The Na+-independent Ca2+ efflux system in mitochondria is a Ca2+/2H+ exchange system

Volume 274, number 1,2, 65-68

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November 1990

The Na+-independent Ca 2+ efflux system in mitochondria is a Ca 2+/2H + exchange system Hagai Rottenberg and Miriam Marbach Pathology Department, Hahnemann University, Philadelphia, PA 19102, USA Received 12 September 1990 The mechanism of the Na+-independent Ca2÷ eftlux system in mitochondria has not been elucidated as yet. With the aid of cyclosporin A, an inhibitor of the Ca2+-induced 'pore', and using a variety of inhibitors, uncouplers and ionophores, it is possible to demonstrate, unequivocally, that this process is driven by ApH. The efflux is not affected by A~u, thus suggesting an electroneutral CaZ+/2H÷ exchange mechanism. Parallel measurements of the rate of Ca2÷ efflux and 3pH, as modulated by valinomycin and nigericin, indicate that the rate of efflux is a function of the magnitude of ApH. Mitochondria; CaZ+; Ca2+/2H+ exchange; 3pH; Cyclosporin A

1. I N T R O D U C T I O N M i t o c h o n d r i a c o n t a i n several distinct C a 2 + t r a n s p o r t systems (for recent reviews see 1,2). T h e m o s t active s y s t e m is the C a 2+ u n i p o r t e r which catalyzes electrogenic Ca 2+ uptake. An electroneutral, Ca 2+/Na + e f f l u x system, which is a c t i v a t e d b y e x t e r n a l N a + is f o u n d in m i t o c h o n d r i a f r o m m o s t tissues, a n d is p a r t i c u l a r l y active in b r a i n a n d m u s c l e m i t o c h o n d r i a . I n a d d i t i o n , a n o t h e r C a 2+ e f f l u x system which is N a + - i n d e p e n d e n t , a p p e a r s to exist in m i t o c h o n d r i a f r o m all tissues. H o w e v e r , the m e c h a n i s m o f this s y s t e m , which was o r i g i n a l l y suggested to be a n elect r o n e u t r a l C a 2 + / 2 H ÷ e x c h a n g e carrier, has n o t been e l u c i d a t e d as yet. D e s p i t e initial suggestions t h a t C a 2 + - e f f l u x is d r i v e n b y H ÷ - u p t a k e [3-5], no uneq u i v o c a l link b e t w e e n C a 2 + -efflux a n d H + - u p t a k e was e s t a b l i s h e d a n d the p r e d i c t e d d e p e n d e n c e o f the efflux o n A p H c o u l d n o t be d e m o n s t r a t e d [1,2]. A c o m p l i c a t i n g f a c t o r , no d o u b t , is the existence o f a n a d d i t i o n a l , n o n s p e c i f i c , p a t h w a y for C a 2 + efflux which can be i n d u c e d in m i t o c h o n d r i a u n d e r a v a r i e t y o f c o n d i t i o n s . H i g h c o n c e n t r a t i o n s o f C a 2 + in the m i t o c h o n d r i a l m a t r i x i n d u c e an increase in the i n n e r m e m b r a n e p e r m e a b i l i t y to small solutes which c o l l a p s e s A#H a n d causes C a 2 + efflux. This process which is a c t i v a t e d b y m a n y ions, s u b s t r a t e s a n d c h e m i c a l s [1] c o n t r i b u t e s to t h e N a + i n d e p e n d e n t C a 2 + e f f l u x a n d c o u l d n o t be s e p a r a t e d f r o m the p u t a t i v e C a 2 ÷ / 2 H ÷ exchange. It is n o t clear as yet w h e t h e r this process represents the acCorrespondence address: H. Rottenberg, Pathology Department (M.S. 435), Hahnemann University, Broad and Vine, Philadelphia, PA 19102, USA Published by Elsevier Science Publishers B. E (Biomedical Division) 00145793/90/$3.50 © 1990 Federation of European Biochemical Societies

