THE JOURNAL OF BIOLOCKXI. CHEMISTRY Vol. 251, No. 16, Issue of August 25, pp. 5069-5077, 1976

Printed in U.S.A.

Relationship between Configuration, Function, and Permeability in Calcium-treated Mitochondria””

(Received for publication, December 1, 1975)

DOUGLAS R. HUNTER,ROBERT A. HAWORTH,~ ANDJAMES H. SOUTHARD

From the Institute of Enzyme Research, University of Wisconsin, Madison, Wisconsin 53706

Low levels of calcium (100 nmol/mg) added to beef heart mitochondria induced a configurational transition from the aggregated to the orthodox state and a simultaneous uncoupling of oxidative phosphorylation. The primary effect of calcium was to cause a nonspecific increase in the permeability of the inner membrane, resulting in entry of sucrose into the matrix space and the observed configurational transition. The uncoupling and permeability change induced by calcium could readily be reversed by lowering the calcium:magnesium ratio in the presence of either substrate or ATP. The configurational state, however, remained orthodox. This, along with studies of hypotonically induced orthodox mitochondria in which the membrane remained coupled and impermeable until after the addition of calcium, led to the conclusion that coupling was related to the permeability state of the inner membrane rather than the configurational state.

Phosphate, arsenate, or oleic acid was found to cause a transition similar to that induced by calcium. Studies with the specific calcium transport inhibitors, EGTA, ruthenium red, and lanthanum revealed that endogenous calcium is required for the anion-induced transitions. A single mechanism was further indicated by a common sensitivity to N-ethylmaleimide.

Strontium was ineffective as an inducer of the transition, even though it is transported by the same mechanism as calcium. This indicates that there are additional calcium-binding sites responsible for triggering the transition. Magnesium and calcium appeared to compete for these additional sites, since magnesium competitively inhibited the calcium-induced transition, but had no effect on calcium uptake.

Calcium was found to potently inhibit the respiration of all NAD+-requiring substrates prior to the transition. Strontium also produced this inhibition without a subsequent transition. ATPase activity was induced at the exact time of transition with calcium and was not induced by strontium. This suggests that calcium-induced ATPase uniquely required the transition for activity, in contrast to the ATPase induced by uncoupler or valinomycin.

The results of this work indicate that mitochondria have a built-in mechanism which responds to low levels of calcium, phosphate, and fatty acids, resulting in simultaneous changes, including increased permeability, induction of ATPase, uncoupling of oxidative phosphorylation, and loss of respiratory control.

The accumulation of calcium by mitochondria takes prece- dence over oxidative phosphorylation (1) and therefore, must be of the utmost importance in the regulation of cellular activity (2). Much is known about the mechanism of calcium transport and its relationship to energy transduction, but little is known about the function of calcium uptake. A major consequence of calcium uptake is the induction of a configura- tional transition which has been observed electron microscopi- cally (3, 4), and by following the decrease in either optical density or light scattering (4-9). This calcium-induced transi- tion has not been accepted as a physiologically important

* This investigation was supported in part by Program Project Grant GM-12847 of the National Institute of General Medical Science of the National Institutes of Health.

$ Recipient of a Wellcome Research Travel Grant.

mechanism because of the difficulty with which it is reversed (3) and the loss of critical mitochondrial functions associated with the transition (3, 6, 10, 11). With these reservations in mind, we have taken a closer, more systematic look at the configurational transition induced by calcium. By studying simultaneously the effect of calcium on structure and function, we have uncovered some of the key events which take place during the transition.

EXPERlMENTAL PROCEDURE

Preparation of Mitochondria-Heavy beef heart mitochondria were prepared daily from fresh beef hearts by the method of Crane et al. (12), as described by Hatefi and Lester (13). The mitochondria were adjusted to a protein concentration of 50 mg/ml and used within 4 h. Protein was determined by the method of Gornall et al. (14).

5069

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5070 Calcium-induced Transition in Mitochondria

EGTA’ mitochondria were prepared as above except that EGTA (1 mM) was present in the suspension buffer during the low speed separation of cell debris from the crude mitochondria and the first of two light-heavy separations.

The transition from aggregated to orthodox configuration was studied by the following standard procedure. Mitochondria (2 mgiml) were suspended in a solution 250 rnM in sucrose and 20 rnM in Tris-Cl, pH 7.4 (standard reaction medium). After 30 s, one of the following inducers was added: calcium chloride, potassium phosphate (pH 7.4), potassium arsenate (pH 7.4), or oleic acid (ethanolic solution). Samples were removed at specific times for electron microscopy and fixed with equal volumes of an ice-cold 2% glutaraldehyde solution which was 250 rnM in sucrose and 50 mM in potassium cacodylate, pH 7.5. The procedure used for staining and thin sectioning was as described earlier (15).

