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THE MITOCHONDRIAL CALCIUM SAGA: IT WAS BRIT WHO STARTED IT VIMM Venetian Institute of Molecular Medicine Padova Department of Biological Chemistry University.

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Presentation on theme: "THE MITOCHONDRIAL CALCIUM SAGA: IT WAS BRIT WHO STARTED IT VIMM Venetian Institute of Molecular Medicine Padova Department of Biological Chemistry University."— Presentation transcript:

1 THE MITOCHONDRIAL CALCIUM SAGA: IT WAS BRIT WHO STARTED IT VIMM Venetian Institute of Molecular Medicine Padova Department of Biological Chemistry University of Padova


3 Ca 2+ UPTAKE BY HEART MITOCHONDRIA (SARCOSOMES) (µmoles per mg protein) Uptake medium EDTA-washed preparation % ∆ Saline preparation % ∆ no Ca mM Ca ~ ~ 40 Heart mitochondria were prepared either in saline, or in saline plus 10 mM EDTA. The reaction medium contained M KCl, 20 mM phosphate, 0.6 mM Ca 2+. Incubation was at 0°. After 30 minutes mitochondria were sedimented by centrifugation, and Ca 2+ was determined in the supernatant and in the ashed residue. E.C. Slater and K.W. Cleland, 1953

4 “ the uptake of Ca 2+ could be brought about by coupling with an energy- yielding mechanism such as respiration however, “ the reaction occurred at 0°, i.e., under conditions in which sarcosomes were not respiring nor carrying out any other obvious metabolic activity” therefore, “the uptake of Ca 2+ was an artifact not representing the state of affairs of the intact fibre” E.C. Slater and K.W. Cleland, 1953

5 B. Chance, 1956

6 “the very fact that successive additions of calcium up to overload gave the same stoichiometry of respiratory stimulation made it apparent to us that calcium was gone, or, in modern terminology, must have been translocated from the external medium, just where in the mitochondria or on the mitochondria was not shown by our studies” B. Chance, 2002


8 N.E. Saris, 1963

9 H.F. De Luca and G.W. Engstrom, 1961

10 F. D. Vasington and J.W. Murphy, 1962


12 C.S. Rossi and A.L. Lehninger, 1964

13 C.C.S. Rossi and A.L. Lehninger, 1964


15 M.Crompton, M. Capano, and E. Carafoli, 1976 Rat heart mitochondria were incubated in a standard reaction medium. Ca 2+ fluxes were measured with a Ca 2+ specific electrode.The substrate (succ) was 5 mM, ruthenium red (RR) was 40 nM, sodium (Na + ) was 5.2 mM. 120 nMol of Ca 2+ were added to the 10 ml medium.

16 M. Crompton, M. Capano, and E. Carafoli, 1976 Rat heart mitochondria were incubated in a standard reaction medium, with succinate as the substrate. Ca 2+ was measured with a specific electrode. 30 nmol Ca 2+ per mg of mitochondrial protein were added,then the uptake was stopped with 60 nM ruthenium red, followed 2 min later by Na +. The efflux rate was linear for at least one min.

17 E. Carafoli and M. Crompton, 1978

18 Distribution of injected 45 Ca 2+ in the subcellular fractions of rat liver ____________________________________________________________________ % distribution FRACTION Control rat preinjected with PCP ____________________________________________________________________ residue 22.6 ±1.12 (9) 21.5±4.33 (3) mitochondria 55.5±1.84 (9) 22.2±2.40 (3) heavy microsomes 4.3±028 (9) 12.8±1.65 (3) microsomes 15.3±290 (9) 30.3±0.46 (3) supernatant 2.5±038 (9) 13.2±052 (3) 45 Ca 2+ (10 µc) was injected intraperitoneally 6 minutes before the death of the animal, and 3 minutes before the injection of pentachlorophenol (20 mg/kg). Liver subcellular fractions were separated with a conventional fractionation scheme. Data are given ± standard errors. The number of experiments is in brackets. E. Carafoli, 1967

19 RR. Rizzuto, M. Brini, M. Murgia, and T. Pozzan, 1993

20 R. Rizzuto, P. Pinton, W. CarringtonF. S. Fay, K. E. Fogarty, L. M. Lifshitz, R. A. Tuft, and T. Pozzan, 1998

21 COMMENTS AND CONCLUSIONS - I The ability of mitochondria to take up Ca 2+ in a reaction driven by the respiratory chain or by ATP was demonstrated directly in However, findings in the preceding decade had shown, albeit indirectly, that mitochondria could indeed accumulate Ca 2+ The affinity of the uniporter turned out to be too low for the efficient regulation of Ca 2+ in the nM concentration of the bulk cytosol at rest.Thus, the process came to be regarded as a means to regulate the activity of 3 Ca 2+ -dependent matrix dehydrogenases that are essential for the synthesis of ATP. In turn, the precipitation of Ca 2+ -phosphate deposits was recognized as a device to enable cells to survive brief periods of cytosolic Ca 2+ overload. The phenomenology of the transport process was established in the 1960s-1970s.That included the stoichiometry of the uptake reaction to the activity of the respiratory chain, the simultaneous uptake of phosphate to precipitate Ca 2+ in the matrix, the uptake of ATP/ADP to stabilize the precipitates, the release of accumulated Ca 2+ via a Na + (or H + ) antiporter.The uptake process was shown to be mediated by an electrophoretic uniporter, which was inhibited by ruthenium red.

