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The Ca2+-Na+ antiporter of heart mitochondria operates electroneutrally
vol.
95, No.
July
16, 1980
BIOCHEMICAL
1, 1980
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
193-196
THE Ca2+-Na+ ANTIPORTER OF HEART MITOCHONDRIA OPERATES ELECTRONEUTRALLY Hubert
Affolter
and Ernest0
Carafoli
Laboratory of Biochemistry Swiss Federal Institute of Technology Universitgtstrasse 16 CH - 8092 Zurich, Switzerland Received
(ETH)
May 6,198O
ABSTRACT The transmembrane potential of heart mitochondria during Na+-induced Ca2+ release has been monitored continuously with a Tetraphenylphosphonium-sensitive electrode. The data obtained indicate that the exchange process does not generate an electrical current, and provides direct support for the concept of a 2 Na+ per 1 Ca2+ exchange. INTRODUCTION In mitochondria nium red
insensitive
(1,2,3).
This
in the and of
of
Ca2+-Na+ antiporter.
(4,5j
presented for
1 Ca2+. that
the
In this
the
operation
recordered
of using
ABBREVIATIONS:
of Ca2+,
Ca2+ content
of the
The kinetic
data
indications direct of
of Crompton
et
for
of
a ratio will is
be
2 Na+
from gxperiments
of mitochondria
the Ca2+-Na+ antiporter
or-
stoichiometry
the antiporter
potential
the
have
evidence
has been reached
a TPP+-selective
of
and thus
difficulties
measurement
article,
a ruthe-
has been described
Experimental
stoicbiometry
transmembrane
tissues
the efflux
of the
indirect
1 Ca2+. The conclusion
in which the
the direct
have provided
b 2 Na+ for
mediates
the cytosol.
prevented
al.
antiporter
regulation
so far the
from various
Ca 2+-Na+
antiporter
participates ganelles
isolated
during
has been continuously
electrode.
TPP+ : Tetraphenylphosphonium - ion : mitochondrial membrane potential AJ, 0006-291 193
X/80/7
30193-04$01.00/O
Copyright 0 1980 by Academic Press, Inc. All righrs of reproduction in anv form reserved.
Vol.
95. No.
1. 1980
BIOCHEMICAL
AND
EXPERIMENTAL
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
PROCEDURES
Mitochondrial Preparation. Rat heart mitochondria were prepared by the methods of Crompton et al. (4). The protein concentration of the suspension was estimated by a modified biuret procedure (6) with bovinaserum albumin (Sigma Chemical CO.) as a standard. Measurement of Ca2'-Fluxes. Ca2+- activitieswere measured with a Ca2+-selective liquid membrane electrode of the neutral ionophore type (7) in a system analogous to that described by Affolter and Sigel (8). Measurement of the Transmembrane Potential (A$). The A$ was monitored continuously with a TTP+-selective electrode as described by Kamo et al. (9) in a system analogous to that used for the Ca*+ electrode measurements. The non-specific binding of TTP+ to the membrane was checked by using appropriate de-energizing inhibitors. RESULTS AND Figure release
1 shows
of
electrode.
have
After
the
protein
nmol
ruthenium
thereafter in
red,
to
a third,
Crompton
et
al.
mitochondria of
lower
2 shows
the
subsequent
of
state
by the
addition
Ca*+
is
0.8
with
mitochonof
induced
The
efflux
300
shortly velocity
Ca 2+ /min/mg
9 nmol
of
agreement
is
ug Antimycin
addition
and
Ca*+-selective Ca 2+/w
of
shown
uptake
25 nmol
15 mM Na'.
of
the
measurement efflux
rapidly
mito-
A/mg mitochon-
Na+ lowers
the
previous
efflux
findings
the
A$,
of
of
of Ca
the
by
A$ during
2+ induced
a transmembrane
the
addition
of
Ca*+
tends
to
approach
which
the
uptake
the
post
is
essentially
Ca2+ A$ level
194
the
by Na+.
developed
inside,
156 mV, when
steady
of
in
165 mV, negative
about
efflux
the
which
(4).
have
den decrease at
to
of
abolished
Addition
velocity
the
is
in
with
accumulation
experiment
prior
and
recorded
addition
protein
take
experiment
and the
protein.
