Dependence of Na+-K+ pump current-voltage
relationship on intracellular Na+, K+, and
Cs+ in rabbit cardiac myocytes
Peter S.
Hansen1,2,
Kerrie A.
Buhagiar1,2,
Benjamin
Y.
Kong3,
Ronald J.
Clarke3,
David F.
Gray1, and
Helge H.
Rasmussen1,2
1 Department of Cardiology, Royal North Shore
Hospital, St. Leonards, NSW 2065; and 2 Department of
Medicine and 3 School of Chemistry, University of
Sydney, Sydney 2006, Australia
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ABSTRACT |
To examine effects of cytosolic
Na+, K+, and Cs+ on the voltage
dependence of the Na+-K+ pump, we measured
Na+-K+ pump current (Ip)
of ventricular myocytes voltage-clamped at potentials
(Vm) from
100 to +60 mV. Superfusates were
designed to eliminate voltage dependence at extracellular pump sites.
The cytosolic compartment of myocytes was perfused with patch pipette solutions with a Na+ concentration ([Na]pip)
of 80 mM and a K+ concentration from 0 to 80 mM or with
solutions containing Na+ in concentrations from 0.1 to 100 mM and K+ in a concentration of either 0 or 80 mM. When
[Na]pip was 80 mM, K+ in pipette solutions
had a voltage-dependent inhibitory effect on Ip
and induced a negative slope of the
Ip-Vm relationship. Cs+ in pipette solutions had an effect on
Ip qualitatively similar to that of
K+. Increases in Ip with increases
in [Na]pip were voltage dependent. The dielectric
coefficient derived from
[Na]pip-Ip relationships at the
different test potentials was 0.15 when pipette solutions included 80 mM K+ and 0.06 when pipette solutions were K+ free.
access channels; ion transport; membrane dielectric; sodium/potassium-exchanging adenosinetriphosphatase
 |
INTRODUCTION |
THE INTERACTION of
Na+ and K+ with extracellular sites of the
membrane Na+-K+ pump has been studied in
considerable detail. However, cytosolic pump sites are difficult to
study in intact cells, and interactions of Na+ and
K+ with these sites remain poorly understood
(27). Studies on isolated
Na+-K+-ATPase reconstituted in lipid membranes
or vesicles indicated that binding of Na+ to the pump in
its E1 conformational state in the Na+
translocation pathway is dependent on membrane voltage (5, 9, 25,
34). The largest effect of voltage dependence occurs at
relatively low, rate-limiting concentrations of cytosolic
Na+ near levels encountered in vivo. If similar voltage
dependence occurs in intact cells, it might be an important target for
cellular regulation of the pump and might be of particular importance
in cells with large sustained changes in membrane potential such as
cardiac myocytes (9).
Voltage-dependent binding of intracellular Na+ in the
pump's Na+ translocation pathway is expected to give rise
to a positive slope in the relationship between electrogenic
Na+-K+ pump current (Ip)
and membrane voltage (Vm). Nakao and Gadsby (23) studied the
Ip-Vm relationships of
internally perfused voltage-clamped single guinea pig ventricular
myocytes with the whole cell patch-clamp technique. Experimental
conditions were designed to eliminate voltage dependence arising from
interaction of Na+ and K+ with the pump at
extracellular sites, and wide-tipped patch pipettes were used to
achieve good control of the intracellular Na+
concentration. Ip-Vm
relationships, determined with four different Na+
concentrations ([Na]pip) in patch pipette solutions, had
a shallow positive slope. However, an increase in [Na]pip
caused an increase in Ip by the same factor at
all Vm tested. A voltage-dependent increase in
Ip with an increase in [Na]pip
would have been expected if activation of the pump by cytosolic
Na+ is voltage dependent. It was concluded that activation
of the pump by cytosolic Na+ seems independent of membrane
potential. The same conclusion was reached in a subsequent study on rat
ventricular myocytes (30).
In agreement with the study of Nakao and Gadsby (23), we
(10) also recorded a shallow positive slope of the
Ip-Vm relationship of
single cardiac myocytes under experimental conditions expected to
eliminate voltage dependence arising at extracellular
Na+-K+ pump sites. However, when myocytes were
exposed to insulin under identical conditions, a negative slope was
recorded (see Fig. 3, Ref. 10). The negative slope is
difficult to explain within the framework of our current understanding
of voltage-dependent steps in the Na+-K+ pump cycle.
A negative slope of the
Ip-Vm relationship may be
generated by voltage-dependent inhibition of the pump by a cytosolic
cation. Although it is firmly established that cytosolic K+
inhibits the pump, such inhibition is not voltage dependent when studied in Na+-K+ pumps in isolated membrane
fragments (1). However, the effect of cytosolic
K+ on Na+-K+ pumps in situ in
intact cells has not been systematically studied. We have examined the
effect of cytosolic K+ on Ip at
different membrane voltages. In an initial series of experiments we
perfused the cytosolic compartment of ventricular myocytes with patch
pipette solution containing Na+ in a high concentration
expected to nearly saturate cytosolic binding at all
Vm. Experimental conditions were designed to
eliminate voltage-dependent steps at extracellular pump sites.
Inclusion of K+ or the K+ congener
Cs+ in pipette solutions inhibited
Ip and induced a negative slope of the
Ip-Vm relationship. The
effect of an increase in the K+ pipette concentration
([K]pip) was voltage dependent. We also examined
activation of the pump by cytosolic Na+. Because
voltage-dependent inhibition by K+ might obscure
voltage-dependent activation by Na+, we used a fixed
[K]pip of 80 or 0 mM and variable levels of [Na]pip to determine
Ip-Vm relationships.
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MATERIALS AND METHODS |
Ventricular myocytes were isolated from 52 male New Zealand
White rabbits as described previously (10). They were
stored at room temperature and used for experimentation on the day of isolation only.