t i v a t i o n o f a p r o t e i n ' p o r e ' [2] or a l i p i d - m e d i a t e d leak [1]. Nevertheless, it has been a r g u e d t h a t u n d e r m o s t C a 2+ t r a n s p o r t assay c o n d i t i o n s , a process o f cont i n u o u s , n o n s y n c h r o n o u s , cycle o f ' p o r e ' o p e n i n g , release o f m a t r i x - C a 2 ÷ c o n t e n t , a n d a resealing with reu p t a k e o f C a 2+, can fully a c c o u n t for the o b s e r v e d N a ÷ - i n d e p e n d e n t efflux [2]. Thus, a c c o r d i n g to this view, the putative, specific, Na ÷-independent C a 2 + - e f f l u x system does n o t exist at all. R e c e n t l y , it was f o u n d t h a t the i m m u n o s u p p r e s s i v e d r u g , C y c l o s p o r i n A , is a p o t e n t i n h i b i t o r o f the C a 2 + - i n d u c e d ' p o r e ' [6,7]. This f i n d i n g m a d e it possible to re-investigate the existence a n d the m e c h a n i s m o f t h e N a + - i n d e p e n d e n t C a 2 +-efflux system. A s s h o w n b e l o w , we f o u n d that once the C a ÷ - i n d u c e d ' p o r e ' is inh i b i t e d b y C y c l o s p o r i n A , the existence o f a specific C a E + / 2 H ÷ e x c h a n g e system can be d e m o n s t r a t e d clearly and unequivocally.

2. M A T E R I A L S A N D M E T H O D S Rat liver mitochondria were prepared by the conventional differential centrifugation method as described [8], except that EGTA (0.1 mM) was included during the washing steps. Rat brain mitochondria were prepared by the conventional ficol gradient method [8], except for the addition of EGTA to the washing steps. Rates of Ca 2+-efflux were measured from the absorbance changes (685-675 nm) of the C a 2 + indicator Arsenazo Ill with an Aminco DW2A double-beam spectrometer, as described previously [10]. The basic medium was composed of 0.22 M mannitol, 80 mM sucrose, 1 mM MgCl2, 0.5 mM KCI, 2 mM Tris-Pi and l0 mM Tris-C1 pH 7.4. Rat liver mitochondria (1 mg/3 ml) were incubated with 50 #M Arsenazo Ill and 2 p.M rotenone. The experiment was started by the addition of 5 mM Trissuccinate and 30 nmoles C a 2 + . When most of the C a 2 + was accumulated from the medium, the efflux was initiated by the addition of 3 nmoles of Ruthenium Red (RR). ApH was calculated from the

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distribution of 14C-DMO and 3H20, using ]4C-sucrose as a marker for non-matrix pellet space, as described previously [11]. 14C-DMO or ]4C-sucrose and 3H20 were added to a suspension of 3 mg protein/ml. Ca 2 ÷ content was also increased to give 30 nmoles Ca z ÷/mg protein. The experiment was carried out exactly as in the efflux assay and the mitochondria separated by centrifugation 2 rain after addition of the ionophores.

3. RESULTS To assess the effect of ApH and A~b on the Na ÷-independent Ca 2 +-efflux we tested the effect of inhibitors, uncouplers and inonophores on the rate of Ca 2+-efflux. Figure 1 shows the effects of (a) Antimycin A (AA), an inhibitor of electron transport; (b) FCCP, a protonophore; (c) Valinomycin, an ionophore that catalyzes electrogenic K ÷ transport; and (d) Nigericin, an ionophore that catalyzes electroneutral K ÷-H ÷ exchange, on Na ÷-induced Ca 2 +-efflux. Rat liver mitochondria were allowed to accumulate Ca 2+, in the presence of 2 mM Pi. Pi greatly reduces the RR

