Ca2+
A N D M E M B R A N E POTENTIAL. I N MITOCHONDRIA
Cebra, J. J., (iivol, D., and Porter, R. R . ( 1 968a), Biochem. J . 107, 69. Cebra, J. J., Steiner, L. A., and Porter, R. R . ( 1 968b), Biochem. J. 107. 79. Edelman, 6 . M., Cunningham, B. A,, Gall, W. E., Gottlieb, P. D., Rutishauer, V., and Waxdel, M. J. (1969), Proc. Nat. Acnd. Sci. U . S. 63, 78. Hill, R. L., Lebovitz, H. E., Fellows, R . E., and Delancy, R. ( I967), Gamma Globulins, Proc. Nobel Svmp. 3rd, 109. Koo, P. H., and Cebra, J. J. (1 974), Biochemistry 13, 184. Minta, J. 0..and Painter, R. H. (1972), Immunochemistry 9, 1041. Oliveira, B., Osler. A. G., Siraganian, R. P., and Sandberg, A. I.. (1970), J . Immunol. 104, 320. Ovary, Z., Renacerraf, B., and Bloch, K. J. (1963), J . E x p .
Med. 117, 951. Pink, J. R. L., Buttery, S. H., DeVries, G. M., and Milstein, C. (1970), Biochem. J. 117. 33. Poljak, R. J., Amzel, L. M., Avey, H. B., Becka, L. N., and Nisonoff, R. ( 1 972), Nature (London), New Biol. 235, 137. Putnam, F. W., Florent, G., Paul, C., Shinoda, T., and Shimizu, A. (l973), Science 182, 287. Tracey, D., and Cebra, J. J. ( 1 974), Biochemistry 13, 48 1 1. Turner, K. J., and Cebra, J. J. (l971), Biochemistry 10, 9. Watanabe, S., Bannikol, H. U., Horn, J., Bertrom, J., and Hilshmann, N. (1973), Hoppe-Seyler's Z . Physiol. Chem. 354, 1505. Yasmeen, D., Ellerson, J. R., Dorrington, K. J., and Painter, R. H. ( 1 973), J . Immunol. 110, 1706.
Calcium Uptake and Membrane Potential in Mitochondria? Hagai Rottenberg* and Antonio Scarpa*
ABSTRACT:
The effect of uncouplers and the ionophores
A23 187, X-537A, nigericin, and valinomycin on calcium
concentration gradients in rat liver mitochondria has been investigated. The calcium gradients under various conditions were compared with rubidium concentration gradients. Calcium concentration gradients were estimated from the extent of uptake as measured by changes in murexide absorption or by 45Ca distribution and the matrix water content, under conditions in which Ca2+ binding and internal precipitation inside mitochondria are minimized. All the tested ionophores in very high concentration cause release of calcium. However, in low concentrations A23187 is without effect, valinomycin causes Ca2+ release (which depends on external potassium), and nigericin increases the rate and extent of Ca2+ uptake. This stimulation is observed only a t
T h e reaction of calcium ions with mitochondria has been the subject of investigation from various standpoints for over a decade. Mainly from the studies of the groups of Lehninger (Lehninger et al., 1967) and Chance (Chance, 1965), it is now well established that Ca2+ accumulation by mitochondria is an energy-linked process in which the energy can be derived from electron transport or A T P hydrolysis. More recently, attention has been focused on the existence of a specific Ca2+ carrier localized in the inner membrane of mitochondria. The Ca2+ binding properties of the proposed carrier (Reynafarje and Lehninger, 1969), its in~
~~
From the Johnson Research Foundation, Department of Biophysics and Physical Biochemistry, llniversity of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 191 74. Received J u n e 24, 1974. Supported by Grants 73-743 from the American Heart Association, I5835 from the National Institutes o f Health, and G M I2202 from the ti. S. Public tlealth Service. A S . is an Investigator of the American Heart Association. Permanent address: Biochemistry Department. Tel Aviv University, Raniat Aviv, Tel Aviv, Israel.
*
low nigericin concentrations, low external calcium concentrations, and in the absence of anions such as acetate or phosphate. In the presence of valinomycin, s6Rb+ distribution shows good correlation with Ca2+ distribution over a wide range of values which were obtained by varying uncoupler concentration or potassium concentration. The C a 2 + concentration ratio is always higher than the Rb+ ratio, approximately obeying the relation log ( [Ca2+],,/ [Ca2f]o,t) = 2 log ( [Rb+]in/[Rb+]o,,t). Assuming that rubidium distribution in the presence of valinomycin is governed by membrane potential obeying the Nernst equation, these results are interpreted as evidence that calcium uptake in mitochondria is an electrogenic process driven by membrane potential with a net charge transfer of 2.
hibition by lanthanides (Mela, 1968), and its kinetic behavior (Vinogradov and Scarpa, 1973) have been studied in great detail. However, the nature of the driving force for the Ca2+ accumulation against concentration gradients in mitochondria has not been established. Several models have been suggested to describe the coupling between the energy producing metabolic reactions and the energy utilizing transport process. The earlier models were based on direct coupling between the metabolic reactions and the transport process, thus forming a calcium pump (Chance, 1965). However, various other models were considered in which the coupling is not direct but mediated through the transport of other ions, either directly or electrically (for recent discussion, see Lehninger, 1973). A gradient of major importance, which is formed in energized mitochondria, is that of the proton electrochemical potential which is composed of a membrane potential (A+) and a concentration gradient (ApH). An important role for this gradient for energy conversion in mitochondria has BIOCHEMISTRY.
