(Received for publication, February 16, 1994; and in revised form, October 24, 1994)
From the
Heart mitochondria contain a nNa/Ca
antiport that
participates in the regulation of matrix
[Ca
]. Based largely on a single study
(Brand, M. D.(1985) Biochem. J. 229, 161-166), there has
been a consensus that this antiport promotes the electroneutral
exchange of two Na
for one Ca
.
However, a recent study in our laboratory (Baysal, K., Jung, D. W.,
Gunter, K. K., Gunter, T. P., and Brierley, G. P. (1994) Am. J.
Physiol. 266, C800-C808) has shown that the
Na
-dependent efflux of Ca
from heart
mitochondria has more energy available to it than can be supplied by a
passive 2Na
/Ca
exchange. We have
therefore re-examined Brand's protocols using fluorescent probes
to monitor matrix pH and free [Ca
].
Respiring heart mitochondria, suspended in KCl and treated with
ruthenium red to block Ca
influx, extrude
Ca
and establish a large
[Ca
]
:
[Ca
]
gradient. The
extrusion of Ca
under these conditions is
Na
-dependent and diltiazem-sensitive and can be
attributed to the nNa
/Ca
antiport. Addition of nigericin increases the membrane potential
(
) and decreases
pH to 0.1 or less, but has virtually
no effect on the magnitude of the [Ca
]
gradient. Under these conditions a gradient maintained by
electroneutral 2Na
/Ca
antiport
should be abolished because the mitochondrial
Na
/H
antiport keeps the
[Na
] gradient equivalent to the
[H
] gradient. The
[Ca
] gradient is abolished, however, when
an uncoupler is added to dissipate
or when the exogenous
electroneutral antiport BrA23187 is added. In addition,
[Ca
] influx via the nNa
/Ca
antiport in
nonrespiring mitochondria is enhanced when
is abolished.
These results are consistent with Ca
extrusion by an
electrophoretic antiport that can respond to
but not with
an electroneutral antiport.
It is now generally accepted that
[Ca] enters the matrix of heart
mitochondria via the ruthenium red-sensitive uniport and is extruded on
a nNa
/Ca
antiport (see (1) and (2) for reviews). Until recently there has
been a consensus that this antiport promotes the electroneutral
exchange of two Na
for one
Ca
(2, 3, 4) . This
conclusion is based largely on a study by Brand (3) that showed
a [Ca
] distribution established by the
endogenous antiport did not differ significantly from that produced by
the ionophore A23187. Because A23187 mediates electroneutral
2H
/Ca
exchange and the endogenous
Na
/H
antiport equilibrates
[H
] and [Na
]
gradients, it was concluded that the nNa
/Ca
antiport must be
electroneutral(3) . This stoichiometry is also supported by the
more recent report of Li et al.(4) that all modes of
transport promoted by a purified and reconstituted
Na
/Ca
antiport are insensitive to
uncouplers and therefore electroneutral. However, there have also been
indications that this antiport could be electrophoretic. The early
report of Crompton et al.(5) established that the
Na
-dependent efflux of Ca
was
dependent on the energy state of the mitochondria and kinetic data
indicate the presence of three independent Na
binding
sites on the antiport(6) . These results could be explained by
a contribution of the membrane potential,
, to an
electrophoretic exchange, such as that found with the
3Na
/Ca
antiport of the
sarcolemma(7) .
Two recent studies from our laboratory
strongly suggest that the concept of an electroneutral nNa/Ca
antiport should be
re-examined. In the first it was noted that the exchanger is regulated
by matrix pH and that high rates of Na
/Ca
antiport can be sustained when
pH (and hence the
Na
gradient) approaches zero(8) . This led us
to a null-point study in which it was established that
[Ca
] gradients maintained by
Na
/Ca
antiport are too large to be
sustained by a passive electroneutral exchange of 2Na
for one Ca
(9) . These observations in
turn led us to the present work in which we examine the protocols of
Brand (3) in more detail using currently available fluorescent
probes to monitor [H
] and
[Ca
] gradients. In contrast to
Brand(3) , we conclude that, when the pH gradient of respiring
mitochondria is collapsed by addition of nigericin, a large
[Ca
]
:[Ca
]
gradient is maintained. Addition of an uncoupler to dissipate
abolishes this gradient, as does addition of the
electroneutral exchanger BrA23187. These experiments lead to the
conclusion that the nNa
/Ca
antiport is not electroneutral under these conditions and that
there is a large contribution of
to the observed
[Ca
] gradient. Experiments showing
Na
-dependent [Ca
] influx
in nonrespiring mitochondria also support the conclusion that the
mitochondrial nNa
/Ca
antiport is electrophoretic.