Figure
been
uptake
particular
chondrial drial
the
by the
the
a typical
Ca*+
drial
DISCUSSION
is
up-
After
potential induces
a sud-
a plateau
complete.
The
evidently
due
Vol.
95. No.
BIOCHEMICAL
1, 1980
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
-30
165 161 c u z ii
--20; \ ‘D 03 s m
I I I I I I I Na
: -10; x E c
S
\\
02- 0 1
-0
fi
to
2 min
Figure
2
The membrane potential during Ca2+ uptake and Naiinduced efflux: same conditions as in figure 1, but instead of 15 PM CaC12, 5 PM TPP-Cl was present. At point "Cd",15 UM CaC12 was added.
nium
red
blocks
in
general,
in
Fig.
however,
Ca2+ after
red
that
does
the
no net
against exchange
not
of
Na+,
in
Ca*+
evidently
upon
slope
of
Na+ 1s . perfectly of
Na+
movement
agreement
It
A+ trace
change
the
the
is
since
experiment
clear
the
superimposable shown).
occurs
during
the
process.
195
from
addition
of
concept
It the of
ruthe-
A$ trace,
clear,
exchange an
shown Fig. of
2, ru-
electrode to
is
and
Na+-induced
TPP+-selective
(not
with
of
after
addition the
(lo),
in
substantially.
the
absence
charge
very
of decrease
steady-state
Indeed,
addition in
it
the
efflux.
served
not
reverses
that
thenium
Ca 2+ induced
the
although
2,
"cycling"
2* E
0
Na+ induced Ca2+ efflux: The incubation medium contains: 130 mM KCl, 10 mM Hepes (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonate), 1 ug Rotenone / ml, 0.5 mg mitochondrial protein / ml, 15 PM CaC12. Final pH = 7.0, temperature 25OC. Further additions are as follows. At the point "R", 150 pmol ruthenium red / ml; at point "Na " , 15 mM NaCl; at point "A", 0.4 vg Antimycin A / ml.
dissipating
131
--
1
energy
: :
112
1 .2-
Fisure
the
142
that
ob-
then, of
electroneutral
Ca*+
Vol.
95, No.
1, 1980
BIOCHEMICAL
ACKNOWLEDGEMENTS for
the
gift
supported
of by the
The authors Ca2+-selective Swiss
AND
BIOPHYSICAL
are
indebted
membranes.
Nationalfonds
RESEARCH
to
COMMUNICATIONS
Dr.
D. Ammann
The work
(Grant
No:
has
been
3.282-0.78).
REFERENCES 1. Crompton, M., Moser, R., Lfidi, H., and (1978) Eur. J. Biochem. 82, 25-31. 2. Crompton, M., and Heid, I. (1978) Eur. 91, 599-608. 3. Nicholls, D.G., and Crompton, M. (1980) FEBS Lett. 111, 261-268. 4. Crompton, M., Capano, M., and Carafoli, Eur. J. Biochem. 69, 453-462. 5. Crompton, M., Kiinzi, M., and Carafoli, Eur. J. Biochem. 79, 549-558. M. (1966) 6. Krijger, A., and Klingenberg, 344, 317-338. 7. Ammann, D., Guggi, M., Pretsch, E., and Anal. Lett. 8 (lo), 709-720. 8. Affolter, H., and Sigel, E. (1979) Anal. 97, 315-319. 9. Kamo, N., Muratsugu, M., and Kobatake, J. Membr. Biol. 49, 105-121. 10. Carafoli, E. (1979) FEBS Lett. 104, l-5.
196
Carafoli, J.
Biochem.
E. E.
E.
(1976) (1977)
Biochem. Simon,
Z. W.
Biochem. Y.