22 COMMENTS AND CONCLUSIONS - II Surprisingly, however, the largest percentage of injected radiocalcium in rat tissues was recovered in the mitochondrial fraction. The percentage of radiocalcium recovered in the mitochondrial fraction dropped dramatically in rats pre-injected with uncouplers of oxidative phosphorylation. Thus, in spite of their poor Ca 2+ affinity, mitochondria could nevertheless still efficiently carry out energy-driven uptake of Ca 2+ in vivo. The paradox was solved in the early 1990s, when it was discovered that the ambient surrounding mitochondria within the cell experienced µM Ca 2+ concentrations, thus satisfying the requirements of the low affinity uniporter. The close association between endoplasmic reticulum and mitochondria enabled the former to create micro-pools of high Ca 2+ concentration when the InsP3 channel was opened in response to InsP3- generating agonists.





27 J. W. Greenawalt, C. S. Rossi, and A. L. Lehninger, 1964

28 J. W. Greenawalt and E. Carafoli, 1966

29 A B C D E F G H A: muscle (mouse poisoned with tetanus toxin) B: kidney tubule (rat poisoned with sublimate) C: kidney tubule (mouse treated with PTH) D: ischemic dog myocardium E: myocardium of a Mg-deficient, cold- stressed rat F:ischemic dog myocardium G: ischemic, reperfused dog myocardium H:myocardium (rat poisoned with isoproterenol) Electron-opaque (Ca-phosphate) granules in mitochondria of variously injured cells E. Carafoli and I. Roman, 1980

30 C.R. Hackenbrock and A.I. Caplan, 1969

31 E. Carafoli, C.S. Rossi, and A.L. Lehninger, 1965 Rat liver mitochondria (1.5 mg protein per ml) in a standard reaction medium with 10 mM succinate, 10 mM Mg 2, 4 mM phosphate, 3 mM 14 C-ATP, 4 mM 45 Ca 2+ at 30°. Adenine nucleotides were separated and identified by paper electrophoresis on perchloric acid extracts of mitochondria.

32 E. Carafoli, C.S. Rossi and A.L. Lehninger, 1965


34 E. Carafoli, R. Tiozzo, G. Lugli, F. Crovetti, and C. Kratzing, 1974 Rat heart mitochondria: Calcium measured isotopically. Substrate succinate, No substrate was present in (a), that contained rotenone. In (b) 10 nM ruthenium red was added 30 sec before sodium. Calcium, 5-10 nmol per mg protein. No calcium added in ©, in which rats had been injected with radioacyive calcium 5 min before being killed. (a), open circles 100mM sodium, triangles 100 mM potassium, closed circles, equal concentration reaction medium. (b), open circles 50 mM sodium, closed circles, reactiion medium. (c ), open circles, 5 mM sodium, closed triangles, 20 mM sodium, open triangles, 50 mM sodium, closed circles, reaction medium.

35 Y.Kirichok, G. Krapivinsky, and D. E. Clapham, 2004


37 R. Palty, W. F. Silverman, M. Hershfinkel,…., D. Khananshvili, and I. Sekler, 2010

38 R. Palty, W. F. Silverman, M. Hershfinkel….. D.Khananshvili, and I. Sekler. 2010

39 R. Palty, W. F. Silverman, M. Hershfinkel…. D. Khananshvili, and I Sekler, 2010

40 AKNOWLEDGMENTS A number of colleagues in Baltimore, Modena, and Zurich have contributed greatly to the original work I have described. Carlo Stefano Rossi and Martin Crompton have been particularly important.

41 …but nothing would have been possible without the invaluable help and support of ALBERT LEHNINGER


43 C. S. Rossi and A. L. Lehninger, 1964

44 COMPETITION BETWEEN MITOCHONDRIA AND “MICROSOMES” OF RAT LIVER FOR CALCIUM UPTAKE cpm in mitochondria % uptake cpm in “microsomes” % uptake - DNP +DNP -DNP +DNP -DNP +DNP +DNP -DNP ,960 8, Rat liver mitochondria in a standard reaction medium containing 10 mM Mg 2+, 10 mM phosphate, 3 mM ATP, 10 mM succinate, 10 mg each ofmitochondrial and “microsomal” protein in a final volume of 10 ml at 25° 0.15 mM Ca 2+labeled with 45 Ca 2+ was added last. DNP was M. After 5 min the system was quickly cooled and cntrifuged at 15,000 g/10 min To sepqarate mitochondria, The supernatant was centrifuged at 150,000 g/45 min to collect “microsomes”. Radioactivity was counted on the pellets. Carafoli, E., 1967

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