©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Sodium-Calcium Antiport of Heart Mitochondria Is Not Electroneutral (*)

(Received for publication, February 16, 1994; and in revised form, October 24, 1994)

Dennis W. Jung (§) Kemal Baysal (¶) Gerald P. Brierley

From the Department of Medical Biochemistry, The Ohio State University, Columbus, Ohio 43210

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Heart mitochondria contain a nNa/Ca antiport that participates in the regulation of matrix [Ca]. Based largely on a single study (Brand, M. D.(1985) Biochem. J. 229, 161-166), there has been a consensus that this antiport promotes the electroneutral exchange of two Na for one Ca. However, a recent study in our laboratory (Baysal, K., Jung, D. W., Gunter, K. K., Gunter, T. P., and Brierley, G. P. (1994) Am. J. Physiol. 266, C800-C808) has shown that the Na-dependent efflux of Ca from heart mitochondria has more energy available to it than can be supplied by a passive 2Na/Ca exchange. We have therefore re-examined Brand's protocols using fluorescent probes to monitor matrix pH and free [Ca]. Respiring heart mitochondria, suspended in KCl and treated with ruthenium red to block Ca influx, extrude Ca and establish a large [Ca]: [Ca] gradient. The extrusion of Ca under these conditions is Na-dependent and diltiazem-sensitive and can be attributed to the nNa/Ca antiport. Addition of nigericin increases the membrane potential (Delta) and decreases DeltapH to 0.1 or less, but has virtually no effect on the magnitude of the [Ca] gradient. Under these conditions a gradient maintained by electroneutral 2Na/Ca antiport should be abolished because the mitochondrial Na/H antiport keeps the [Na] gradient equivalent to the [H] gradient. The [Ca] gradient is abolished, however, when an uncoupler is added to dissipate Delta or when the exogenous electroneutral antiport BrA23187 is added. In addition, [Ca] influx via the nNa/Ca antiport in nonrespiring mitochondria is enhanced when Delta is abolished. These results are consistent with Ca extrusion by an electrophoretic antiport that can respond to Delta but not with an electroneutral antiport.


INTRODUCTION

It is now generally accepted that [Ca] enters the matrix of heart mitochondria via the ruthenium red-sensitive uniport and is extruded on a nNa/Ca antiport (see (1) and (2) for reviews). Until recently there has been a consensus that this antiport promotes the electroneutral exchange of two Na for one Ca(2, 3, 4) . This conclusion is based largely on a study by Brand (3) that showed a [Ca] distribution established by the endogenous antiport did not differ significantly from that produced by the ionophore A23187. Because A23187 mediates electroneutral 2H/Ca exchange and the endogenous Na/H antiport equilibrates [H] and [Na] gradients, it was concluded that the nNa/Ca antiport must be electroneutral(3) . This stoichiometry is also supported by the more recent report of Li et al.(4) that all modes of transport promoted by a purified and reconstituted Na/Ca antiport are insensitive to uncouplers and therefore electroneutral. However, there have also been indications that this antiport could be electrophoretic. The early report of Crompton et al.(5) established that the Na-dependent efflux of Ca was dependent on the energy state of the mitochondria and kinetic data indicate the presence of three independent Na binding sites on the antiport(6) . These results could be explained by a contribution of the membrane potential, Delta, to an electrophoretic exchange, such as that found with the 3Na/Ca antiport of the sarcolemma(7) .

Two recent studies from our laboratory strongly suggest that the concept of an electroneutral nNa/Ca antiport should be re-examined. In the first it was noted that the exchanger is regulated by matrix pH and that high rates of Na/Ca antiport can be sustained when DeltapH (and hence the Na gradient) approaches zero(8) . This led us to a null-point study in which it was established that [Ca] gradients maintained by Na/Ca antiport are too large to be sustained by a passive electroneutral exchange of 2Na for one Ca(9) . These observations in turn led us to the present work in which we examine the protocols of Brand (3) in more detail using currently available fluorescent probes to monitor [H] and [Ca] gradients. In contrast to Brand(3) , we conclude that, when the pH gradient of respiring mitochondria is collapsed by addition of nigericin, a large [Ca]:[Ca] gradient is maintained. Addition of an uncoupler to dissipate Delta abolishes this gradient, as does addition of the electroneutral exchanger BrA23187. These experiments lead to the conclusion that the nNa/Ca antiport is not electroneutral under these conditions and that there is a large contribution of Delta to the observed [Ca] gradient. Experiments showing Na-dependent [Ca] influx in nonrespiring mitochondria also support the conclusion that the mitochondrial nNa/Ca antiport is electrophoretic.