Assay of Uncoupler Respimtory Control Index-Mitochondria (3 mg) were added to a water-jacketed reaction cell containing 4 ml of solution which was 20 mM in Tris-HCl, pH 7.4, and 250 rnM in sucrose. Further additions were made after 30 s and the suspension was continuously stirred. Fifty microliters of a saturated ethanolic solution of durohydroquinone (63 mM) were added and respiration was moni- tored, using a Beckman oxygen analyzer. After % to 1 min, FCCP (ethanolic solution) was added to a concentration of 1 JIM. Respiration was recorded until the remaining oxygen was exhausted. For these samples, the uncoupler respiratory control index was then calculated as the ratio of the rate of durohydroquinone respiration with FCCP to the rate of respiration without FCCP.

Measurement of Sucrose-impermeable Space-The method was based upon the dual isotope procedure of Hunter and Brierley (16). *Hz0 and [“C]sucrose were added to a solution 250 rnM in sucrose (10 rnM for hypotonic experiments) and 20 rnM in Tris-Cl, pH 7.4, to specific activities of 0.4 &i/ml and 0.8 rCi/ml respectively. Calcium chloride or potassium arsenate was added at this stage when appropriate. The reaction was initiated by addition of mitochondria (50 mg/ml in 250 rnM sucrose/l0 rnM Tris-Cl, pH 7.8, at O”) to a final concentration of 10 mg/ml. Twin l-ml aliquots were removed at time intervals and spun down in a high speed Beckman bench centrifuge. The spin time was 4 min. but a pellet was formed within 5 s. The supernatants from a single time sequence were pooled and the pellets were blotted with filter paper. Each pellet was dispersed by sonication in 1 ml of 0.5 M perchloric acid. The samples were spun again for 2 min. The clear supernatant (0.7 ml) was added to 10 ml of Aquasol (New England Nuclear) for counting.

In experiments where sucrose penetration measurements and config- uration were both measured, l-ml aliquots for electron microscopy were removed from the incubation mixture at the same time as aliquots for isotope analysis. These were fixed as stated above except that when hypotonic samples were taken, 60 mM sucrose was used in the fixation buffer.

Oxidatiue Phosphotylation Assay-Mitochondria (1 mg/ml) were suspended in a solution 250 rnM in sucrose, 10 rnM in Tris-HCl, pH 7.4, 5 mM in glucose, 10 mM in [?‘]phosphate, and 1 rnM in ADP, containing 5 units/ml of hexokinase. Durohydroquinone was added to start the reaction. When all the oxygen was consumed (measured with an oxygen analyzer), the reaction was stopped with perchloric acid and the amount of esterified inorganic phosphate was determined as previously described (17, 18).

ATPase Assay-The reaction medium (1 ml) contained the follow- ing reagents: sucrose (250 mM); Tris-HCl (20 mM), pH 7.4; ATP (7 mM); and mitochondria (1 mg). The reaction was started with either ATP or mitochondria and was stopped after 1% min by the addition of 4.4 ml of the silica tungstate reagent for the separation of inorganic phosphate from organic phosphate by method D of Lindberg and Ernster (17). Inorganic phosphate was determined according to the method of Martin and Doty (19).

Measurement of Proton, Calcium, and Magnesium Mooements- The pH of a mitochondrial suspension was monitored continuously as previously described (20). The reaction medium was similar to that used to measure the uncoupler respiratory control index, except that the concentration of Tris buffer was lowered to 2 mM.

To determine the magnesium and calcium content of mitochondria, samples were removed and centrifuged in an Eppendorf microcentri-

‘The abbreviations used are: EGTA, ethylene glycol bis(&amino- ethyl ether)-N,N’-tetraacetic acid; FCCP, carbonylcyanide p-tri- fluoromethoxyphenylhydrazone; P/O, phosphate esterified to oxygen consumed; MalNEt, N-ethylmaleimide.

fuge. The pellet was rinsed once with 0.25 M sucrose and dispersed by sonication in 1.0 ml of H,O. The protein was precipitated by HClO, and removed by centrifugation. The supernatant was diluted to the appropriate level with 1% La(NOJ)a for determination of Mg’+ and Ca*+ by atomic absorption spectrophotometry (21). All assays were carried out at 30”.