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matrix free Ca 2 + -concentration, thus reducing the role of the Ca 2 + concentration gradient as a driving force for Ca 2 +-efflux (see below). Then the electrogenic uptake by the uniporter was inhibited completely by excess Ruthenium red (RR) and Ca2+-efflux (0.8-1.1 nmol/mg protein/min) was observed. In the absence of Cyclosporin A (Fig. 1A), the addition of Antimycin A (trace a) or FCCP (trace b) strongly stimulated the efflux. Valinomycin (trace c) was without significant effect and Nigericin (trace d) inhibited the efflux. Under these conditions, the effects of FCCP, AA and Valinomycin are not consistent with a role for ApH as a driving force in Ca 2 + -efflux, while the Nigericin effect is. This inconsistency is most probably due to the activating effect of uncouplers and electron transport inhibitors on the Ca 2 +-induced 'pore'. When Cyclosporin A, which inhibits the opening of the 'pore', is included in the incubation medium (Fig. 1B), the results are quite different. Antimycin A, FCCP, and Nigericin all inhibit the efflux. Since Antimycin A and FCCP collapse both ApH and A~b, while Nigericin increases A~b but collapses ApH, it is evident that ApH drives the efflux while A~ is without effect. This is further confirmed by the effect of Valinomycin, which in the presence of Cyclosporin A stimulates the efflux. Since Valinomycin collapses A~bbut increases ApH, it is apparent that ApH drives the efflux while A~ is without

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Fig. 1. The effects of ApH and A~bon Na ÷ -independent Ca 2 + -efflux. Ca2+-efflux rates were measured as described in Materials and Methods. Part A is without Cyclosporin A and part B is with the addition of 3 #g Cyclosporin A. Trace a, Antimycin A (AA) 30 ng/mg; Trace b FCCP 0.33 ~M. Tract c Valinomycin 0.1 p.M; and trace d, Nigericin 0.1 /~M. Efflux rates (nmol/mg protein/rain) are shown above the traces. Rates were calculated from the slopes of the curve 60 s after the addition of reagents. All the results of Fig. 1 are from the same mitochondrial batch.

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Fig. 2. The effects of Pi (A) and KCI (B) on Ha +-independent Ca 2 + -efflux. In A, medium and other assay conditions were the same as in Fig. 1 except for the addition of 100/zM ADP and 1 #g/mg Oligomycin. Pi concentration was varied from 0 to 5 mM as indicated. o, with Cyclosporin A (3/zg/mg); e, with Cyclosporin A and Nigericin (0.1/zM). In B (inset), medium and assay conditions are the same as in Fig. 2A, except for KCI concentrations that were varied from 0 to 32 mM as indicated, and Pi which was 0.2 mM. o, with Cyclosporin A (3/zg/mg); A, with Cyclosporin A and Nigericin (0.1/~M); o with Cyclosporin A and Valinomycin (0.1 /zM). All data points are the averages of 4 rate determinations. The standard errors in these rate determinations were approximately 15%. The experiments of A and B were each performed on the same mitochondrial batch. Similar experiments with other mitochondrial preparations produced similar results.

effect. These results are compatible with the suggestion that the efflux is mediated by a Ca 2 ÷ / 2 H ÷ exchange carrier. Similar results were obtained with brain mitochondria (not shown). We have previously shown that in brain mitochondria, Pi at very low concentrations ( < 200 #M), greatly inhibits the Na ÷-independent Ca 2 ÷-efflux while stimulating the rate o f the electrogenic uptake o f Ca 24, an effect which was attributed to lowering of the matrix free Ca 2 ÷ concentration. The lowering of matrix free Ca 2 ÷ by Pi, greatly reduces the magnitude of the Ca 2 ÷ concentration gradient, and thus, enhances the role of the pH gradient as a driving force for the efflux. At higher concentration of Pi (0.2-5 mM) further inhibition of Ca 2 ÷-efflux was observed and the latter effect correlated with the well known Pi-dependent reduction o f ApH [12]. Fig. 2A shows that Na+-independent Ca 2 ÷ efflux in liver mitochondria in the presence of Cyclosporin A is also inhibited by Pi. The inhibition of efflux at high concentrations of Pi ( > 0.2 mM) is correlated with the reduction o f ApH [13]. Nigericin further inhibited the efflux and the combination of high Pi and Nigericin inhibited the efflux almost completely. Similar effects of Pi on Ca 2 ÷ efflux in liver mitochondria were reported previously [14]. The dependence of the Ca 2 ÷ efflux on the medium potassium concentrations in control, Valinomycin- and Nigericin-treated liver mitochondria is shown in Fig. 2B. There was no dependence on K ÷ -concentration in the absence of ionophores. The Valinomycin enhance7