EXPERIMENTAL PROCEDURES

Equilibration of Mitochondria with Fluorescent Probes

Beef heart mitochondria were prepared as described previously (10) and suspended at 25 mg/ml in sucrose (0.25 M), containing TES (^1)(10 mM, K salt, pH 7.4). Mitochondria were equilibrated with fluorescent probes as follows. Mitochondria (12.5 mg/ml) were incubated at 24 °C in a medium of sucrose (0.25 M), TES (10 mM), NaCl (20 mM), ATP (1.6 mM), EGTA (0.2 mM) neutralized to pH 7.4 with KOH and containing either BCECF/AM (7.2 µM), cSNARF-1/AM (8.8 µM), SBFI/AM (11.1 µM), or fura-2/AM (5 µM). Pluronic F-127 was also added when SBFI/AM was used(14) . A second suspension of mitochondria, incubated in the same medium without the fluorescent probe, was used to determine autofluorescence. After 20 min, 3 volumes of ice-cold wash medium (0.2 M sucrose, 10 mM K TES, 30 µM EGTA, pH 7.4) were added, and the mitochondria were isolated by centrifugation and resuspended in the same medium. After an additional 5-min incubation at 24 °C, the mitochondria were again diluted, reisolated by centrifugation, and suspended at 25 mg/ml in the wash medium. These loading conditions deplete the mitochondria of almost all endogenous Ca. Virtually none of the fura-2 is outside the matrix since addition of near saturating concentrations of [Ca] to suspended mitochondria results in an insignificant increase in fura-2 fluorescence. ADP-stimulated respiration and respiratory control ratios of probe-loaded mitochondria are 90% of control mitochondria(17) .

Incubation Conditions

For most experiments, the mitochondria containing sequestered fluorescent probes were incubated at 0.5 mg/ml in a 3-ml cuvette at 25 °C in a standard medium consisting of: KCl (100 mM), HEPES (15 mM, K salt, pH 7.35), cyclosporin A (1 µM), rotenone (1 µg/ml), and oligomycin (2 µg/ml). The EGTA carried over with the addition of mitochondria was 0.6 µM. Further additions were made as described in the legends. The experiments shown in Fig. 3were carried out in the mannitol-sucrose medium described by Brand(3) . [Ca] buffers were prepared by the addition of 2 mM EGTA (for 0-2 µM [Ca] buffers) or 2 mM NTA (5-50 µM [Ca] buffers) and variable amounts of CaCl(2) to the standard medium. The free [Ca] for each buffer was calculated using the Fabiato computer program (11) using apparent stability constants for 25 °C and pH 7.4 of 1.146 times 10^4 for Ca/NTA, 1.592 times 10^7 for Ca/EGTA, and 15.75 for Ca/succinate. The absolute log K(1) for Ca/NTA was taken as 6.45 with log protonation constants of 9.71, 2.49, 1.86, and 0.8(12) . The pH(o) was measured with a glass electrode.


Figure 3: Effect of nigericin on matrix pH of respiring and nonrespiring heart mitochondria in a mannitol-sucrose medium. BCECF-equilibrated mitochondria were suspended in the medium used by Brand (3) which consisted of mannitol (210 mM), sucrose (70 mM), HEPES (10 mM), NTA (1 mM), P(i) (1 mM), NaCl (4 mM), rotenone (1 µg/ml), and oligomycin (2 µg/ml) neutralized to pH 7.0 with TMA hydroxide. Where indicated TMA succinate (5 mM) was also present or added at 300 s. Nigericin (1 µM) was added at 100 s, except where indicated. Matrix pH was calculated from net fluorescence intensity as described (8) and external pH was measured with a glass electrode.



Fluorescence Measurements

Fluorescence was measured using a Perkin-Elmer LS-5B fluorimeter interfaced with a computer as described previously(9, 13) . The monochromators were driven to obtain ratios approximately every 7 s for a single probe (13, 14) or every 30 s when two probes were used(9) . BCECF fluorescence was recorded at 550 nm with excitation at 509 and 450 nm, fura-2 fluorescence at 510 nm with excitation at 340 and 365 nm, SBFI fluorescence at 510 nm with excitation at 350 and 390 nm excitation, and cSNARF-1 emission at 630 and 604 nm with excitation at 534 nm. For each experiment the exact protocol was repeated using nonloaded mitochondria to record autofluorescence. The records of autofluorescence were subtracted from the experimental records before [Ca], [Na], and pH(i) were calculated using the ratioing procedure(8, 9, 13, 14) .

Matrix [Ca] was calculated after determining R(min), R(max), and S/S values using the following expression(15) .

To determine R(min), BrA23187 (2 µM) was added to respiring mitochondria suspended in the standard medium containing 2 mM EGTA to deplete mitochondrial Ca, leaving only uncomplexed fura-2 in the matrix (Fig. 1). R(max) was obtained by allowing respiring mitochondria to accumulate Ca and saturate matrix fura-2 (Fig. 1). S/S is the ratio of fluorescence intensities at 365 nm with zero and with excess Ca. R(min), R(max), and S/S values were determined for each mitochondrial preparation. The means ± S.E. for the experiments presented here (n = 6) were 0.626 ± 0.001 R(min), 2.653 ± 0.113 R(max), and 1.858 ± 0.242 S/S. The solid line in Fig. 1represents the relationship between calculated [Ca] and the 340/365 ratio and shows that fura-2 approaches saturation above 2 µM matrix [Ca]. With this in mind our experiments were designed to keep matrix [Ca] below 2 µM.