Chemicals-Durohydroquinone was purchased in the reduced form from K & K Laboratories and dissolved in absolute ethanol to a concentration of 63 mM. One drop of 0.1 N HCl per 3 ml was added to prevent autooxidation. FCCP was purchased from Pierce Chemicals, ruthenium red from Sigma, and [“C]sucrose, [“Clcholine and 3H,0 were purchased from New England Nuclear.

RESULTS

Effect of Calcium on Configuration of Mitochondria-The

addition of calcium to beef heart mitochondria resulted in a

configurational transition, as was found with rat liver (3) and

adrenal cortex mitochondria (4). Within 15 s after the addition

of calcium, over 80% (or 80 nmol/mg) was accumulated by the

mitochondria and an equal amount of protons was ejected (1,

22). Although no substrate was added, rotenone completely

inhibited both calcium uptake and proton ejection, indicating

that endogenous NAD+-requiring substrates were oxidized.

Samples of mitochondria were taken at various times after the

addition of calcium and fixed for electron microscopy. Fig. IA

shows that immediately after calcium was accumulated, the

configurational state of the mitochondria was aggregated; 3 %

min later, (Fig. lB), the mitochondria were in either the

aggregated or orthodox state. Seven minutes later (Fig. lC),

the mitochondria were all in the orthodox configuration. In a

control experiment, mitochondria incubated for 7 min in the

absence of calcium remained in the aggregated configuration.

Judging from the absence of any configuration other than

aggregated or orthodox, we conclude that the transition for an

individual mitochondrion must consist of a lag phase during

which it is aggregated, followed by a rapid change to the

orthodox state. The gradual nature of the overall transition,

shown in Fig. 2, was therefore due to some heterogeneity of the

mitochondrial population which resulted in a range of lag

phases.

Effect of Calcium on Coupling of Inner Membrane-For the

study of the effect of calcium on mitochondrial function, an

assay of respiratory control in the standard system was used.

Durohydroquinone was chosen as a substrate because it could

by-pass the calcium-induced inhibition of respiration (23), and

the oxidation showed good respiratory control. Uncoupler

release of respiration was used instead of release by ADP in

order to avoid using phosphate or ADP which both complicate

the calcium-induced transition. That the uncoupler respiratory

control index was an accurate indicator of the phosphorylating

ability was confirmed by us in numerous experiments. In Fig.

2, the uncoupler respiratory control index (UK0 is compared

to the configurational state in samples taken at various times.

At each point, the percentage of aggregated mitochondria

correlated directly with the uncoupler respiratory control

index. The significance of this correlation is that the all-or-

nothing transition from the aggregated to the orthodox state for

each separate mitochondrion has as a functional counterpart,

an all-or-nothing coupling transition from a state of maximal

respiratory control (3 to 4), to one of minimal respiratory

control (1 to 1.5).

Effect of Calcium on Membrane Permeability-During the transition, the sucrose-permeable space of the mitochondrial

pellet was found to increase from about 85% to about 96% (Fig.

3). The dextran- (M, = 16,000) or inulin- (M, = 5,000)

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Calcium-induced Transition in Mitochondria

.

FIG. 1. Calcium-induced configurational transition. Aliquots were fixed after different times of incubation in the presence of 100 nmol of calcium/mg of protein: A, 15 s; B, 3% min; C, 7 min. See “Experimental Procedure” for details.

Time (min) After a&Iii Calcium (100 nmllmg)

FIG. 2. Effect of calcium on configuration and coupling. This experiment was the same experiment from which the electron micro- scopic pictures for Fig. 1 were taken. Samples (1.5 ml) for uncoupler respiratory control index (URCZ) determination were taken at the same time as the samples for electron microscopy. See “Experimental Procedure” for details.

permeable space remained constant at about 70% (data not shown). Identifying the inulin-permeable space as extramito- chondrial pellet water, we find that during the transition there is an approximate doubling of the mitochondrial sucrose-

I:~~~~~ II $66- 0 8 0 Oh

E l I I I

8 2 4 6 6 ‘I 30 0 z Time (min)

FIG. 3. Correlation between calcium-induced configurational change and change in permeability to sucrose. See “Experimental Procedure” for details.

permeable space. Furthermore, when mitochondria were turned orthodox in unlabeled sucrose and then [“Clsucrose was added, equilibration with the matrix space was found to be complete within 1 min (Fig. 4, curue A). From these experi- ments, we conclude that calcium has the novel effect on the mitochondria of increasing the permeability of the inner membrane to sucrose. Water accompanying the entrance of sucrose into the matrix compartment would result in the observed configurational transition. Our results thus do not support the contention of Hackenbrock and Caplan (3) that the calcium-induced transition is nonosmotic.