Figure 3:
Effect
of nigericin on matrix pH of respiring and nonrespiring heart
mitochondria in a mannitol-sucrose medium. BCECF-equilibrated
mitochondria were suspended in the medium used by Brand (3) which consisted of mannitol (210 mM), sucrose (70
mM), HEPES (10 mM), NTA (1 mM), P (1 mM), NaCl (4 mM), rotenone (1 µg/ml),
and oligomycin (2 µg/ml) neutralized to pH 7.0 with TMA
hydroxide. Where indicated TMA
succinate (5
mM) was also present or added at 300 s. Nigericin (1
µM) was added at 100 s, except where indicated. Matrix pH
was calculated from net fluorescence intensity as described (8) and external pH was measured with a glass
electrode.
Matrix
[Ca] was calculated after determining R
, R
, and S
/S
values using the
following
expression(15) .
To determine R, BrA23187 (2
µM) was added to respiring mitochondria suspended in the standard medium containing 2 mM EGTA to deplete
mitochondrial Ca
, leaving only uncomplexed fura-2 in
the matrix (Fig. 1). R
was obtained by
allowing respiring mitochondria to accumulate Ca
and
saturate matrix fura-2 (Fig. 1). S
/S
is the ratio of
fluorescence intensities at 365 nm with zero and with excess
Ca
. R
, R
, and S
/S
values were determined for each mitochondrial preparation. The
means ± S.E. for the experiments presented here (n = 6) were 0.626 ± 0.001 R
,
2.653 ± 0.113 R
, and 1.858 ± 0.242 S
/S
. The solid line in Fig. 1represents the relationship between calculated
[Ca
] and the 340/365 ratio and shows that
fura-2 approaches saturation above 2 µM matrix
[Ca
]. With this in mind our experiments
were designed to keep matrix [Ca
] below 2
µM.
Figure 1:
Estimation
of R and R
for fura-2 sequestered in the
matrix of heart mitochondria. Mitochondria were loaded with fura-2 as
described under ``Experimental Procedures'' and added to the standard medium containing 2 mM EGTA for the R
determination and 15 µM EGTA for
the R
determination. Succinate (5 mM)
was added at 100 s. CaCl
(2 mM) was added at 200 s
for the R
determination and BrA23187 (2
µM) for the R
determination.
Fluorescence was corrected for autofluorescence and ratios
(340/365)
were calculated from net fluorescence
intensities. The relative net fluorescence intensities and calculated
ratios were: R
(340/530) = 0.64, R
(760/254) = 3.0, and S
/S
(530/254) =
2.09. The solid line shows the relationship between the ratios
and [Ca
] calculated according to . The initial ratio value of 1.55 indicates a matrix
[Ca
] of 0.25
µM.
All chemicals used were of reagent grade purity or higher. Ruthenium red was obtained from Fluka and recrystallized according to Luft(19) . The AM esters were obtained from Molecular Probes, Inc. (Eugene, OR) and were dissolved in dimethyl sulfoxide (silylation grade) obtained from Pierce.
A passive nNa/Ca
antiport will exchange Ca
for Na
until the energy contained in the electrochemical gradient of
Ca
is balanced by the proper stoichiometric factor (n) multiplied by the energy in the electrochemical gradient
of Na
(20, 21, 22) . Because
Na
/H
exchange is rapid in isolated
mitochondria and effectively equilibrates the
[Na
] and [H
]
gradients, the following expression should hold for a passive nNa
/Ca
antiport at
equilibrium.
At 25 °C this relationship can be expressed as follows(20, 21) .
This thermodynamic relationship predicts that when pH is
collapsed, no [Ca
] gradient can be
maintained at equilibrium if the antiport is electroneutral (n = 2), since 10
= 1 under these
conditions. However, a [Ca
] gradient can be
maintained if the antiport promotes electrophoretic exchange (n > 2) and responds to
. The term
``electrophoretic'' is used to indicate that the transport
occurs in response to an existing electrical potential. This is in
contrast to a transport reaction that generates a potential that would
be termed ``electrogenic''(23) .