(1979)
(1975)
95, No.
July
16, 1980
BIOCHEMICAL
1, 1980
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
193-196
THE Ca2+-Na+ ANTIPORTER OF HEART MITOCHONDRIA OPERATES ELECTRONEUTRALLY Hubert
Affolter
and Ernest0
Carafoli
Laboratory of Biochemistry Swiss Federal Institute of Technology Universitgtstrasse 16 CH - 8092 Zurich, Switzerland Received
(ETH)
May 6,198O
ABSTRACT The transmembrane potential of heart mitochondria during Na+-induced Ca2+ release has been monitored continuously with a Tetraphenylphosphonium-sensitive electrode. The data obtained indicate that the exchange process does not generate an electrical current, and provides direct support for the concept of a 2 Na+ per 1 Ca2+ exchange. INTRODUCTION In mitochondria nium red
insensitive
(1,2,3).
This
in the and of
of
Ca2+-Na+ antiporter.
(4,5j
presented for
1 Ca2+. that
the
In this
the
operation
recordered
of using
ABBREVIATIONS:
of Ca2+,
Ca2+ content
of the
The kinetic
data
indications direct of
of Crompton
et
for
of
a ratio will is
be
2 Na+
from gxperiments
of mitochondria
the Ca2+-Na+ antiporter
or-
stoichiometry
the antiporter
potential
the
have
evidence
has been reached
a TPP+-selective
of
and thus
difficulties
measurement
article,
a ruthe-
has been described
Experimental
stoicbiometry
transmembrane
tissues
the efflux
of the
indirect
1 Ca2+. The conclusion
in which the
the direct
have provided
b 2 Na+ for
mediates
the cytosol.
prevented
al.
antiporter
regulation
so far the
from various
Ca 2+-Na+
antiporter
participates ganelles
isolated
during
has been continuously
electrode.
TPP+ : Tetraphenylphosphonium - ion : mitochondrial membrane potential AJ, 0006-291 193
X/80/7
30193-04$01.00/O
Copyright 0 1980 by Academic Press, Inc. All righrs of reproduction in anv form reserved.
Vol.
95. No.
1. 1980
BIOCHEMICAL
AND
EXPERIMENTAL
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
PROCEDURES
Mitochondrial Preparation. Rat heart mitochondria were prepared by the methods of Crompton et al. (4). The protein concentration of the suspension was estimated by a modified biuret procedure (6) with bovinaserum albumin (Sigma Chemical CO.) as a standard. Measurement of Ca2'-Fluxes. Ca2+- activitieswere measured with a Ca2+-selective liquid membrane electrode of the neutral ionophore type (7) in a system analogous to that described by Affolter and Sigel (8). Measurement of the Transmembrane Potential (A$). The A$ was monitored continuously with a TTP+-selective electrode as described by Kamo et al. (9) in a system analogous to that used for the Ca*+ electrode measurements. The non-specific binding of TTP+ to the membrane was checked by using appropriate de-energizing inhibitors. RESULTS AND Figure release
1 shows
of
electrode.
have
After
the
protein
nmol
ruthenium
thereafter in
red,
to
a third,
Crompton
et
al.
mitochondria of
lower
2 shows
the
subsequent
of
state
by the
addition
Ca*+
is
0.8
with
mitochonof
induced
The
efflux
300
shortly velocity
Ca 2+ /min/mg
9 nmol
of
agreement
is
ug Antimycin
addition
and
Ca*+-selective Ca 2+/w
of
shown
uptake
25 nmol
15 mM Na'.
of
the
measurement efflux
rapidly
mito-
A/mg mitochon-
Na+ lowers
the
previous
efflux
findings
the
A$,
of
of
of Ca
the
by
A$ during
2+ induced
a transmembrane
the
addition
of
Ca*+
tends
to
approach
which
the
uptake
the
post
is
essentially
Ca2+ A$ level
194
the
by Na+.
developed
inside,
156 mV, when
steady
of
in
165 mV, negative
about
efflux
the
which
(4).
have
den decrease at
to
of
abolished
Addition
velocity
the
is
in
with
accumulation
experiment
prior
and
recorded
addition
protein
take
experiment
and the
protein.