Figure 1: Estimation of R(min) and R(max) for fura-2 sequestered in the matrix of heart mitochondria. Mitochondria were loaded with fura-2 as described under ``Experimental Procedures'' and added to the standard medium containing 2 mM EGTA for the R(min) determination and 15 µM EGTA for the R(max) determination. Succinate (5 mM) was added at 100 s. CaCl(2) (2 mM) was added at 200 s for the R(max) determination and BrA23187 (2 µM) for the R(min) determination. Fluorescence was corrected for autofluorescence and ratios (340/365) were calculated from net fluorescence intensities. The relative net fluorescence intensities and calculated ratios were: R(min) (340/530) = 0.64, R(max) (760/254) = 3.0, and S/S (530/254) = 2.09. The solid line shows the relationship between the ratios and [Ca] calculated according to . The initial ratio value of 1.55 indicates a matrix [Ca] of 0.25 µM.



Estimation of Delta

Membrane potential (Delta) was estimated using [^3H]TPP distribution calibrated with Rb(16) . Succinate respiration was inhibited with malonate to vary Delta(16) . Mitochondrial volumes and water spaces were determined using [^14C]sucrose and [^3H]H(2)O as described previously(17) . Following the recent suggested convention (18) , Delta and DeltapH are calculated as the difference between the matrix and medium values. This means that Delta is negative and DeltapH is positive in normal respiring mitochondria. The reader should be aware that this convention was not used by Brand (3) or in many of the other references cited.

All chemicals used were of reagent grade purity or higher. Ruthenium red was obtained from Fluka and recrystallized according to Luft(19) . The AM esters were obtained from Molecular Probes, Inc. (Eugene, OR) and were dissolved in dimethyl sulfoxide (silylation grade) obtained from Pierce.


RESULTS

A passive nNa/Ca antiport will exchange Ca for Na until the energy contained in the electrochemical gradient of Ca is balanced by the proper stoichiometric factor (n) multiplied by the energy in the electrochemical gradient of Na(20, 21, 22) . Because Na/H exchange is rapid in isolated mitochondria and effectively equilibrates the [Na] and [H] gradients, the following expression should hold for a passive nNa/Ca antiport at equilibrium.

At 25 °C this relationship can be expressed as follows(20, 21) .

This thermodynamic relationship predicts that when DeltapH is collapsed, no [Ca] gradient can be maintained at equilibrium if the antiport is electroneutral (n = 2), since 10 = 1 under these conditions. However, a [Ca] gradient can be maintained if the antiport promotes electrophoretic exchange (n > 2) and responds to Delta. The term ``electrophoretic'' is used to indicate that the transport occurs in response to an existing electrical potential. This is in contrast to a transport reaction that generates a potential that would be termed ``electrogenic''(23) .

The assumption that DeltapH and DeltapNa are very near equivalent is essential to this investigation and is supported by several studies. Crompton and Heid (24) using isotope distribution procedures showed that the [Na] and [H] gradients are nearly at equilibrium for nonrespiring rat heart mitochondria suspended in a KCl medium. This equilibrium results from the high rate of Na/H exchange relative to the Na/Ca antiport(24) . Studies from this laboratory (14) using SBFI-equilibrated pig heart mitochondria strongly support this conclusion. When the [Na] gradients reported (14) are compared with the appropriate [H] gradients measured in related studies using the fluorescent probes BCECF (25) or cSNARF-1(8) , the mean difference between DeltapH and DeltapNa calculates to 0.11 ± 0.06 (n = 12).

Isolated beef heart mitochondria are difficult to load with SBFI (or the analogous probe Sodium Green). The fluorescence signal obtained with these preparations is no greater than 1.5 times the autofluorescence. This makes it very difficult to distinguish between changes in redox components and changes in matrix [Na] and has prevented us from following the [Na] gradient directly in the respiring beef heart mitochondria used in these studies. However, we were able to follow the spontaneous decay of the pH and pNa gradients in nonrespiring beef heart mitochondria that had been equilibrated with SBFI and cSNARF-1 (Fig. 2). In this protocol the decay of DeltapNa approximates the DeltapH decay with a maximum difference of 0.17. These results support the concept that DeltapH and DeltapNa are maintained nearly equal by the Na/H antiport (14, 24


Figure 2: Spontaneous decay of DeltapH and DeltapNa in nonrespiring heart mitochondria. Mitochondria equilibrated with both SBFI and cSNARF-1 were added to the standard medium containing ruthenium red (1 µM), NaCl (19 mM), EGTA (1 mM), and CaCl(2) (0.9 mM). Net fluorescence intensities were used to calculate matrix pH (8) and matrix [Na](14) . Medium pH was 7.37.



In his influential 1985 study of the stoichiometry of this antiport, Brand (3) used a Ca-electrode to follow medium [Ca] in a suspension of rat heart mitochondria respiring in a low K medium. After addition of nigericin he found the matrix to be acid with a DeltapH of about -1.0. Under these conditions he calculated that external [Ca] for an n = 2 stoichiometry should differ from that for n = 3 by 0.634 µM at equilibrium(3) . At equilibrium Brand (3) reported a DeltapH of -0.99 (using the conventions of (18) ), a DeltapNa of -0.86, Delta of -161 mV, external [Ca] of 6.37 µM, and a calculated matrix [Ca] of nearly 500 µM for mitochondria containing approximately 3 nmol of Ca/mg of protein.