The assumption
that pH and
pNa are very near equivalent is essential to this
investigation and is supported by several studies. Crompton and Heid (24) using isotope distribution procedures showed that the
[Na
] and [H
]
gradients are nearly at equilibrium for nonrespiring rat heart
mitochondria suspended in a KCl medium. This equilibrium results from
the high rate of Na
/H
exchange
relative to the Na
/Ca
antiport(24) . Studies from this laboratory (14) using SBFI-equilibrated pig heart mitochondria strongly
support this conclusion. When the [Na
]
gradients reported (14) are compared with the appropriate
[H
] gradients measured in related studies
using the fluorescent probes BCECF (25) or
cSNARF-1(8) , the mean difference between
pH and
pNa
calculates to 0.11 ± 0.06 (n = 12).
Isolated
beef heart mitochondria are difficult to load with SBFI (or the
analogous probe Sodium Green). The fluorescence signal obtained with
these preparations is no greater than 1.5 times the autofluorescence.
This makes it very difficult to distinguish between changes in redox
components and changes in matrix [Na] and
has prevented us from following the [Na
]
gradient directly in the respiring beef heart mitochondria used in
these studies. However, we were able to follow the spontaneous decay of
the pH and pNa gradients in nonrespiring beef heart mitochondria that
had been equilibrated with SBFI and cSNARF-1 (Fig. 2). In this
protocol the decay of
pNa approximates the
pH decay with a
maximum difference of 0.17. These results support the concept that
pH and
pNa are maintained nearly equal by the
Na
/H
antiport (14, 24
Figure 2:
Spontaneous decay of pH and
pNa
in nonrespiring heart mitochondria. Mitochondria equilibrated with both
SBFI and cSNARF-1 were added to the standard medium containing
ruthenium red (1 µM), NaCl (19 mM), EGTA (1
mM), and CaCl
(0.9 mM). Net fluorescence
intensities were used to calculate matrix pH (8) and matrix
[Na
](14) . Medium pH was
7.37.
In his influential 1985 study of the
stoichiometry of this antiport, Brand (3) used a
Ca-electrode to follow medium
[Ca
] in a suspension of rat heart
mitochondria respiring in a low K
medium. After
addition of nigericin he found the matrix to be acid with a
pH of
about -1.0. Under these conditions he calculated that external
[Ca
] for an n = 2
stoichiometry should differ from that for n = 3 by
0.634 µM at equilibrium(3) . At equilibrium Brand (3) reported a
pH of -0.99 (using the conventions of (18) ), a
pNa of -0.86,
of -161
mV, external [Ca
] of 6.37 µM,
and a calculated matrix [Ca
] of nearly 500
µM for mitochondria containing approximately 3 nmol of
Ca
/mg of protein.
These values are out of line
with our experience with respiring beef heart mitochondria under quite
similar conditions. Using BCECF fluorescence to monitor pH we see a large negative
pH (interior acid) only for
nonrespiring mitochondria following nigericin addition in the low
K
medium used by Brand (Fig. 3). Under these
conditions a
pH of about -0.8 is established immediately and
decays to about -0.5 after 500 s (Fig. 3). Mitochondria
respiring with succinate in the same medium show a transient
pH of
about -0.5 following nigericin addition, but respiration rapidly
establishes a condition in which
pH is about 0.15 (Fig. 3).
This is probably the result of electrophoretic uptake of TMA
in response to the high
established by nigericin.
Addition of succinate to nonrespiring mitochondria maintaining a large
negative
pH results in an alkaline shift to a final
pH near
0.1 (Fig. 3). In the KCl medium used for the studies reported
here there is no discernible
pH after nigericin addition whether
or not respiration is taking place because nigericin equilibrates the
[H
] gradient with the
[K
] gradient(23) . The BCECF-loaded
mitochondria lose some endogenous K
during
equilibration with the probe and average just over 100 ng ion
K
mg
protein. The incubation
medium contains about 120 mM K
, so the
[K
] gradient is minimal under these
conditions.
These results suggest that Brand's estimate of
pH for respiring mitochondria treated with nigericin (3) is too large. The distribution of
[
C]methylamine and
Na
was used to estimate the gradients in his study (3) and
it is possible that either redistribution of the probe at anaerobiosis
(following centrifugation), nonohmic uptake of the cationic probes, or
changes in probe binding during energization in the low ionic strength
suspending medium could have affected the results (see (26) ).
