THE MITOCHONDRIAL
CALCIUM SAGA: IT WAS
BRIT WHO STARTED IT
VIMM
Venetian Institute of Molecular Medicine
Padova
Department of Biological Chemistry
University of Padova
TABLE 1
Ca 2+ UPTAKE BY HEART MITOCHONDRIA (SARCOSOMES)
Ca 2+ UPTAKE BY HEART MITOCHONDRIA
(µmoles per mg protein)
Ca
2+
CONTENT OF MITOCHONDRIA
(µmoles per mg protein)
EDTA-washed preparation
Uptake medium
no Ca
2+
Uptake medium
no Ca 2+
0.1 mM Ca 2+ 0.1 mM
Ca 2+
% ∆
EDTA-washed preparation % ∆
0.015
Saline preparation
0.0015
0.068
0.068
Saline preparation
% ∆
% ∆
0.083
0.083
+
~ 450 +
~ 450
0.127
- ~ 50
0.127
+ ~ 40
Heart mitochondria were prepared either in saline, or in saline plus 10 mM EDTA. The
Heart mitochondria
were prepared either in saline, or in saline plus 10 mM EDTA. The
reaction medium contained 0.135 M KCl, 20 mM phosèhate, 0.6 mM Ca Incubation
reaction medium
contained 0.135 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 at 0°. After
30 minutes
mitochondria
were
was determined
in the supernatant.
Modified from
(9). sedimented by centrifugation, and Ca
was determined in the supernatant and in the ashed residue.
2+.
2+
E.C. Slater and K.W. Cleland, 1953
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 Ca2+ was an artifact not representing the state of
affairs of the intact fibre”
E.C. Slater and K.W. Cleland, 1953
B. Chance, 1956
“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
1
3
5
7
9
N.E. Saris, 1963
H.F. De Luca and G.W. Engstrom, 1961
F. D. Vasington and J.W. Murphy, 1962
A. L. LEHNINGER
C.S. Rossi and A.L. Lehninger, 1964
C.C.S. Rossi and A.L. Lehninger, 1964
C.C.S. Rossi and A.L. Lehninger, 1964
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.
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.
M. Crompton, M. Capano, and E. Carafoli , 1976
E. Carafoli and M. Crompton, 1978
Distribution of injected 45Ca2+ in the subcellular fractions of rat liver
DISTRIBUTION OF INJECTED 45CA2+ IN THE SUBCELLULAR FRACTIONS OF
RAT LIVER
____________________________________________________________________
______________________________________________________________________
% distribution
% DISTRIBUTION
FRACTION
FRACTION
Control
rat preinjected with PCP
CONTROL
RAT PREINJECTED WITH DNP
____________________________________________________________________
_______________________________________________________________________
residue
22.6 ±1.12 (9)
21.5±4.33 (3)
22.655.5±1.84
±1.12 (9)
21.5±4.33 (3)
mitochondria RESIDUE
(9)
22.2±2.40 (3)
MITOCHONDRIA
55.5±1.84
(9)
22.2±2.40 (3 )
heavy microsomes
4.3±028
(9)
12.8±1.65 (3)
4.3±028
(9)
12.8±1.65 (3)
microsomes HEAVY MICROSOMES
15.3±290
(9)
30.3±0.46 (3)
15.3±290
(9 )
30.3±0.46 (3) 13.2±052 (3)
supernatant MICROSOMES
2.5±038
(9)
SUPERNATANT
2.5±038 (9)
13.2±052 (3)
45Ca2+
(10 µc) was injected intraperitoneally 6 minutes before the death of the animal,
Ca (10 µc)
were
injected intraperitoneally
6 minutes before the death
o f mg/kg).
the animal, Liver subcellular
and 3 minutes before
the
injection
of pentachlorophenol
(20
3 minutes before the injection of pentachlorophenol (20 mg/kg). Livers were
fractions were and
separated
with a conventional fractionation scheme. Data are given ±
in a Potter–Elvehjem homogenizer and subc ellular fractions separated with
standard errors.homogenized
The number
of experiments is in brackets.
45
2+
a conventional fractionation scheme. Data are given ± standard errors. The nu mber of
experiments is in brackets. Modified from (72).
E. Carafoli, 1967
RR. Rizzuto, M. Brini, M. Murgia,
and T. Pozzan, 1993
R. Rizzuto, P. Pinton, W. CarringtonF. S. Fay, K. E. Fogarty,
L. M. Lifshitz, R. A. Tuft, and T. Pozzan, 1998
COMMENTS AND CONCLUSIONS - I
The ability of mitochondria to take up Ca2+ in a reaction driven by the
respiratory chain or by ATP was demonstrated directly in 1961-1962.
However, findings in the preceding decade had shown, albeit indirectly,
that mitochondria could indeed accumulate Ca2+
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 Ca2+ in the matrix, the uptake of ATP/ADP to stabilize the
precipitates, the release of
accumulated Ca2+ via a Na+ (or H+)
antiporter.The uptake process was shown to be mediated by an
electrophoretic uniporter, which was inhibited by ruthenium red.
The affinity of the uniporter turned out to be too low for the efficient
regulation of Ca2+ 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
Ca2+-dependent matrix dehydrogenases that are essential for the
synthesis of ATP. In turn, the precipitation of Ca2+-phosphate deposits was
recognized as a device to enable cells to survive brief periods of cytosolic
Ca2+ overload.
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
Ca2+affinity, mitochondria could nevertheless still efficiently carry
out energy-driven uptake of Ca2+ in vivo.
The paradox was solved in the early 1990s, when it was discovered
that the ambient surrounding mitochondria within the cell experienced
µM Ca2+ 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 Ca2+ concentration when the InsP3 channel was opened in
response to InsP3- generating agonists.
Y
THANK
YOU
J. W. Greenawalt, C. S. Rossi, and A. L. Lehninger, 1964
J. W. Greenawalt and E. Carafoli, 1966
Electron-opaque (Ca-phosphate) granules
in mitochondria of variously injured cells
A
B
D
C
F
E
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, coldstressed rat
F : ischemic dog myocardium
G : ischemic, reperfused dog myocardium
H : myocardium (rat poisoned with
isoproterenol)
E. Carafoli and I. Roman, 1980
C.R. Hackenbrock and A.I. Caplan, 1969
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 45Ca2+ at 30°. Adenine
nucleotides were separated and identified by paper electrophoresis on perchloric acid extracts
of mitochondria.
E. Carafoli, C.S. Rossi, and A.L. Lehninger, 1965
E. Carafoli, C.S. Rossi and A.L. Lehninger, 1965
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.
E. Carafoli, R. Tiozzo, G. Lugli, F. Crovetti, and C. Kratzing, 1974
Y.Kirichok, G. Krapivinsky, and D. E. Clapham, 2004
Y. Kirichok, G. Krapivinsky, and D. E. Clapham, 2004
R. Palty, W. F. Silverman, M. Hershfinkel,…., D. Khananshvili,
and I. Sekler, 2010
R. Palty, W. F. Silverman, M. Hershfinkel…..
D.Khananshvili, and I. Sekler. 2010
R. Palty, W. F. Silverman, M. Hershfinkel….
D. Khananshvili, and I Sekler, 2010
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.
…but nothing would have been possible without the
invaluable help and support of
ALBERT LEHNINGER
C. S. Rossi and A. L. Lehninger, 1964
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
----------------------------------------------------------------------------------------------------14,960 8,170
92
45.4
218
585
0.10 0.3
-----------------------------------------------------------------------------------------------------
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 10 -4 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