The change in membrane permeability induced by calcium was found to be unspecific. When choline chloride (0.15 M)

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50’72 Calcium-induced Transition in Mitochondria

Time (min)

FIG. 4. Restoration of coupling and membrane impermeability. The incubation medium was as for the experiment shown in Fig. 3 except the [“Clsucrose was initially omitted. After a 5-min incubation in the presence of 150 nmol of calcium/mg of protein, the additions listed were made. After a further 1-min incubation, the [“Clsucrose was added. Aliquots were removed at 1-min intervals thereafter and treated as described (see “Experimental Procedure”). Aliquots were also removed at 10 min for measurement of P/O ratios in a medium lacking added magnesium, using durohydroquinone as substrate. Final concentrations of additions were: ATP, 6 mM; MgCl,, 6 mM; EGTA, 3 mM.

Time (min) cltbsr adding Calcium (loo nmdhl)

FIG. 5. Effect of the calcium-induced transition on the magnesium content of mitochondria. To mitochondria suspended in the standard assay mixture (2 mg/ml) were added 100 nmol/mg of calcium chloride. Samples were taken at the times shown and the uncoupler respiratory control index (URCI) and the magnesium content of mitochondria was determined as described under “Experimental Procedure.” The zero time point was done on samples taken before calcium addition.

replaced sucrose as the osmotic support, a similar calcium- induced configurational transition was observed and [%]cho- line permeability studies showed that choline entered the matrix space. When sucrose was replaced by glucose, the transition occurred at the same rate as in sucrose. This shows that mitochondria do not become permeable to carbon mole- cule 6 before carbon molecule 12, but rather that the permea- bility changes abruptly to a level which allows sucrose and smaller molecules to freely permeate. The upper limit on size of permeable molecules has not yet been established.

Consistent with the permeability change above, ions inside the matrix space equilibrated with the medium. In Fig. 5, the magnesium content of mitochondria is compared to the uncou- pler respiratory control index at different times after the addition of calcium. Magnesium was lost exactly parallel to the transition. We also found equilibration of calcium and protons

FIG. 6. Correlation between configuration and movement of cal- cium, magnesium, and protons. See “Experimental Procedure” for details.

0 2 4 6 10 Time (min)

FIG. 7. Effect of calcium on sucrose-permeable space of mitochon- dria suspended in 250 rnM sucrose and 60 rnr.4 sucrose. The amount of calcium used was 100 nmol/mg of protein. See “Experimental Proce- dure” for details.

to occur at the time of transition (Fig. 6). This is in disagree- ment with others, who claim that the magnesium loss precedes the loss of calcium (24). The question arises whether the change in function is related to the change in configuration or to the change in permeability. This has been resolved by the following two experiments.

Hypotonically Treated Mitochondria-Mitochondria sus- pended in a hypotonic medium (60 mM sucrose) become orthodox in configuration (25) with no accompanying change in the permeability of the inner membrane. In fact, the sucrose- impermeable space is twice as large, due to an expansion of the matrix space and contraction of the intercristal space (Fig. 7). Upon the addition of calcium, sucrose was found to enter the swollen matrix space, while the configuration remained ortho- dox. In the absence of calcium, the inner membrane remained impermeable to sucrose. Coupling in hypotonically swollen mitochondria was compared with coupling in aggregated mitochondria. Hypotonically swollen mitochondria were found to have only a slightly lower uncoupler respiratory control index; as with aggregated mitochondria, these mitochondria became uncoupled after the addition of calcium (Fig. 6). These results demonstrate that a configurational transition without a membrane permeability change does not lead to a change in

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Calcium-induced Transition in Mitochondria 5073

coupling. Any calcium-induced membrane permeability

change, whether accompanied by a configurational transition

or not, is paralleled by a coupling transition.

Reoersal of Calcium-induced Membrane Transition-The

conclusion that coupling parallels permeability rather than

configuration was further supported by the experiments on

reversal of the calcium-induced orthodox transition (10, 26).