At pH 7 methylamine (pK
10.5) is >99%
cationic. Regardless of the reason for the discrepancy, a decrease in
the
pNa from -0.86 as estimated by Brand (3) to
-0.50 or less would result in loss of ability to distinguish
between n = 2 and n = 3 stoichiometry
in his experiment. As shown in Fig. 4the difference in external
[Ca
] predicted for these two cases becomes
less than 0.1 µM at -0.5
pNa. If
pH (and
therefore
pNa) were as small as the experiments of Fig. 3indicate, the difference between n = 2 and n = 3 would disappear completely (Fig. 4). If in
fact the
pH in Brand's experiment (3) was much less
than his estimate, then he would have seen no measurable change in
external [Ca
] when A23187 was added to
provide an exogenous electroneutral exchanger, even if the endogenous
antiport were electrophoretic.
Figure 4:
Predicted values for extramitochondria
[Ca] versus the
Na
gradient as a function of the stoichiometry of the nNa
/Ca
antiport. Values
were calculated from the data of Table I (sample 1) of (3) using Equation 1 in (3) , a stoichiometry of either
2 or 3 Na
/Ca
, and various values of
pNa. Due to uncertainties in some of the values these calculations
vary slightly when compared with those of Brand(3) . For
example, our value for n = 2 and
pNa of
-0.86 is 6.66 µM as opposed to 6.53 given
in(3) .
Figure 5:
Effects of nigericin on matrix
[Ca] and matrix pH of respiring heart
mitochondria. A, mitochondria equilibrated with fura-2 were
added to the standard medium containing NaCl (20 mM),
succinate (5 mM), and [Ca
]
buffered at either 0.8 µM or 5 µM. The 0.8
µM [Ca
] was established by
adding CaCl
(1.86 mM) and EGTA (2 mM),
whereas the 5 µM buffer contained NTA (2 mM) and
CaCl
(0.115 mM). Ruthenium red (1 µM)
was added 10 s after the mitochondria and fluorescence measurements
were then started. Nigericin (1 µM) was added at 100 s. B, mitochondria equilibrated with cSNARF-1 were incubated in
the same medium and under the same conditions as A. Matrix pH was
calculated from net fluorescence intensity as described(8) .
The records shown are from a different mitochondrial preparation than
that used in A. Records from mitochondria loaded with both
fura-2 and cSNARF-1 show essentially the same picture when
[Ca
] and pH are monitored
simultaneously.
Heart mitochondria were equilibrated
with the fluorescent pH indicator cSNARF-1 or the
[Ca] indicator fura-2 and suspended in a
KCl medium containing either 0.8 or 5 µM buffered
[Ca
] and 20 mM NaCl. The respiring
mitochondria were allowed to take up Ca
for 10 s. At
this point ruthenium red was added to block further influx via the
uniport and fura-2 fluorescence reported a matrix
[Ca
] of about 0.4 µM for 0.8
µM external [Ca
] and 0.65
µM for 5 µM external
[Ca
] (Fig. 5A). The
fluorescence then indicated a loss of matrix
[Ca
] which was completely
Na
-dependent and blocked by diltiazem (100
µM), properties consistent with efflux via the nNa
/Ca
antiport(2) . Cyclosporin A was included in all of the
experiments reported here to prevent possible contributions of the
Ca
-dependent permeability pore(1) . Addition
of nigericin under these conditions decreases
pH to 0.1 or less (Fig. 5B), but has no significant effect on
Ca
efflux (Fig. 5A). When a steady
state is reached (600 s), matrix [Ca
] is
0.185 and 0.130 µM in the presence of nigericin, and 0.130
and 0.110 µM in its absence in the 5 µM and
0.8 µM [Ca
] buffers,
respectively. This means that the mitochondria are maintaining
comparable
[Ca
]
:[Ca
]
gradients in the presence and absence of nigericin (27 versus 38 in 5 µM and 6.2 versus 7.3 in 0.8
µM [Ca
]). In the presence of
nigericin,
pH is near zero and indicates that
Ca
gradients of 27 and 6.2 could be maintained by
values of -85 and -47 mV, respectively, if the
antiport stoichiometry were 3Na
for one
Ca
. If n = 2, pH (or pNa) gradients
of 0.7 and 0.4 would be required to maintain these Ca
gradients, but since
pH is 0.1 or less, these results are
not compatible with electroneutral exchange. The larger Ca
gradients seen in the absence of nigericin (
pH and
present) than in its presence (
pH = 0) (Fig. 5A) follow according to where the
pH term is multiplied by n but the
/59 term by
only (2 - n). It does not necessarily follow that the
conversion of
pH to
by nigericin should increase the
rate of Ca
efflux via an electrophoretic antiport.
