11.
Brierley, G. P.,
E. S. Panzeter,
and
D. W. Jung.
Regulation of mitochondrial K+/H+ antiport activity by hydrogen ions.
Arch. Biochem. Biophys.
288:
358-367,
1991[Medline].
12.
Brown, G. C.,
and
M. D. Brand.
Changes in permeability to protons and other cations at high protonmotive force in rat liver mitochondria.
Biochem. J.
234:
75-81,
1986[Medline].
13.
Corkey, B. E.,
J. Duszynski,
T. L. Rich,
B. Matschinsky,
and
J. R. Williamson.
Regulation of free and bound magnesium in rat hepatocytes and isolated mitochondria.
J. Biol. Chem.
261:
3567-3574,
1986.
14.
Garlid, K. D.
On the mechanism of regulation of the mitochondrial K+/H+ exchanger.
J. Biol. Chem.
255:
11273-11279,
1980[Free Full Text].
15.
Garlid, K. D.
Mitochondrial volume control.
In: Integration of Mitochondrial Function, edited by J. J. Lemasters,
C. R. Hackenbrock,
R. G. Thurman,
and H. V. Westerhoff. New York: Plenum, 1988, p. 259-278.
16.
Garlid, K. D.
Cation transport in mitochondria
the potassium cycle.
Biochim. Biophys. Acta
1275:
123-126,
1996[Medline].
17.
Garlid, K. D.,
D. J. DiResta,
A. D. Beavis,
and
W. H. Martin.
On the mechanism by which dicyclohexylcarbodiimide and quinine inhibit K+ transport in rat liver mitochondria.
J. Biol. Chem.
261:
1529-1535,
1986[Abstract/Free Full Text].
18.
Grubbs, R. D.,
and
M. E. Maguire.
Magnesium as a regulatory cation: criteria and evaluation.
Magnesium
6:
113-127,
1988.
19.
Grynkiewicz, G.,
M. Poenie,
and
R. Y. Tsien.
A new generation of Ca2+ indicators with greatly improved fluorescence properties.
J. Biol. Chem.
260:
3440-3450,
1985[Abstract].
20.
Gunter, T. E.,
and
D. R. Pfeiffer.
Mechanisms by which mitochondria transport calcium.
Am. J. Physiol.
258 (Cell Physiol. 27):
C755-C786,
1990[Abstract/Free Full Text].
21.
Halestrap, A. P.
The regulation of the matrix volume of mammalian mitochondria in vivo and in vitro and its role in the control of mitochondrial metabolism.
Biochim. Biophys. Acta
973:
355-382,
1989[Medline].
22.
Hallows, K. R.,
and
P. A. Knauf.
Principles of cell volume regulation.
In: Cellular and Molecular Physiology of Cell Volume Regulation, edited by K. Strange. Boca Raton, FL: CRC, 1994, p. 3-29.
23.
Hoffmann, E. K.,
and
P. B. Dunham.
Membrane mechanisms and intracellular signalling in cell volume regulation.
Int. Rev. Cytol.
161:
173-262,
1995[Medline].
24.
Huser, J.,
C. E. Rechenmacher,
and
L. A. Blatter.
Imaging the permeability pore transition in single mitochondria.
Biophys. J.
74:
2129-2137,
1998[Abstract/Free Full Text].
25.
Jung, D. W.,
L. Apel,
and
G. P. Brierley.
Matrix free Mg2+ changes with metabolic state in isolated heart mitochondria.
Biochemistry
29:
4121-4128,
1990[Medline].
26.
Jung, D. W.,
and
G. P. Brierley.
Determination of free Mg2+ in isolated heart mitochondria using fluorescent probes.
Magnes. Trace Elem.
10:
151-164,
1992.
27.
Jung, D. W.,
and
G. P. Brierley.
Magnesium transport by mitochondria.
J. Bioenerg. Biomembr.
26:
527-535,
1994[Medline].
28.
Jung, D. W.,
C. J. Chapman,
K. Baysal,
D. R. Pfeiffer,
and
G. P. Brierley.
On the use of fluorescent probes to estimate free Mg2+ in the matrix of heart mitochondria.
Arch. Biochem. Biophys.
332:
19-29,
1996[Medline].
29.
Jung, D. W.,
E. Panzeter,
K. Baysal,
and
G. P. Brierley.
On the relationship between matrix free Mg2+ concentration and total Mg2+ in heart mitochondria.
Biochim. Biophys. Acta
1320:
310-320,
1997[Medline].
30.
Kakar, S. S.,
F. Mahdi,
X. Li,
and
K. D. Garlid.
Reconstitution of the mitochondrial non-selective Na+/H+ (K+/ H+) antiporter into proteoliposomes.