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ment o f efflux was strongly dependent on external K + The enhancement increased with K÷-concentration over the mM range in parallel with the effect of K + on ApH [15]. The inhibition of efflux by Nigericin was stronger at low K + and was diminished at high K + These differential effects of K + on efflux are correlated with its efect on ApH. The Nigericin effect on ApH is enhanced by increasing the Kin/Kout ratio while the Valinomycin effect on ApH is enhanced by increasing Ko,t. Similar dependence on KCI of the Valinomycin and Nigericin effects on Ca 2 +-efflux were also observed in brain mitochondria (not shown). To obtain a quantitative correlation between the magnitude o f ApH and the rate of Na +-independent Ca 2 ÷-efflux, we measured in parallel the effect of different concentrations of Nigericin and Valinomycin on the rate of Ca 2 +-efflux and ApH. To maximize the effect o f Valinomycin and Nigericin on ApH, we used 4 m M KC1 in the Valinomycin experiments and 0.5 mM KC1 in the Nigericin experiments. Fig. 3 shows that increasing the valinomycin concentration increased ApH and Ca 2 ÷ -efflux rate while increasing Nigericin concentrations reduced ApH and inhibited the efflux. Fig. 4 shows the relationship between ApH and Ca 2 +-efflux obtained from the results of Fig. 3 and a similar experiment with a different batch of mitochondria. It is clearly observed that the rate of Ca 2 + -efflux depends on the magnitude of ApH over the entire experimental range. The dependence is particularly strong at high ApH where 2 ApH >> ApCa. At lower value of 2 ApH, where ApH = ApCa, the dependence is more moderate, This observation strongly supports the notion that (a) the Na ÷ -independent Ca 2+-efflux system does exist and (b) the process is catalyzed by an electroneutral Ca 2 ÷ / 2 H + exchange carrier.

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Fig. 3. The concentration dependence of the Valinomycin and Nigericin effects on ApH and Ca 2 ÷ -efflux. Na ÷ -independent efflux was measured as in Fig. 2B. ApH was estimated as described in Materials and Methods. Medium and assay conditions were the same as in Fig. 2B, except that 4 mM KCI was used for the Valinomycin experiments and 0.5 mM KC1 was used for the Nigericin experiments. Full triangles ( • ) show the effect o f Nigericin on ApH and empty triangles (zx) show the effect of Nigericin on Ca 2+ efflux. Full squares ( • ) show the effect of Valinomycin on ApH and empty squares ([]) show the effect of Valinomycin on Ca 2 ÷ efflux. Data points of efflux rates are the averages o f 4 determinations (standard errors were approximately 15%). Data points of ApH determinations are the averages of 3 determinations (standard errors were approximately 10%). All data are from the same mitochondrial batch. Similar experiments with other mitochondrial preparations produced similar results (see Fig. 4).