The kinetics of the nNa
/Ca
antiport appear to depend on many factors on either side of the
membrane including pH, [Na
],
[Ca
], [Mg
], and
adenine nucleotides(2, 6, 8, 9) .
The present study is concerned with the
[Ca
] gradients maintained at equilibrium
and not with the kinetics of Ca
transport.
If the
exogenous electroneutral exchanger BrA23187 is added 130 s after
nigericin in the protocol of Fig. 5, there is a rapid increase
in matrix [Ca] to nearly that of the
external [Ca
] (Fig. 6A).
Thus, in contrast to the results of Brand(3) , we see a steady
state [Ca
] gradient established by the
endogenous nNa
/Ca
antiport
that is far from the value seen in the presence of the electroneutral
ionophore BrA23187. BrA23187 promotes electroneutral exchange of
Ca
for 2H
, so that the equilibrium
[Ca
] gradient should equal the square of
the [H
] gradient. A record of cSNARF-1
fluorescence shows that addition of Br23187 produces no change in
matrix pH and that
pH remains near zero after this addition (not
shown). BrA23187 will also promote loss of matrix Mg
in exchange for H
. When 1 mM [Mg
] is added to the medium of Fig. 6A to approximate matrix
[Mg
], matrix
[Ca
] rises to nearly the same concentration
(86% of control) on addition of BrA23187 as in the absence of external
[Mg
] (not shown). In the high
[K
] medium used in these studies,
respiration-dependent H
extrusion, in combination with
nigericin, keeps
pH near zero and allows no
[Ca
] or [Mg
]
gradient to be maintained in the presence of BrA23187.
Figure 6:
The electroneutral exchanger BrA23187 and
uncoupler equilibrate the [Ca] gradient
maintained by nNa
/Ca
antiport in respiring mitochondria. A, mitochondria were added
to the standard KCl medium containing NaCl (20 mM),
K
succinate (5 mM), EGTA (2 mM), and
CaCl
(1.94 mM). The calculated
[Ca
] of this medium is 2 µM.
Ruthenium red (1 µM) was added 10 s after the
mitochondria, and recording of fluorescence was begun. Nigericin (1
µM) was added at 100 s, and efflux of matrix
[Ca
] was followed. At 230 s, BrA23187 (2
µM) was added, and the rapid equilibration of matrix
[Ca
] with external
[Ca
] was followed. In a parallel experiment
FCCP (1 µM) was added at 230 s to produce a slower
equilibration via endogenous antiport. A control without further
addition after nigericin is also shown. The net 340/365 nm ratios (left ordinate) were used to calculate matrix
[Ca
] (right ordinate) using 0.615 R
, 2.62 R
, and 1.86 S
/S
. Note that the
[Ca
] scale is nonlinear in this panel. B, conditions identical to A with BrA23187 and
nigericin added where indicated. C, effect of diltiazem (100
µM) and TPP
(3.1 and 6.6 µM)
after a steady state [Ca
] gradient has been
established in the protocol of A.
A similar but
slower decay of the steady state [Ca]
gradient is seen following addition of the uncoupler FCCP (Fig. 6A). The uncoupler collapses the protonmotive
force to zero eliminating
as well
pH. This allows
matrix [Ca
],
[Na
], and [H
] to
equilibrate with the corresponding external ions via endogenous
antiporters regardless of the value of n for the nNa
/Ca
antiport.
Addition of BrA23187 to respiring mitochondria in the absence of
nigericin results in a decrease in matrix
[Ca] to a new steady state near 0.7
µM (Fig. 6B). Addition of nigericin to
these mitochondria abolishes the
pH and results in rapid influx of
Ca
to bring matrix [Ca
]
to very nearly the same concentration as external
[Ca
].