Addition of either EGTA or magnesium, in the presence of an

energy source (durohydroquinone or ATP), resulted in a rapid

reversal of the permeability of the inner membrane to sucrose. In the absence of reversing agents, [“Clsucrose has fully

permeated the orthodox mitochondria within 1 min of its

addition (Fig. 4, curue A), whereas the addition of reversing

agents 1 min before the [“Clsucrose is seen to prevent

permeation (Fig. 4, curues B and C). These additions also cause a rapid recoupling of durohydroquinone oxidase activity

(Fig. 9). However, the mitochondria remained in the orthodox

configuration. This rapid recoupling of the inner membrane

was also observable with measurements of the P/O ratio. The P/O ratio of calcium-treated orthodox mitochondria ( <O. 1)

increased with EGTA treatment (0.5) or with magnesium

addition (1.0) (Fig. 4). The lower P/O obtained with EGTA is

not a result of incomplete recoupling (Fig. 9), but probably due

s 2-

,1 0 33 67 100 33

CaCl2 added (nmollmg)

FIG. 8. Comparison of the effect of calcium on the uncoupler FIG. 9. Recoupling induced by either EGTA or magnesium. Mito- respiratory control index (URCn of normal and hypotonically swollen chondria (0.75 mg/ml) were preincubated for 5 min with 100 nmol/mg mitochondria. Mitochondria (0.75 mg/ml) were incubated for 30 s in 20 of calcium chloride under standard conditions before the addition of mre Tris-HCl buffer, pH 7.4, in 60 rnM or 250 mM sucrose. The amount durohydroquinone. The traces shown are taken directly from the of calcium indicated was added and the suspension was stirred for 4 oxygraph. Numbers in parentheses indicate rate of respiration in min more, at which time the uncoupler respiratory control index was microgram atoms of oxygen/minute/mg protein. Final concentrations measured. of additions were: FCCP, 1 PM; MgCl,, 2 mM; EGTA, 0.5 mM.

to the loss of endogenous magnesium which is required for the

phosphorylation mechanism.

Other Inducers of Transition-Reagents capable of inducing

the transition include phosphate, arsenate, and fatty acids.

These reagents have been shown previously to cause swelling

(5-7). The simultaneous uncoupling and configurational tran- sition induced by these reagents was similar to that induced by

calcium (Fig. 10). As with calcium, the configurational transi-

tion induced by arsenate, for example, is paralleled by the loss

of the sucrose-impermeable space (Fig. 11).

The similarities between the calcium-induced transition and

the transition induced by these anions led us to look for

common denominators between the transitions induced by

each agent. Phosphate- or fatty acid-induced swelling (7, 27)

has been shown to be prevented by chelators of calcium. We

have confirmed this and also find a common sensitivity of the

transitions to ruthenium red and lanthanum, specific inhibi-

tors of high affinity calcium transport (28, 29) (Figs. 12 and 13).

Further proof of the requirement for endogenous calcium in the

anion-induced transition came from an experiment with EGTA

mitochondria. EGTA mitochondria were found to be insensi-

tive to arsenate (or phosphate), but completely sensitive to

calcium (Fig. 14). The calcium content of EGTA mitochondria

was compared to the calcium content of normal mitochondria

and found to be lower by over 80%.

A second common denominator for the various inducers of a

membrane transition was found by studying the effect of

MalNEt, the inhibitor of the phosphate-hydroxyl exchange

system (30). MalNEt was expected to inhibit the phosphate- or

FIG. 10 (left). Effect of arsenate, phosphate, and oleic acid on configura- tion and coupling. This experiment was done the same way as the experiment described in the legend to Fig. 2 except that 1 mM arsenate, 10 mM phosphate, or oleic acid (10 nmol/mg) was added in place of calcium. +

FIG. 11 (right). Correlation between u

,

arsenate-induced configurational change 3 and change in permeability to sucrose. Oxygen was bubbled through the incuba- tion vessel during the course of the ex- periment. See “Experimental Proce- dure” for details. Tlmelmm) Tlme(min) Time(mln)

Time (min)

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Calcium-induced Transitioiz in Mitochondria

- 6 Time (min)

After aedng Arsenate(lmY)

I- O 2 4

Time (min) After addmQ c*ium wo inmtlms)

FIG. 12 (left). Effect of inhibitors on arsenate-induced uncoupling. Mitochondria (0.75 mg/ml) were preincubated under standard condi- tions (see “Experimental Procedure” for details) with the inhibitor added 30 s before the addition of 1 rn~ arsenate. Each point represents a separate experiment. Final concentration of inhibitors was: EGTA (0.5 mM), LaCl, (2 nmol/mg), ruthenium red (3 nmol/mg), MalNEt (15 nmol/ml). URCI, uncoupler respiratory control index.