These values are out of line with our experience with respiring beef heart mitochondria under quite similar conditions. Using BCECF fluorescence to monitor pH(i) we see a large negative DeltapH (interior acid) only for nonrespiring mitochondria following nigericin addition in the low K medium used by Brand (Fig. 3). Under these conditions a DeltapH of about -0.8 is established immediately and decays to about -0.5 after 500 s (Fig. 3). Mitochondria respiring with succinate in the same medium show a transient DeltapH of about -0.5 following nigericin addition, but respiration rapidly establishes a condition in which DeltapH is about 0.15 (Fig. 3). This is probably the result of electrophoretic uptake of TMA in response to the high Delta established by nigericin. Addition of succinate to nonrespiring mitochondria maintaining a large negative DeltapH results in an alkaline shift to a final DeltapH near 0.1 (Fig. 3). In the KCl medium used for the studies reported here there is no discernible DeltapH after nigericin addition whether or not respiration is taking place because nigericin equilibrates the [H] gradient with the [K] gradient(23) . The BCECF-loaded mitochondria lose some endogenous K during equilibration with the probe and average just over 100 ng ion Kbulletmg protein. The incubation medium contains about 120 mM K, so the [K] gradient is minimal under these conditions.

These results suggest that Brand's estimate of DeltapH for respiring mitochondria treated with nigericin (3) is too large. The distribution of [^14C]methylamine and Na was used to estimate the gradients in his study (3) and it is possible that either redistribution of the probe at anaerobiosis (following centrifugation), nonohmic uptake of the cationic probes, or changes in probe binding during energization in the low ionic strength suspending medium could have affected the results (see (26) ). At pH 7 methylamine (pK(a) 10.5) is >99% cationic. Regardless of the reason for the discrepancy, a decrease in the DeltapNa from -0.86 as estimated by Brand (3) to -0.50 or less would result in loss of ability to distinguish between n = 2 and n = 3 stoichiometry in his experiment. As shown in Fig. 4the difference in external [Ca] predicted for these two cases becomes less than 0.1 µM at -0.5 DeltapNa. If DeltapH (and therefore DeltapNa) were as small as the experiments of Fig. 3indicate, the difference between n = 2 and n = 3 would disappear completely (Fig. 4). If in fact the DeltapH in Brand's experiment (3) was much less than his estimate, then he would have seen no measurable change in external [Ca] when A23187 was added to provide an exogenous electroneutral exchanger, even if the endogenous antiport were electrophoretic.


Figure 4: Predicted values for extramitochondria [Ca] versus the Na gradient as a function of the stoichiometry of the nNa/Ca antiport. Values were calculated from the data of Table I (sample 1) of (3) using Equation 1 in (3) , a stoichiometry of either 2 or 3 Na/Ca, and various values of DeltapNa. Due to uncertainties in some of the values these calculations vary slightly when compared with those of Brand(3) . For example, our value for n = 2 and DeltapNa of -0.86 is 6.66 µM as opposed to 6.53 given in(3) .



Use of Fluorescent Probes to Estimate the [Ca] Gradient Maintained by the nNa/Ca Antiport

Because mitochondria respiring in a KCl medium do not maintain a significant pH gradient following treatment with nigericin (Fig. 5B), predicts that no [Ca] gradient will be maintained if the stoichiometry is n = 2. On the other hand, if it is electrophoretic a [Ca]:[Ca] gradient of 10 will be maintained by the nNa/Ca antiport at equilibrium.


Figure 5: Effects of nigericin on matrix [Ca] and matrix pH of respiring heart mitochondria. A, mitochondria equilibrated with fura-2 were added to the standard medium containing NaCl (20 mM), succinate (5 mM), and [Ca] buffered at either 0.8 µM or 5 µM. The 0.8 µM [Ca] was established by adding CaCl(2) (1.86 mM) and EGTA (2 mM), whereas the 5 µM buffer contained NTA (2 mM) and CaCl(2) (0.115 mM). Ruthenium red (1 µM) was added 10 s after the mitochondria and fluorescence measurements were then started. Nigericin (1 µM) was added at 100 s. B, mitochondria equilibrated with cSNARF-1 were incubated in the same medium and under the same conditions as A. Matrix pH was calculated from net fluorescence intensity as described(8) . The records shown are from a different mitochondrial preparation than that used in A. Records from mitochondria loaded with both fura-2 and cSNARF-1 show essentially the same picture when [Ca] and pH are monitored simultaneously.