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i A23187 1.6pghl
\ITP F I G L R E 1: The effect of FCCP and the ionophores X-537A, A23187, valinomycin, and nigericin on calcium release from rat liver mitochondria. The reaction mixture contained 50 mM KCI, 100 mM sucrose, I O mM Mops (pH 7.2), 5 mM sodium succinate, 1 p M rotenone, 3 miv MgC12. 100 p M murexide, and 0.8 mg/ml of mitochondrial protein. T h e other additions a r e indicated in the figure. Temperature, 24'.
been suggested by Mitchell (1961, 1966), and its possible role in ion movements in mitochondria and submitochondrial particles was discussed by Chance and Montal (1971). It has been proposed that the driving force in the uptake of monovalent cations is the membrane potential which is formed in energized mitochondria (Mitchell, 1968; Rottenberg. 1973). It has been recently shown (Rottenberg, 1973) that, when the membrane becomes permeable to both sodiu m and potassium (as in the presence of gramicidin). the concentration gradients of these cations in steady state are identical, suggesting that both gradients a r e being held by equivalent membrane potential. In the present study, we present measurements of calciurn concentration gradients in mitochondria as affected by uncouplers and ionophores and we compare these gradients with rubidium gradients. A careful use of uncouplers and ionophores resulted in either a stimulation of Ca2+ release or uptake, which could be correlated with the expected effect of these compounds on energization, ApH, and membrane potential. The results of these studies are interpreted as an indication that calcium uptake by mitochondria is driven by a membrane potential and that the net charge transfer during this electrogenic process is 2. Materials and Methods Mitochondria were prepared from rat liver homogenates, as described previously (Vinogradov et al., 1972) in an isolation medium containing 0.25 mM sucrose and 1 mM sodiu m ethylenediaminetetraacetate (pH 7.2). The same medium was used for the first two washings and a 0.25 M sucrose solution, which was deionized and contained less than 0.2 I.~MCa2+, was used for the last washing and final suspension of mitochondria. -Calcium content in this preparation ranged from I O to 15 nmol of calcium/mg of protein. Protein was measured by the biuret method with crystalline
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bovine serurn albumin used as a standard. C a 2 + uptake and release by isolated mitochondria were measured either radiochemically or spectrophotometrically. In the latter case, kinetics of Ca2+ movements were followed by recording the absorption changes of the metallochromic indicator murexide through an Aminco DW2 dual wavelength spectrophotometer (540-507 nm), as described previously (Scarpa, 1972). Water content ( 3 H 2 0 ) and 45Ca2+ and x6Rb+ distribution were calculated from the relative activities of these isotopes in the pellets and supernatants after centrifugation of mitochondria through a Coleman 8-61 1 desk microcentrifuge. 14C-Sucrosedistribution was used to correct the water volume of the total pellet from that of the nonmatrix space. This analysis was carried out as described previously in great detail (Padan and Rottenberg, 1973), except that the pellet was dissolved overnight in 2 N formic acid. Calcium-45 (18 Ci/g), rubidium-86 (5 C i / g ) , tritiumlabeled water ( 1 mCi/g), and [14C(U)]sucrose (3 Ci/mol) were purchased from New England Nuclear, Boston, Mass. Murexide was obtained from K and K Chemicals, Plainview, N. J., and was crystallized twice. Valinomycin was from Calbiochem, Los Angeles, Calif., and rotenone from Sigma Co., St. Louis, Mo. FCCP' (carbonyl cyanide p - trifluoromethoxyphenylhydrazone) was a gift from Dr. P. G . Heytler of Du Pont Co., Wilmington, Del.; X-537A and nigericin were from Dr. J. Berger of Hoffman-La Roche. Nutley, N . J.; A23187 was from Dr. R. Hamill of Eli Lilly arid Co., Indianapolis, Ind. All the ionophores were dissolved in ethanol and the volume of ethanol added for each experiment did not exceed 3 Kl/ml of reaction mixture. ~
' Abbreviatinns
~
_
_
used are: FCCP, carbonyl cyanide p - trifluoromethoxyphenylhydrazone; Mops, morpholinopropanesulfonic acid.
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Ca2+
AND MEMBRANE POTENTIAL I N MITOCHONDRIA
X - 537A