J. Biol. Chem.
264:
5846-5851,
1989[Abstract/Free Full Text].
31.
LaNoue, K. F.,
and
A. C. Schoolwerth.
Metabolite transport in mitochondria.
Annu. Rev. Biochem.
48:
871-922,
1979[Medline].
32.
Lattanzio, F. A.,
and
D. K. Bartschat.
Effect of pH on rate constant, ion selectivity and thermodynamic properties of fluorescent Ca and Mg indicators.
Biochem. Biophys. Res. Commun.
177:
184-191,
1991[Medline].
33.
Li, X.,
M. G. Hegazy,
F. Mahdi,
P. Jezek,
R. D. Lane,
and
K. D. Garlid.
Purification of a reconstitutively active K+/H+ antiporter from rat liver mitochondria.
J. Biol. Chem.
265:
15316-15322,
1990[Abstract/Free Full Text].
34.
Minton, A. P,
G. C. Colclasure,
and
J. C. Parker.
Model for the role of macromolecular crowding in regulation of cellular volume.
Proc. Natl. Acad. Sci. USA
89:
10504-10506,
1992[Abstract].
35.
Mitchell, P.
Chemiosmotic Coupling and Energy Transduction. Bodmin, Cornwall, UK: Glynn Research, 1968.
36.
Nakashima, R. A.,
R. S. Dordick,
and
K. D. Garlid.
On the relative roles of Ca2+ and Mg2+ in regulating the endogenous K+/H+ exchanger of rat liver mitochondria.
J. Biol. Chem.
257:
12540-12545,
1982[Abstract/Free Full Text].
37.
Nakashima, R. A.,
and
K. D. Garlid.
Quinine inhibition of Na+ and K+ transport provides evidence for two cation/H+ exchangers in rat liver mitochondria.
J. Biol. Chem.
257:
9252-9254,
1982[Abstract/Free Full Text].
38.
Petronilli, V.,
G. Miotto,
M. Canton,
M. Brini,
R. Colonna,
P. Bernardi,
and
F. Di Lisa.
Transient and long-lasting openings of the mitochondrial permeability transition pore can be monitored directly in intact cells by changes in mitochondrial calcein fluorescence.
Biophys. J.
76:
725-734,
1999[Abstract/Free Full Text].
39.
Raju, B.,
E. Murphy,
L. A. Levy,
R. D. Hall,
and
R. E. London.
A fluorescent indicator for measuring cytosolic free magnesium.
Am. J. Physiol.
256 (Cell Physiol. 25):
C540-C548,
1988.
40.
Rutter, G. A.,
J.-M. Theler,
M. Murgia,
C. B. Wollheim,
T. Pozzan,
and
R. Rizzuto.
Stimulated Ca2+ influx raises mitochondrial free Ca2+ to supramicromolar levels in a pancreatic cell line.
J. Biol. Chem.
268:
22385-22390,
1993[Abstract/Free Full Text].
41.
Sarkadi, B.,
and
J. C. Parker.
Activation of ion transport pathways by changes in cell volume.
Biochim. Biophys. Acta
1071:
407-427,
1991[Medline].
42.
Schoenmakers, T. J. M.,
G. J. Visser,
G. Flink,
and
A. P. R. Theuvenet.
CHELATOR: an improved method for computing metal ion concentrations in physiological solutions.
Biotechniques
12:
870-879,
1992[Medline].
43.
Shi, G.-Y.,
D. W. Jung,
K. D. Garlid,
and
G. P. Brierley.
Induction of respiration-dependent net efflux of K+ from heart mitochondria by depletion of endogenous divalent cations.
J. Biol. Chem.
255:
10299-10305,
1980[Abstract/Free Full Text].
44.
Silverman, H. S.,
F. Di Lisa,
R. C. Hui,
H. Miyata,
S. J. Sollott,
R. G. Hansford,
E. G. Lakatta,
and
M. D. Stern.
Regulation of intracellular free Mg2+ and contraction in single adult mammalian cardiac myocytes.
Am. J. Physiol.
266 (Cell Physiol. 35):
C222-C233,
1994[Abstract/Free Full Text].
45.
Sun, X.,
H. Zhu,
N. Xu,
V. P. Zinchenko,
and
K. D. Garlid.
Cloning and partial sequence reveals nucleotide regulation of bovine heart mitochondrial K+/H+ antiporter protein (Abstract).
Biophys. J.
66:
A335,
1994.
Am J Physiol Cell Physiol 277(6):C1194-C1201
0002-9513/99 $5.00
Copyright © 1999 the American Physiological Society