Heart mitochondria were equilibrated with the fluorescent pH indicator cSNARF-1 or the [Ca] indicator fura-2 and suspended in a KCl medium containing either 0.8 or 5 µM buffered [Ca] and 20 mM NaCl. The respiring mitochondria were allowed to take up Ca for 10 s. At this point ruthenium red was added to block further influx via the uniport and fura-2 fluorescence reported a matrix [Ca] of about 0.4 µM for 0.8 µM external [Ca] and 0.65 µM for 5 µM external [Ca] (Fig. 5A). The fluorescence then indicated a loss of matrix [Ca] which was completely Na-dependent and blocked by diltiazem (100 µM), properties consistent with efflux via the nNa/Ca antiport(2) . Cyclosporin A was included in all of the experiments reported here to prevent possible contributions of the Ca-dependent permeability pore(1) . Addition of nigericin under these conditions decreases DeltapH to 0.1 or less (Fig. 5B), but has no significant effect on Ca efflux (Fig. 5A). When a steady state is reached (600 s), matrix [Ca] is 0.185 and 0.130 µM in the presence of nigericin, and 0.130 and 0.110 µM in its absence in the 5 µM and 0.8 µM [Ca] buffers, respectively. This means that the mitochondria are maintaining comparable [Ca]:[Ca] gradients in the presence and absence of nigericin (27 versus 38 in 5 µM and 6.2 versus 7.3 in 0.8 µM [Ca]). In the presence of nigericin, DeltapH is near zero and indicates that Ca gradients of 27 and 6.2 could be maintained by Delta values of -85 and -47 mV, respectively, if the antiport stoichiometry were 3Na for one Ca. If n = 2, pH (or pNa) gradients of 0.7 and 0.4 would be required to maintain these Ca gradients, but since DeltapH is 0.1 or less, these results are not compatible with electroneutral exchange. The larger Ca gradients seen in the absence of nigericin (DeltapH and Delta present) than in its presence (DeltapH = 0) (Fig. 5A) follow according to where the DeltapH term is multiplied by n but the Delta/59 term by only (2 - n). It does not necessarily follow that the conversion of DeltapH to Delta by nigericin should increase the rate of Ca efflux via an electrophoretic antiport. The kinetics of the nNa/Ca antiport appear to depend on many factors on either side of the membrane including pH, [Na], [Ca], [Mg], and adenine nucleotides(2, 6, 8, 9) . The present study is concerned with the [Ca] gradients maintained at equilibrium and not with the kinetics of Ca transport.

If the exogenous electroneutral exchanger BrA23187 is added 130 s after nigericin in the protocol of Fig. 5, there is a rapid increase in matrix [Ca] to nearly that of the external [Ca] (Fig. 6A). Thus, in contrast to the results of Brand(3) , we see a steady state [Ca] gradient established by the endogenous nNa/Ca antiport that is far from the value seen in the presence of the electroneutral ionophore BrA23187. BrA23187 promotes electroneutral exchange of Ca for 2H, so that the equilibrium [Ca] gradient should equal the square of the [H] gradient. A record of cSNARF-1 fluorescence shows that addition of Br23187 produces no change in matrix pH and that DeltapH remains near zero after this addition (not shown). BrA23187 will also promote loss of matrix Mg in exchange for H. When 1 mM [Mg] is added to the medium of Fig. 6A to approximate matrix [Mg], matrix [Ca] rises to nearly the same concentration (86% of control) on addition of BrA23187 as in the absence of external [Mg] (not shown). In the high [K] medium used in these studies, respiration-dependent H extrusion, in combination with nigericin, keeps DeltapH near zero and allows no [Ca] or [Mg] gradient to be maintained in the presence of BrA23187.


Figure 6: The electroneutral exchanger BrA23187 and uncoupler equilibrate the [Ca] gradient maintained by nNa/Ca antiport in respiring mitochondria. A, mitochondria were added to the standard KCl medium containing NaCl (20 mM), K succinate (5 mM), EGTA (2 mM), and CaCl(2) (1.94 mM). The calculated [Ca] of this medium is 2 µM. Ruthenium red (1 µM) was added 10 s after the mitochondria, and recording of fluorescence was begun. Nigericin (1 µM) was added at 100 s, and efflux of matrix [Ca] was followed. At 230 s, BrA23187 (2 µM) was added, and the rapid equilibration of matrix [Ca] with external [Ca] was followed. In a parallel experiment FCCP (1 µM) was added at 230 s to produce a slower equilibration via endogenous antiport. A control without further addition after nigericin is also shown. The net 340/365 nm ratios (left ordinate) were used to calculate matrix [Ca] (right ordinate) using 0.615 R(min), 2.62 R(max), and 1.86 S/S. Note that the [Ca] scale is nonlinear in this panel. B, conditions identical to A with BrA23187 and nigericin added where indicated. C, effect of diltiazem (100 µM) and TPP (3.1 and 6.6 µM) after a steady state [Ca] gradient has been established in the protocol of A.



A similar but slower decay of the steady state [Ca] gradient is seen following addition of the uncoupler FCCP (Fig. 6A). The uncoupler collapses the protonmotive force to zero eliminating Delta as well DeltapH. This allows matrix [Ca], [Na], and [H] to equilibrate with the corresponding external ions via endogenous antiporters regardless of the value of n for the nNa/Ca antiport.

Addition of BrA23187 to respiring mitochondria in the absence of nigericin results in a decrease in matrix [Ca] to a new steady state near 0.7 µM (Fig. 6B). Addition of nigericin to these mitochondria abolishes the DeltapH and results in rapid influx of Ca to bring matrix [Ca] to very nearly the same concentration as external [Ca].

Addition of diltiazem or TPP to block nNa/Ca antiport after a steady state [Ca] gradient has been established results in a slow increase in matrix [Ca] (Fig. 6C). The increase amounts to only about 0.07 pmol of Cabulletminbulletmg and probably results from a slow inward leak of Ca in response to the high Delta that is maintained under these conditions (see 27). This apparent leak increases with increasing external [Ca] and requires Delta (data not shown). The presence of an inward leak of [Ca] in response to Delta does not affect the conclusion that the nNa/Ca antiport is electrophoretic. However, it does mean that the steady state matrix [Ca] seen in protocols such as those in Fig. 5and Fig. 6represents a steady state balance between extrusion on the antiport and influx through the leak and is not due to the nNa/Ca equilibrium alone.