FIG. 13 (center). Effect of inhibitors on calcium-induced transition.

arsenate-induced transitions (Fig. 12), but the inhibition of the calcium- (Fig. 13) and oleic acid-induced transitions was unexpected. We cannot say whether the sensitivity to MalNEt means that the phosphate carrier is involved in all transitions or whether there is another as yet unknown MalNEt-sensitive protein required for the transition.

Antagonism between Calcium and Magnesium-The speci- ficity for induction of the transition by a divalent metal was greater than the specificity for active divalent metal uptake. Strontium can be transported as readily as calcium (31). However, for inducing the transition, we found that strontium was completely ineffective and even inhibited the transition when added in concentrations equal to calcium. This inhibi- tion was expected, since strontium competes with calcium for transport by the ruthenium red-sensitive carrier (31). Magne- sium was also found to be an inhibitor of the transition (10,32), but unlike that by strontium, the inhibition by magnesium at concentrations below 3 mM was not at the level of calcium uptake. This inhibition by magnesium of the calcium-induced transition was determined to be competitive from the data shown in Fig. 15. Here, the concentration of calcium required to cause uncoupling within a set time is shown to increase proportionally to the magnesium concentration. From the slope of the line, a relative affinity for magnesium was determined to he approximately ‘/a the affinity of the mito- chondria for calcium.

Another important difference between the inhibition by magnesium and the inhibition by strontium of the calcium- induced transition was found by changing the order of addition of reagents (Table I). The uncoupler respiratory control index of the membrane was found to be equally high whether mag- nesium was added before calcium or after the calcium transition was completed. Strontium, on the other hand, was almost with- out effect when added after the transition. Both ruthenium red

and MalNEt behaved similarly to strontium in being effective only when added before calcium (see “Discussion”).

1 2 3 4 Time (min)

After ae-ag Arsenate (Imy,

Conditions were the same as described in the legend to Fig. 12 except that calcium (100 nmol/mg) was added instead of arsenate. URCI, uncoupler respiratory control index.

FIG. 14 (right). Effect of arsenate on EGTA mitochondria. Either regular or EGTA mitochondria (see “Experimental Procedure”) were incubated (0.75 mg/ml) under standard conditions with 1 mM arsenate. The uncoupler respiratory control index (URCI) was determined in separate experiments for the different time points shown on the curves.

/

Magnesium Calcium

Ratio = 4.4

A-J---- 100 200

Calcium (nmol/mg) Required to gave 50% transition

FIG. 15. Effect of magnesium on calcium-induced transition. Mito- chondria (0.75 mg/ml) were preincubated for 15 s under standard conditions with magnesium before the addition of calcium. The uncoupler respiratory control index was determined 4 min after the addition of calcium (see “Experimental Procedure”). For each magne- sium concentration indicated, separate assays were performed with various calcium concentrations. From the data for each magnesium concentration, a curve was drawn similar to the one shown in Fig. 8. The amount of calcium required to give 50% uncoupling (uncoupler respiratory control index x2.5) was obtained from each curve and re- plotted against the respective magnesium concentration.

Effect of Calcium on Substrate Oxidation-The uncoupling effect of the calcium-induced transition on durohydroquinone oxidation tells us a great deal about the state of the inner mem- brane, but little about physiological changes in mitochondrial function as a result of the transition. Kun’s laboratory has studied the inhibition of glutamate oxidase activity by low

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Calcium-induced Transition in Mitochondria 5075

levels of calcium (23). We now extend his results to include

pyruvate/malate and P-hydroxybutyrate respiration and sug- gest that effect of calcium to be a general inhibition of the oxi-

dation of all NAD+-requiring substrates. This inhibition was

found to take place before the transition was induced and took

place even in the absence of a transition when strontium was

used in place of calcium (Fig. 16). With succinate as a substrate,

a different and more complicated result was found (33). Suc-

cinic oxidase was only partially inhibited by calcium at the

time of transition and the remaining activity became uncoupled

similar to durohydroquinone respiration. When strontium was

used in place of calcium, no uncoupling resulted, but partial

inhibition still took place.