Addition of diltiazem or
TPP to block nNa
/Ca
antiport after a
steady state [Ca
] gradient has been
established results in a slow increase in matrix
[Ca
] (Fig. 6C). The
increase amounts to only about 0.07 pmol of
Ca
min
mg
and probably results from a slow inward leak of Ca
in response to the high
that is maintained under
these conditions (see 27). This apparent leak increases with increasing
external [Ca
] and requires
(data not shown). The presence of an inward leak of
[Ca
] in response to
does not
affect the conclusion that the nNa
/Ca
antiport is
electrophoretic. However, it does mean that the steady state matrix
[Ca
] seen in protocols such as those in Fig. 5and Fig. 6represents a steady state balance
between extrusion on the antiport and influx through the leak and is
not due to the nNa
/Ca
equilibrium alone.
Figure 7:
Matrix [Ca]
of respiring mitochondria treated with ruthenium red (1
µM) and suspended in media with different external
[Ca
]. Mitochondria equilibrated with fura-2
were added to the standard medium containing NaCl (20 mM),
succinate (5 mM), ruthenium red (1 µM), NTA (2
mM), and CaCl
(0.115, 0.215, 0.565, or 0.78
mM) to produce a final external
[Ca
] of 5, 10, 31.5, or 50 µM,
respectively. Nigericin (1 µM) was added at 100 s and
matrix [Ca
] allowed to come to a steady
state. Matrix [Ca
] at 600 s was calculated
to be 0.32, 0.49, 0.72, and 0.95 µM for external
[Ca
] of 5, 10, 31.6, and 50
µM, respectively.
Figure 8:
Stimulation of Ca influx
by FCCP, nigericin, and valinomycin in nonrespiring heart mitochondria.
Mitochondria were added to the standard medium containing ruthenium red
(1 µM), NaCl (20 mM), and EGTA (10
µM). CaCl
(40 nmol) was added at 130 s and as
indicated FCCP (1 µM), nigericin (1 µM),
valinomycin (1 µM), and succinate (5 mM) were
added. After the CaCl
addition the calculated
[Ca
] of the medium is approximately 3
µM.
These observations
suggest that Ca is entering the matrix by passive
exchange on the electrophoretic antiporter, since optimal rates are
obtained only when both
pH and
are collapsed.
Eliminating
pH would permit matrix [Na
]
to be elevated and provide ample Na
for exchange with
entering [Ca
]. Eliminating
would remove the electrical constraint that favors Ca
efflux over Ca
influx on the electrophoretic
antiport.
The present studies have established that the nNa/Ca
antiport of heart
mitochondria promotes an electrophoretic exchange of Na
for Ca
rather than the electroneutral antiport
indicated by previous work(2, 3, 4) . The
experimental basis for the conclusion that the antiport is
electroneutral (3) appears to be flawed and three different
lines of evidence now support the concept that this antiport is
electrophoretic. First, respiring mitochondria maintain a very
significant [Ca
] gradient (external
[Ca
] > matrix
[Ca
]) under conditions in which an
electroneutral antiport should support no gradient ( Fig. 5and Fig. 6). Addition of an exogenous electroneutral exchanger, such
as BrA23187, under these conditions discharges this gradient. These
steady state [Ca
] gradients can be produced
either by extrusion of accumulated Ca
from the matrix
or by uptake of extramitochondrial Ca
( Fig. 6and Fig. 7). Second, passive uptake of
Ca
by nonrespiring mitochondria via the
diltiazem-sensitive antiport is rapid only when both the pH gradient
and the
opposing Ca
influx is discharged (Fig. 8). Third, a recent null-point study from our laboratory
has shown that the nNa
/Ca
antiport has more energy available to it than can be provided by
a passive 2Na
/Ca
exchange(9) .
Early studies from Crompton's
laboratory (5) suggested that the nNa/Ca
antiport was
electrophoretic. The rate of Na
-dependent
Ca
efflux was increased by respiration and inhibited
by uncouplers(5) , results compatible with a contribution of
to the exchange reaction. In addition, the presence of
three independent binding sites for Na
was indicated
by kinetic studies(6) . However, Affolter and Carafoli (29) concluded that the antiport was electroneutral, because a
TPP
electrode showed no change in
when
Na
-dependent Ca
efflux was
initiated. Their study (29) has been largely dismissed (1, 2, 3) because respiratory compensation of
was not considered and TPP
has been shown
to be a potent inhibitor of Na
/Ca
antiport(1, 28) . In the present studies
TPP
was used to estimate
in parallel
incubations and was never present when nNa
/Ca
activity was
measured.