Equilibration of the [Ca] Gradient by Ca Influx

The [Ca] gradients just discussed were established by allowing efflux of accumulated Ca to reach a steady state with buffered external [Ca]. A comparable picture is obtained when Ca influx is allowed to proceed until it is balanced by Ca efflux. In this protocol respiring mitochondria are suspended in a KCl medium containing ruthenium red, 20 mM Na, and external [Ca] buffered at either 5, 10, 31, or 50 µM (Fig. 7). Under these conditions fura-2 fluorescence reports an increase in matrix [Ca] that is more rapid at higher external [Ca] (Fig. 7). Under these conditions Ca influx also occurs via the leak pathway just noted. Addition of nigericin abolishes DeltapH (and DeltapNa) and accelerates Ca entry when external [Ca] is low. A steady state in which influx is balanced by efflux is approached in all four cases. The [Ca]:[Ca] gradient established is 16, 20, 44, and 53 for the increasing values of external [Ca] (Fig. 7). If the stoichiometry of the antiport is n = 3, these gradients would require a Delta of between -71 and -102 mV in order to be maintained. The average measured Delta under these conditions is -115 mV, and the calculated n values using for the observed gradients ranges from 2.62 to 2.88.


Figure 7: Matrix [Ca] of respiring mitochondria treated with ruthenium red (1 µM) and suspended in media with different external [Ca]. Mitochondria equilibrated with fura-2 were added to the standard medium containing NaCl (20 mM), succinate (5 mM), ruthenium red (1 µM), NTA (2 mM), and CaCl(2) (0.115, 0.215, 0.565, or 0.78 mM) to produce a final external [Ca] of 5, 10, 31.5, or 50 µM, respectively. Nigericin (1 µM) was added at 100 s and matrix [Ca] allowed to come to a steady state. Matrix [Ca] at 600 s was calculated to be 0.32, 0.49, 0.72, and 0.95 µM for external [Ca] of 5, 10, 31.6, and 50 µM, respectively.



Na/Ca Antiport in Nonrespiring Mitochondria

Another test of the dependence of the nNa/Ca antiport on the pH and electrical gradients can be made using mitochondria treated with rotenone and oligomycin to block regeneration of DeltapH and Delta (Fig. 8). The mitochondria are suspended in KCl containing 20 mM NaCl, treated with ruthenium red, and challenged with Ca (13 µM). Under these conditions [Ca] entry is linked to nNa efflux and the presence of a residual Delta (matrix negative) will oppose [Ca] entry if the antiport is electrophoretic. Fura-2 fluorescence reports little Ca uptake until an uncoupler is added to dissipate residual DeltapH and Delta (Fig. 8). The uncoupler FCCP increases the rate of Ca influx by 4.6-fold under these conditions. Addition of nigericin dissipates DeltapH and accelerates Ca uptake by a little over two fold (Fig. 8), and a similar enhancement of passive Ca uptake is seen when valinomycin is added to abolish Delta (Fig. 8). Addition of both nigericin and valinomycin results in a rate of Ca influx nearly identical to that seen with FCCP (Fig. 8). The FCCP-stimulated entry of Ca under these conditions is inhibited 78% by diltiazem (0.1 mM) and 98% by the omission of Na, but only 11% by TPP (6.6 µM) (not shown). This suggests that TPP inhibition of Na/Ca antiport depends on Delta for accumulation into the matrix(28) , or TPP inhibition specifically occurs on the matrix side of the inner membrane.


Figure 8: Stimulation of Ca influx by FCCP, nigericin, and valinomycin in nonrespiring heart mitochondria. Mitochondria were added to the standard medium containing ruthenium red (1 µM), NaCl (20 mM), and EGTA (10 µM). CaCl(2) (40 nmol) was added at 130 s and as indicated FCCP (1 µM), nigericin (1 µM), valinomycin (1 µM), and succinate (5 mM) were added. After the CaCl(2) addition the calculated [Ca] of the medium is approximately 3 µM.



These observations suggest that Ca is entering the matrix by passive exchange on the electrophoretic antiporter, since optimal rates are obtained only when both DeltapH and Delta are collapsed. Eliminating DeltapH would permit matrix [Na] to be elevated and provide ample Na for exchange with entering [Ca]. Eliminating Delta would remove the electrical constraint that favors Ca efflux over Ca influx on the electrophoretic antiport.