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4. D I S C U S S I O N The results of this study clearly demonstrate that the Na ÷-independent Ca 2 ÷-efflux in mitochondria is an electroneutral process driven by ApH. It is necessary to emphasize that this could only be demonstrated when all alternative Ca 2 ÷ transport systems were blocked. The electrogenic uniporter was blocked by excess Ruthenium red, the 2Na ÷ / C a 2 ÷ exchange system was inhibited by eliminating Na ÷ from the suspension medium and the Ca 2 ÷ induced ' p o r e ' was inhibited by excess Cyclosporin A. Mg 2÷ and A D P , both of which also inhibit the 'pore' formation, were also included in most experiments. Although Cyclosporin A does not appear to completely inhibit the phospholipase A2 mediated permeabilization [1], the latter process is very slow and does not affect the efflux when measured immediately after Ca 2÷ accumulation, as done in our assay. It is not necessary to review here the numerous conflicting studies of Na ÷-independent Ca 2 ÷ efflux of the last decade (reviewed in Refs. 1,2). It is now clear that most of the earlier confusion arises f r o m the inability to separate the C a 2 + / 2 H ÷ efflux f r o m the Ca 2 +-induced ' p o r e ' . Cyclosporin A, when used properly, allows complete inhibition of the ' p o r e ' and reveals the existence of the C a 2 + / 2 H ÷ exchange system. The suggestion that all the Na ÷-independent Ca 2 + -efflux is due to a cycle of 'pore' activation and inactivation [2] is shown to be incorrect. However, under a variety of conditions, Cyclosporin A inhibited f r o m 5°70 to 95070 of the Na+-independent efflux and under some assay conditions the cycle of pore opening and closing appeared to be a significant and even a dominant component of the efflux. In agreement with our conclusion is the demonstration that in the presence of Ruthenium red the equilibrium distribution o f Ca 2 ÷ in liver mitochondria is not altered by the ionophore A23187 (which catalyzes Ca 2 ÷ / 2 H ÷ exchange) [16]. We have previously shown that Nigericin enhances Ca 2÷ retention in liver mitochondria even in the absence o f Ruthenium red [17] and this effect too is probably due to inhibition of ApH. Because of the inability to demonstrate a flux stoichiometry of 2H ÷ / C a 2 ÷ [18,19], alternative mechanisms have been

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suggested. However, we believe that because of the presence of many, very active, H ÷-coupled transport systems in mitochondria, it is not reasonable to expect to be able to measure the slow proton flux of the Ca 2 + / 2 H ÷ exchange system isolated from all other H ÷ fluxes as attempted in previous studies. In contrast, the calcium flux of the carrier could be isolated, as demonstrated here, and shown to be driven by the p H gradient.

Acknowledgements: We thank Dr William S. Thayer for critical reading of this manuscript. Supported by PHS Grants GM-28173 and AA-07238.

REFERENCES [1] Gunter, T.E. and Pfeiffer, D.R. (1990) Am. J. Physiol. 258, C755-C786. [2] Crompton, M. (1990) in: Intracellular Calcium Regulation (Bronner, F. ed), pp. 181-209, Wiley-Liss, New York. [3] Akerman, K.E.O. (1978) Arch. Biochem. Biophys. 189, 256-262. [4] Fiskum, G. and Cockrell, R.S. (1978) FEBS Lett. 92, 125-128. [5] Fiskum, G. and Lehninger, A.L. (1979) S. Biol. Chem. 254, 6236-6239. [6] Crompton, M., Elinger, H. and Costi, A. (1988) Biochem. J. 255, 357-360. [7] Brockemeir, K.M., Dempsy, M.E. and Pfeiffer, D.R. (1989) J. Biol. Chem. 268, 7826-7830. [8] Hashimoto, K. and Rottenberg, H. (1983) Biochemistry 22, 5738-5745. [9] Lai, J.C.K. and Clark, J.B. (1979) Methods Enzymol. LV, 51-60. [10] Rottenberg, H. and Marbach, M. (1990) Biochem. Biophys. Acta 1016, 79-86. [11] Rottenberg, H. (1979) Methods Enzymol. LV, 547-569. [12] Rottenberg, H. and Marbach, M. (1990) Biochim. Biophys. Acta 1016, 87-98. [13] Klingenberg, M. and Rottenberg, H. (1977) Europ. J. Biochem. 73, 125-130. [14] Zoccarato, F. and Nicholls, D. (1982) Eur. J. Biochem. 127, 333-338. [15] Padan, E. and Rottenberg, H. (1973) Europ. J. Biochem. 40, 431-437. [16] Brand, M. (1985) Biochem. J. 225, 413-419. [17] Rottenberg, H. and Scarpa, A. (1974) Biochemistry 13, 4811-4817. [18] Cockrell, R.S. (1985) Arch. Biochem. Biophys. 243, 70-79. [19] Gunter, T.E., Chace, J.H., Puskin, J.S. and Gunter, K.K. (1983) Biochemistry 22, 6341-6351.