TABLE I

Effect of inhibitors on calcium-induced uncoupling

Beef heart mitochondria (0.75 m&n!) were preincubated 30 s in the standard assay mixture before the addition of calcium (100 nmol/mg). In the prevention experiments, the inhibitors were added immediately after the mitochondria and 30 s before the calcium. The mitochondria were then incubated for 5 min after calcium addition and the uncoupler respiratory control index was determined. In the reversal experiments, the inhibitors were added after a 5-min incubation period with calcium. Fifteen seconds later, the uncoupler respiratory control index was determined. See “Experimental Procedure” for details.

Uncoupler respiratory control Index

Inhibitor Control Prevention RWerSal

(no calcium) (before (5 min after

calcium) calcium)

None 3.72 1.38 1.38

Magnesium (2 mM) 4.44 3.68 3.27 Strontium (2 mM) 3.21 3.29 1.91

Ruthenium red (3 nmol/mg) 4.06 3.82 1.38 MalNEt (15 nmol/mg) 3.52 3.27 1.24

Sri2 ---

<\ _ _ - - - - - -

-\

J ‘Li -r f

I I I 2 4 6 8

3.5

3.0

Effect of Configurational Transition on ATPase-The only

activity induced by the transition was ATPase (Fig. 17). This ATPase was magnesium-dependent, as judged by the stimula-

tion of activity by magnesium and by the selective inhibition

by EDTA over EGTA (Fig. 18). The fact that EDTA was able

to inhibit the activity is further evidence that the membrane of

orthodox mitochondria is permeable to magnesium. In contrast,

uncoupler- or valinomycin plus K+-induced ATPase were each

found to be insensitive to EDTA. The point to be made here

is that calcium does not simply induce an ATPase, like un-

coupler or valinomycin, but calcium induces a membrane transition which carries an ATPase with it. Further evidence

for this came from the following two observations: rotenone, which inhibited the transition, also inhibited the ATPase (Fig.

-.-

0 5 10 15

Concentration of metal chelator(mM)

FIG. 18. Effect of divalent metal chelators on the calcium-induced ATPase activity. Mitochondria (1 mg/ml) were incubated for 5 min in the standard assay mixture with 100 a~ calcium before the addition of EDTA or EGTA. ATP was added 15 s later and ATPase was assayed as described under “Experimental Procedure.”

4

Time (min) After adding metdl

FIG. 16 (left). Comparison of effects of calcium and strontium on oxidation of pyruvate/malate and durohydroquinone. To mitochondria suspended at 1 mg/ml in the standard assay mixture was added either calcium (100 nmol/mg) or strontium (100 nmol/mg). Pyruvate plus malate was added .to a final concentration of 5 mM each at the times shown in the figure. FCCP (final concentration 0.5 PM) was added 30 s later and the respiratory rate was determined. The effect of calcium and strontium on the uncoupler respiratory control index (URCI) with durohydroquinone as substrate was determined in separate experi-

0 5 lo 15 20 Time (min)

ments (see “Experimental Procedure” for details). FIG. 17 (right). Effect of calcium-induced transition on ATPase

activity. Mitochondria (2 mg/ml) were incubated in the standard assay mixture for 30 s before calcium (100 nmol/mg) was added. Samples were removed and assayed separately for the uncoupler respiratory control index (URCZ) and ATPase activity at the times shown as described under “Experimental Procedure.” Rotenone (5 nmol/mg) was added 30 s before calcium.

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5076 Calcium-induced Transition in Mitochondria

17). and strontium, which could not induce a transition, cannot

induce ATPase (34).

DISCUSSION

We have given evidence that the calcium-induced configura-

tional transition from the aggregated to the orthodox state is

a consequence of a primary effect of the increased permeability

of the inner membrane which goes parallel with the transition.

Azzone et al. (35) have reported a similar reversible effect by

calcium on increasing the permeability of the inner membrane

to potassium.

Lehninger previously recognized that swollen mitochondria

were permeable to sucrose but believed this to be a result of

swelling and not the cause (36). Azzone and co-workers also

found that calcium phosphate-treated mitochondria were

highly permeable (37), and maintained that such mitochondria

were still capable of energy conservation (38). We, however, have found here that an increase in permeability is paralleled

by an increased uncoupling in calcium-treated mitochondria,

and conditions which restore coupling also restore imperme-

ability to sucrose. This is not to say that an impermeable mem-

brane is the sine qua non for energy conservation. Work in this

laboratory (18, 39) on lysolecithin-treated submitochondrial

particles has shown that coupling can be retained in a case

where the membrane is completely disrupted. Our results on hypotonically swollen mitochondria dismiss

the long held view that swollen mitochondria must be damaged

and uncoupled. The correlation between uncoupling and swell-

ing arose because swollen mitochondria were commonly pro-

duced by agents which promoted the transition studied here.