DISCUSSION

The present studies have established that the nNa/Ca antiport of heart mitochondria promotes an electrophoretic exchange of Na for Ca rather than the electroneutral antiport indicated by previous work(2, 3, 4) . The experimental basis for the conclusion that the antiport is electroneutral (3) appears to be flawed and three different lines of evidence now support the concept that this antiport is electrophoretic. First, respiring mitochondria maintain a very significant [Ca] gradient (external [Ca] > matrix [Ca]) under conditions in which an electroneutral antiport should support no gradient ( Fig. 5and Fig. 6). Addition of an exogenous electroneutral exchanger, such as BrA23187, under these conditions discharges this gradient. These steady state [Ca] gradients can be produced either by extrusion of accumulated Ca from the matrix or by uptake of extramitochondrial Ca ( Fig. 6and Fig. 7). Second, passive uptake of Ca by nonrespiring mitochondria via the diltiazem-sensitive antiport is rapid only when both the pH gradient and the Delta opposing Ca influx is discharged (Fig. 8). Third, a recent null-point study from our laboratory has shown that the nNa/Ca antiport has more energy available to it than can be provided by a passive 2Na/Ca exchange(9) .

Early studies from Crompton's laboratory (5) suggested that the nNa/Ca antiport was electrophoretic. The rate of Na-dependent Ca efflux was increased by respiration and inhibited by uncouplers(5) , results compatible with a contribution of Delta to the exchange reaction. In addition, the presence of three independent binding sites for Na was indicated by kinetic studies(6) . However, Affolter and Carafoli (29) concluded that the antiport was electroneutral, because a TPP electrode showed no change in Delta when Na-dependent Ca efflux was initiated. Their study (29) has been largely dismissed (1, 2, 3) because respiratory compensation of Delta was not considered and TPP has been shown to be a potent inhibitor of Na/Ca antiport(1, 28) . In the present studies TPP was used to estimate Delta in parallel incubations and was never present when nNa/Ca activity was measured.

As discussed above, the report of Brand (3) was most influential in establishing the consensus that the Na/Ca antiport promotes electroneutral exchange (see (2) ). In his study conditions were chosen that should have produced a significant difference in the steady state Ca gradient if n were 2 as opposed to 3 (see Fig. 4), and no change in external [Ca] was seen with a Ca electrode when the exogenous antiport A23187 was added(3) . However, the current work establishes that DeltapH (and hence DeltapNa) as reported by the continuous readout of a fluorescent probe (Fig. 3) is considerably different from the value obtained by Brand using distribution of labeled probes and centrifugation(3) . If DeltapH is not as large as estimated by Brand (3) the ability to distinguish between n = 2 and n = 3 is lost (Fig. 4). The present study also follows matrix [Ca] using a fluorescent probe and finds values under 2 µM for this parameter in most experiments as opposed to the 500 µM calculated in the Brand report(3) . A value of 500 µM for matrix [Ca] is far out of line with estimates from other laboratories (see (30) and (31) , for example).

In addition, Brand (3) used only 67 pmol of ruthenium red/mg of mitochondrial protein to block infux via the Ca uniporter. We have demonstrated a K(i) of approximately 40 pmolbulletmg for inhibition of the uniport in beef heart mitochondria as assayed by Ca-induced K release and inhibition was not complete until 200 pmolbulletmg were added(32) . This suggests that the amount of ruthenium red used by Brand may not have been sufficient to completely block Ca uniport. In his protocol(3) , Ca influx via the uniport would decrease the measured external [Ca] and result in an erroneous lower apparent value for the Na/Ca antiport at equilibrium. In this study we use 1 µM ruthenium red, equivalent to 2000 pmol/mg protein, and although the uniporter is maximally inhibited, when nigericin is present other Delta-dependent pathways may become available for Ca influx.

In a recent study, Li et al.(4) report the isolation of a 110-kDa fraction with the properties of a Na/Ca antiport from mitochondria. When reconstituted into liposomes this fraction promoted transport that was not affected by uncouplers and it was concluded that the antiport was electroneutral(4) . This reconstituted antiporter, however, displays several unusual properties not observed for the native protein, such as K/Na exchange and inhibition by low concentrations of TPP in the absence of Delta(33) . In contrast, we find that Delta does affect the rate of passive influx of Ca on the antiport in intact, nonrespiring mitochondria (Fig. 8). The basis for these discrepancies between the native protein and the reconstituted fraction is not clear and requires further study.

It may be possible that the Na/Ca antiport can operate in either an electrophoretic or an electroneutral mode depending on environmental conditions(9) . A slow rate of Na-dependent Ca extrusion is seen in nonrespiring mitochondria (5, 8) that may reflect electroneutral exchange under these conditions. In a recent study (34) the sarcolemmal nNa/Ca antiport was reported to promote electroneutral Na for Ca exchange at low pH and electrophoretic antiport at physiological pH. Relative rates of Ca and Na movement were modulated by the state of protonation of the carrier(34) , and it was suggested that other regulatory components may control relative movements of these ions according to the physiological needs of the cell. There is ample precedent for modification of a mitochondrial transporter in such a way as to change its stoichiometry (see (35) , for example).

