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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

MITOCHONDRIA IN SITU maintain a high negative membrane potential (Delta psi ) 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 Delta psi 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
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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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 beta  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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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.


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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 (beta ) 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 Delta 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 (beta /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.


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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+].


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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).


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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).


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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 Delta pH to Delta psi 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 Delta pH component.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

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 Delta pH was -0.3 to -0.4 (matrix acid). However, direct measurement with fluorescent probes shows that the Delta 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 Delta pH and does not depend on equilibration of Mg2+ by the ionophore. When the measured Delta 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).


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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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 277(6):C1194-C1201
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