As discussed above, the report of Brand (3) was
most influential in establishing the consensus that the
Na/Ca
antiport promotes
electroneutral exchange (see (2) ). In his study conditions
were chosen that should have produced a significant difference in the
steady state Ca
gradient if n were 2 as
opposed to 3 (see Fig. 4), and no change in external
[Ca
] was seen with a Ca
electrode when the exogenous antiport A23187 was
added(3) . However, the current work establishes that
pH
(and hence
pNa
) as reported by the continuous
readout of a fluorescent probe (Fig. 3) is considerably
different from the value obtained by Brand using distribution of
labeled probes and centrifugation(3) . If
pH is not as
large as estimated by Brand (3) the ability to distinguish
between n = 2 and n = 3 is lost (Fig. 4). The present study also follows matrix
[Ca
] using a fluorescent probe and finds
values under 2 µM for this parameter in most experiments
as opposed to the 500 µM calculated in the Brand
report(3) . A value of 500 µM for matrix
[Ca
] is far out of line with estimates from
other laboratories (see (30) and (31) , for example).
In addition, Brand (3) used only 67 pmol of ruthenium red/mg
of mitochondrial protein to block infux via the Ca uniporter. We have demonstrated a K
of
approximately 40 pmol
mg
for inhibition of the
uniport in beef heart mitochondria as assayed by
Ca
-induced
K
release
and inhibition was not complete until 200 pmol
mg
were added(32) . This suggests that the amount of
ruthenium red used by Brand may not have been sufficient to completely
block Ca
uniport. In his protocol(3) ,
Ca
influx via the uniport would decrease the measured
external [Ca
] and result in an erroneous
lower apparent value for the Na
/Ca
antiport at equilibrium. In this study we use 1 µM ruthenium red, equivalent to 2000 pmol/mg protein, and although
the uniporter is maximally inhibited, when nigericin is present other
-dependent pathways may become available for Ca
influx.
In a recent study, Li et al.(4) report the isolation of a 110-kDa fraction with the
properties of a Na/Ca
antiport from
mitochondria. When reconstituted into liposomes this fraction promoted
transport that was not affected by uncouplers and it was concluded that
the antiport was electroneutral(4) . This reconstituted
antiporter, however, displays several unusual properties not observed
for the native protein, such as K
/Na
exchange and inhibition by low concentrations of TPP
in the absence of
(33) . In contrast, we find
that
does affect the rate of passive influx of
Ca
on the antiport in intact, nonrespiring
mitochondria (Fig. 8). The basis for these discrepancies between
the native protein and the reconstituted fraction is not clear and
requires further study.
It may be possible that the
Na/Ca
antiport can operate in either
an electrophoretic or an electroneutral mode depending on environmental
conditions(9) . A slow rate of Na
-dependent
Ca
extrusion is seen in nonrespiring mitochondria (5, 8) that may reflect electroneutral exchange under
these conditions. In a recent study (34) the sarcolemmal nNa
/Ca
antiport was
reported to promote electroneutral Na
for
Ca
exchange at low pH and electrophoretic antiport at
physiological pH. Relative rates of Ca
and
Na
movement were modulated by the state of protonation
of the carrier(34) , and it was suggested that other regulatory
components may control relative movements of these ions according to
the physiological needs of the cell. There is ample precedent for
modification of a mitochondrial transporter in such a way as to change
its stoichiometry (see (35) , for example).