The [Ca] gradients established and the response to BrA23187 shown in Fig. 6represent powerful evidence that the antiport promotes an electrophoretic rather than an electroneutral exchange. The [Ca] gradients measured in the present study in the presence of nigericin with DeltapH near zero range from 6 to 53 (Fig. 5Fig. 6Fig. 7). To account for these gradients for an n = 2 antiport DeltapNa would have to be 0.39 to 0.86. Although it has not been conclusively shown that DeltapH = DeltapNa under all conditions, our measurements suggest a maximum difference between DeltapH and DeltapNa of approximately 0.2. This is well below the values of DeltapNa needed to accomodate an n = 2 antiport. An n = 3 antiport would require Delta values of -46 to -102 mV to maintain the [Ca] gradients as calculated from . Our estimates of Delta by TPP distribution average -115 mV (range -103 to -127 mV) over a wide range of external [Ca] in the KCl buffers used in our protocols indicate there is clearly sufficient potential available to produce the observed gradients. The n values calculated from when Delta is -115 mV for [Ca] gradients of 6-53 range from 2.4 to 2.88. Because there appears to be a small inward leak of Ca in response to Delta under our conditions (Fig. 6C), the steady state matrix [Ca] is higher than the true nNa/Ca equilibrium value and reflects a balance between the leak and extrusion on the antiport. The presence of this leak does not affect the conclusion that the antiport is electrophoretic, but it does complicate attempts to evaluate n with certainty from . Although it is attractive to suggest that n is equal to 3 as found for the sarcolemma antiport, the possibility remains that the stoichiometry is variable and can operate in either the electrophoretic or electroneutral mode(4, 34) . Further experiments are needed, especially with the isolated and reconstituted protein, to determine the mechanism and stoichiometry of the exchange conclusively.

The membrane potentials measured in this study with nigericin present (-115 mV average) seem relatively low when compared with literature values approaching -200 mV(23, 27) . However, in contrast to most of these measurements, the present investigation was performed in a high K medium. In the presence of nigericin DeltapH is collapsed and Delta elevated in the KCl medium, and this can result in nonohmic influx of K (and Ca)(27) . When coupled to nigericin-mediated K/H exchange such an influx would create a futile cycle and decrease Delta and the protonmotive force.

The possibility has been raised that the antiport is indeed electroneutral but that heterogeneity of the mitochondrial preparation contributes to the appearance of electrophoretic exchange. However, it appears unlikely that differential loading of [Ca] or fura-2 into well coupled and poorly coupled fractions could account for the [Ca] gradients observed in the presence of nigericin ( Fig. 5and Fig. 6). When nigericin is added (Fig. 5A) matrix [Ca] continues to decrease (i.e. the [Ca] gradient increases). This is incompatible with electroneutral antiport because the pH gradient is near zero (Fig. 5B), and no energy is available to an n = 2 antiport to increase the gradient. Under these conditions matrix [Ca] should increase for an electroneutral antiport and approach thermodynamic equilibrium at matrix [Ca] = medium [Ca]. This holds for all mitochondria that maintain a functioning endogenous antiporter, since both well coupled and poorly coupled mitochondria will approach thermodynamic equilibrium.

An electrophoretic antiport may have physiological advantages over an electroneutral exchanger in that Ca efflux can be driven by the energy in both the electrical and pH gradients making it asymmetric and essentially irreversible. For example, if respiring mitochondria maintain a Delta of -150 mV and a DeltapH gradient of 0.4, predicts that an n = 2 mechanism can maintain a [Ca]:[Ca] gradient of only 6.3 at equilibrium as compared to 5526 for an n = 3 mechanism. This may be particularly important during steady state Ca cycling in situ when the transmembrane gradients of Na and Ca may be small(22) . Mitochondria in situ may have only a small Na gradient due to the presence of metabolites such as P(i) and CO(2) that tend to equilibrate the mitochondrial pH gradient.

Recently, an active mechanism has been proposed for the Na-independent Ca efflux seen in liver mitochondria(21) . Because nNa/Ca exchange can be demonstrated in mitochondria where respiration is blocked by rotenone and ATPase activity is blocked by oligomycin (Fig. 7), and sufficient energy is available in the Na electrochemical gradient to account for the Ca gradients measured, it does not seem necessary to postulate that the nNa/Ca antiport represents an active transport(21) . The results appear compatible with a passive electrophoretic mechanism with an n value of approximately 3 that mediates ``downhill'' exchange of Na and Ca.

The present studies, in conjunction with our previous report(9) , establish that the mitochondrial antiport, like that in the plasmalemma (7) , is capable of supporting an electrophoretic exchange. Further work will be necessary to establish the stoichiometry with certainty and whether or not the antiport can also operate as an electroneutral exchange under some conditions.


FOOTNOTES

*
This work was supported in part by United States Public Health Services Grant HL09364-29. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Medical Biochemistry, Ohio State University, 333 Hamilton Hall, Columbus, OH 43210-1218. Tel.: 614-292-3917; Fax: 614-292-4118.

Present address: Division of Cardiovascular Research, St. Elizabeth's Medical Center, 736 Cambridge St., Boston, MA 02135.

(^1)
The abbreviations used are as follows: TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; BCECF, 2`,7`-bis(carboxyethyl)-5(6)-carboxyfluorescein; AM, acetoxymethyl ester; cSNARF-1(TM), a pH indicator marketed by Molecular Probes, Inc.; pH, extramitochondrial pH; pH, matrix pH; Delta, membrane potential; TPP, tetraphenylphosphonium ion; NTA, nitrilotriacetic acid; FCCP, carbonyl cyanide-4-trifluoromethoxyphenylhydrazone; TMA, tetramethylammonium ion.


ACKNOWLEDGEMENTS

We thank Lynn Apel for expert technical assistance.


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