We have shown that the swelling per se is not a significant

factor in determining the state of coupling. Even mitochondria

grossly swollen (5 mM sucrose/20 mM Tris-Cl) were found to

be almost fully capable of coupled respiration, even with

NAD’-linked substrates.2

It remains to be seen what is responsible for the uncoupling

of orthodox mitochondria. Since ruthenium red and strontium

did not inhibit respiration in calcium-induced orthodox mito-

chondria, we can eliminate cycling of calcium by way of the

high affinity transport system. The uncoupling induced by a

membrane transition is therefore different from uncoupling

induced by the addition of the calcium ionophore A23187 (40).

Our results indicate that the orthodox configurational transi-

tion is induced by calcium in all cases studied here. The

configurational transition induced by thyroxine (41) and di-

amide (42) also appear to be dependent on calcium. We found

the amount of calcium required to induce a transition to be

dependent on the concentration of other reagents. With no

other additions, 100 nmol of calcium/mg of mitochondrial pro-

tein was routinely used, but in the presence of either phosphate,

arsenate, or fatty acids, endogenous calcium was found to be

sufficient. To date, the lowest amount of total calcium (includ-

ing endogenous) we have found to induce a transition was just 10 nmol/mg.

We can divide the calcium-induced membrane transition

into three distinct steps. The first step is the energized uptake

of calcium through the ruthenium red-sensitive carrier. The energy required for this step comes from the oxidation of en-

dogenous NAD+-linked substrates, as rotenone completely

prevented both the calcium uptake and the transition. The

‘D. R. Hunter, R. A. Haworth, and J. H. Southard, unpublished observations.

second step is the inhibition of respiration of all NAD+-linked

substrates. For these first two steps, strontium can replace

calcium, but for the third step, which is the membrane transi-

tion itself, only calcium works. In the simplest model, the third

step involves sites that respond to calcium, which results in

uncoupling and changing the permeability of the membrane.

Magnesium may be tightly bound at these sites and the dis-

placement of magnesium by calcium leads to the transition.

Other laboratories have noted a similar critical role for magne-

sium in maintaining the integrity of the membrane (43, 44).

Once the transition has taken place, the mitochondrial mem-

brane can readily be recoupled and made impermeable again

by the addition of ATP or substrate, along with some reagent,

such as EGTA or magnesium, which will lower the calcium:

magnesium ratio. The reversal of sucrose permeability traps

the sucrose inside and thus explains the failure of the orthodox

mitochondria to return to the aggregated configuration (5).

These recoupled mitochondria did not, however, regain the

ability to oxidize NAD+-linked substrates.* At the present

time, we cannot say if this function is irreversibly lost. Others

have claimed to find conditions for reversal (45).

The data we have presented show the calcium-induced mem-

brane transition to be closely connected to high affinity calcium

transport. Most workers who studied calcium uptake (46-48)

did their assays in the presence of a large amount of magnesium

so that the transition was either inhibited, or the membrane

was rapidly recoupled after the transition had taken place. For

the studies done on calcium-stimulated ATPase (48, 49), we

believe that a membrane transition was a prerequisite.

Possible roles of the transition in the control of mitochondrial

and cellular function are presently emerging. The stimulation

of heat production in brown adipose tissue cells by hormones

is thought to occur by the stimulation of uncoupled respiration

in the mitochondria. Reed and Fain (50) have proposed that

fatty acids mobilized by hormone action cause this stimulation

by the induction of permeability changes in the mitochondrial

inner membrane, allowing potassium ions to pump in a futile

cycle. Others (51) have criticized this hypothesis on the grounds

that there is no evidence for the induction of such changes by

fatty acid in mitochondria from other tissues. However, such a

change has been demonstrated here.

Bygrave and his collaborators (52, 53) found that mitochon-

dria isolated from ascites tumor cells were resistant to uncou-

pling by either calcium or phosphate, and postulated that the

defect must be located at some site other than the transport

systems for calcium or phosphate. This resistance must reflect

a difference in susceptibility to the transition studied here.

Acknowledgments-We thank Dr. David E. Green for his

advice and encouragement. The skilled technical assistance of

Ms. Ghousia Nizamuddin and Ms. Karen Senzig is gratefully

acknowledged.

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D R Hunter, R A Haworth and J H Southardmitochondria.

Relationship between configuration, function, and permeability in calcium-treated

1976, 251:5069-5077.J. Biol. Chem. 

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