The
[Ca] gradients established and the response
to BrA23187 shown in Fig. 6represent powerful evidence that the
antiport promotes an electrophoretic rather than an electroneutral
exchange. The [Ca
] gradients measured in
the present study in the presence of nigericin with
pH near zero
range from 6 to 53 (Fig. 5Fig. 6Fig. 7). To
account for these gradients for an n = 2 antiport
pNa would have to be 0.39 to 0.86. Although it has not been
conclusively shown that
pH =
pNa under all conditions,
our measurements suggest a maximum difference between
pH and
pNa of approximately 0.2. This is well below the values of
pNa needed to accomodate an n = 2 antiport. An n = 3 antiport would require
values of
-46 to -102 mV to maintain the
[Ca
] gradients as calculated from . Our estimates of
by TPP
distribution average -115 mV (range -103 to
-127 mV) over a wide range of external
[Ca
] in the KCl buffers used in our
protocols indicate there is clearly sufficient potential available to
produce the observed gradients. The n values calculated from when
is -115 mV for
[Ca
] gradients of 6-53 range from 2.4
to 2.88. Because there appears to be a small inward leak of
Ca
in response to
under our conditions (Fig. 6C), the steady state matrix
[Ca
] is higher than the true nNa
/Ca
equilibrium value
and reflects a balance between the leak and extrusion on the antiport.
The presence of this leak does not affect the conclusion that the
antiport is electrophoretic, but it does complicate attempts to
evaluate n with certainty from . Although it is
attractive to suggest that n is equal to 3 as found for the
sarcolemma antiport, the possibility remains that the stoichiometry is
variable and can operate in either the electrophoretic or
electroneutral mode(4, 34) . Further experiments are
needed, especially with the isolated and reconstituted protein, to
determine the mechanism and stoichiometry of the exchange conclusively.
The membrane potentials measured in this study with nigericin
present (-115 mV average) seem relatively low when compared with
literature values approaching -200 mV(23, 27) .
However, in contrast to most of these measurements, the present
investigation was performed in a high K medium. In the
presence of nigericin
pH is collapsed and
elevated in
the KCl medium, and this can result in nonohmic influx of K
(and Ca
)(27) . When coupled to
nigericin-mediated K
/H
exchange such
an influx would create a futile cycle and decrease
and the
protonmotive force.
The possibility has been raised that the
antiport is indeed electroneutral but that heterogeneity of the
mitochondrial preparation contributes to the appearance of
electrophoretic exchange. However, it appears unlikely that
differential loading of [Ca] or fura-2 into
well coupled and poorly coupled fractions could account for the
[Ca
] gradients observed in the presence of
nigericin ( Fig. 5and Fig. 6). When nigericin is added (Fig. 5A) matrix [Ca
]
continues to decrease (i.e. the
[Ca
] gradient increases). This is
incompatible with electroneutral antiport because the pH gradient is
near zero (Fig. 5B), and no energy is available to an n = 2 antiport to increase the gradient. Under these
conditions matrix [Ca
] should increase for
an electroneutral antiport and approach thermodynamic equilibrium at
matrix [Ca
] = medium
[Ca
]. This holds for all mitochondria that
maintain a functioning endogenous antiporter, since both well coupled
and poorly coupled mitochondria will approach thermodynamic
equilibrium.
An electrophoretic antiport may have physiological
advantages over an electroneutral exchanger in that Ca efflux can be driven by the energy in both the electrical and pH
gradients making it asymmetric and essentially irreversible. For
example, if respiring mitochondria maintain a
of -150
mV and a
pH gradient of 0.4, predicts that an n = 2 mechanism can maintain a
[Ca
]
:[Ca
]
gradient of only 6.3 at equilibrium as compared to 5526 for an n = 3 mechanism. This may be particularly important
during steady state Ca
cycling in situ when
the transmembrane gradients of Na
and Ca
may be small(22) . Mitochondria in situ may have
only a small Na
gradient due to the presence of
metabolites such as P
and CO
that tend to
equilibrate the mitochondrial pH gradient.
Recently, an active
mechanism has been proposed for the Na-independent
Ca
efflux seen in liver mitochondria(21) .
Because nNa
/Ca
exchange can
be demonstrated in mitochondria where respiration is blocked by
rotenone and ATPase activity is blocked by oligomycin (Fig. 7),
and sufficient energy is available in the Na
electrochemical gradient to account for the Ca
gradients measured, it does not seem necessary to postulate that
the nNa
/Ca
antiport
represents an active transport(21) . The results appear
compatible with a passive electrophoretic mechanism with an n value of approximately 3 that mediates ``downhill''
exchange of Na
and Ca
.
The present studies, in conjunction with our previous report(9) , establish that the mitochondrial antiport, like that in the plasmalemma (7) , is capable of supporting an electrophoretic exchange. Further work will be necessary to establish the stoichiometry with certainty and whether or not the antiport can also operate as an electroneutral exchange under some conditions.