Figure
been
uptake
particular
chondrial drial
the
by the
the
a typical
Ca*+
drial
DISCUSSION
is
up-
After
potential induces
a sud-
a plateau
complete.
The
evidently
due
Vol.
95. No.
BIOCHEMICAL
1, 1980
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
-30
165 161 c u z ii
--20; \ ‘D 03 s m
I I I I I I I Na
: -10; x E c
S
\\
02- 0 1
-0
fi
to
2 min
Figure
2
The membrane potential during Ca2+ uptake and Naiinduced efflux: same conditions as in figure 1, but instead of 15 PM CaC12, 5 PM TPP-Cl was present. At point "Cd",15 UM CaC12 was added.
nium
red
blocks
in
general,
in
Fig.
however,
Ca2+ after
red
that
does
the
no net
against exchange
not
of
Na+,
in
Ca*+
evidently
upon
slope
of
Na+ 1s . perfectly of
Na+
movement
agreement
It
A+ trace
change
the
the
is
since
experiment
clear
the
superimposable shown).
occurs
during
the
process.
195
from
addition
of
concept
It the of
ruthe-
A$ trace,
clear,
exchange an
shown Fig. of
2, ru-
electrode to
is
and
Na+-induced
TPP+-selective
(not
with
of
after
addition the
(lo),
in
substantially.
the
absence
charge
very
of decrease
steady-state
Indeed,
addition in
it
the
efflux.
served
not
reverses
that
thenium
Ca 2+ induced
the
although
2,
"cycling"
2* E
0
Na+ induced Ca2+ efflux: The incubation medium contains: 130 mM KCl, 10 mM Hepes (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonate), 1 ug Rotenone / ml, 0.5 mg mitochondrial protein / ml, 15 PM CaC12. Final pH = 7.0, temperature 25OC. Further additions are as follows. At the point "R", 150 pmol ruthenium red / ml; at point "Na " , 15 mM NaCl; at point "A", 0.4 vg Antimycin A / ml.
dissipating
131
--
1
energy
: :
112
1 .2-
Fisure
the
142
that
ob-
then, of
electroneutral
Ca*+
Vol.
95, No.
1, 1980
BIOCHEMICAL
ACKNOWLEDGEMENTS for
the
gift
supported
of by the
The authors Ca2+-selective Swiss
AND
BIOPHYSICAL
are
indebted
membranes.
Nationalfonds
RESEARCH
to
COMMUNICATIONS
Dr.
D. Ammann
The work
(Grant
No:
has
been
3.282-0.78).
REFERENCES 1. Crompton, M., Moser, R., Lfidi, H., and (1978) Eur. J. Biochem. 82, 25-31. 2. Crompton, M., and Heid, I. (1978) Eur. 91, 599-608. 3. Nicholls, D.G., and Crompton, M. (1980) FEBS Lett. 111, 261-268. 4. Crompton, M., Capano, M., and Carafoli, Eur. J. Biochem. 69, 453-462. 5. Crompton, M., Kiinzi, M., and Carafoli, Eur. J. Biochem. 79, 549-558. M. (1966) 6. Krijger, A., and Klingenberg, 344, 317-338. 7. Ammann, D., Guggi, M., Pretsch, E., and Anal. Lett. 8 (lo), 709-720. 8. Affolter, H., and Sigel, E. (1979) Anal. 97, 315-319. 9. Kamo, N., Muratsugu, M., and Kobatake, J. Membr. Biol. 49, 105-121. 10. Carafoli, E. (1979) FEBS Lett. 104, l-5.
196
Carafoli, J.
Biochem.
E. E.
E.
(1976) (1977)
Biochem. Simon,
Z. W.
Biochem. Y.
(1979)
(1975)