Composition of superfusates.
For measurement of Ip, myocytes were initially
superfused with modified Tyrode solution warmed to 35 ± 0.5°C.
The solution contained (in mM) 140 NaCl, 5.6 KCl, 2.16 CaCl2, 1 MgCl2, 0.44 NaH2PO4, 10 glucose, and 10 HEPES. It had a pH
of 7.40 ± 0.01 at 35°C. When the whole cell configuration had
been established, we switched to a superfusate that was nominally
Ca2+ free and contained 0.2 mM Cd2+ and 2 mM
Ba2+. To eliminate voltage dependence arising at
extracellular Na+-K+ pump sites, this
superfusate contained K+ in concentrations at or above
physiological levels and was Na+ free or contained
Na+ in a low concentration (27). It contained
15 mM K+ and was Na+ free in most experiments.
We used 140 mM N-methyl-D-glucamine chloride (NMG-Cl) to
maintain osmotic balance and ionic strength of the superfusate. In some
experiments we used a K+ concentration of 5.6 mM and a
Na+ concentration of 1.5 mM. These solutions were used to
allow comparisons of results with data published by others.
Composition of pipette filling solutions.
Solutions with high fixed Na+ and variable K+
concentration contained (in mM) 79 Na glutamate, 1 NaH2PO4, 0-80 KOH, 5 HEPES, 2 MgATP, 5 EGTA, 20 tetraethylammonium chloride (TEA-Cl), 31-45 aspartic
acid, and 0-66 tetramethylammonium hydroxide (TMA-OH). Solutions
with high fixed K+ and variable Na+
concentration contained (in mM) 5-80 NaOH, 79 K glutamate, 1 KH2PO4, 5 HEPES, 5 EGTA, 2 MgATP, 20 TEA-Cl,
0-66 TMA-OH, and 45-31 aspartic acid. K+-free
solutions with variable Na+ concentrations contained (in
mM) 0-99 NaOH, 0.1-1 NaH2PO4, 5 HEPES, 2 MgATP, 5 EGTA, 20 TEA-Cl, 66-145 TMA-OH, and 45 aspartic acid. Cs+-containing solution included (in mM) 79 Na
glutamate, 1 NaH2PO4, 5 HEPES, 5 EGTA, 2 MgATP,
20 TEA-Cl, 70 CsCl, and 50 aspartic acid. All pipette filling solutions
were titrated to a pH of 7.2 at 22°C with 1 M HCl. Osmolarities
measured with a vapor pressure osmometer (model 5500 osmometer, Wescor,
Logan, UT) varied by <10% between solutions. We nulled offset
potentials by placing both ground electrode and patch pipette in
pipette filling solution (24). Jpcalc (P. Barry,
University of New South Wales, Sydney, Australia) was used to estimate
junction potentials between ground electrode and superfusates.
They were <3 mV for all solutions and were not corrected for. Patch
pipettes had resistances of 0.8-1.1 M
before the whole cell
configuration was established. The resistance of pipettes always
increases with seal formation. The
Ip-Vm relationships were
not always reproducible between experiments performed with identical
solutions when the series resistance exceeded 2.2 M
. We therefore
only proceeded with experiments when the series resistance in the whole
cell configuration was at or below this level. To reduce effects of
interrabbit variability, we used pipette filling solutions of different
compositions in experiments on cells from each rabbit.
Measurement of Ip.
Ip was identified as the shift in holding
current induced by Na+-K+ pump blockade with
100 µM ouabain. Ouabain was always superfused 8-10 min after the
whole cell configuration had been established. Holding currents were
recorded during voltage steps of 320-ms duration in 20-mV increments to
test potentials from
100 to +60 mV. Each test potential was bracketed
by a return to a holding potential of
40 mV for 2 s. Recordings
were averaged from three applications of the voltage-clamp protocol and
sampled at 5-ms intervals for the last 50 ms at each test potential.
The holding current was taken as the mean value of the samples.
Membrane currents were recorded with the single-electrode voltage-clamp
mode of an Axoclamp-2B amplifier, and voltage-clamp protocols were
generated with pCLAMP (Axon Instruments, Foster City, CA). Unless
otherwise specified, Ip is reported normalized
for membrane capacitance. The capacitance for myocytes in this study
was 127 ± 30 pF (mean ± SD).
Statistical analysis.
Results are expressed as means ± SE unless otherwise indicated.
Statistical comparisons were made using the Mann-Whitney rank-sum test
and linear regression. We considered a P value <0.05
significant. The Hill and Boltzmann equations were fitted to data with
nonlinear regression.
 |
RESULTS |
Effect of pipette K+ and
Cs+ on membrane currents.
The voltage dependence of the Na+-K+ pump in
cardiac myocytes must be studied under conditions that markedly reduce
nonpump membrane currents and virtually eliminate time-dependent
currents. This can be achieved with appropriate compositions of
superfusates and patch pipette solutions. Membrane K+
currents are blocked by including Ba2+ in the superfusate
and TEA-Cl and Cs+ in pipette solutions. In an initial
series of experiments, we examined whether using K+ rather
than Cs+ in pipette solutions altered the membrane
conductance. Myocytes were patch-clamped with a pipette filling
solution that included 80 mM K+. The superfusate was
nominally Na+ free and contained 15 mM K+.
Figure 1 shows currents elicited in
response to steps in command voltages from
100 to +60 mV. They were
small, largely time independent, and similar to the currents elicited
when pipette solutions were K+ free and contained 70 mM
Cs+ as illustrated previously (10).

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Fig. 1.
Holding currents at test potentials from 100 to +60 mV
before and after exposure of a myocyte to 100 µM ouabain. Traces
shown represent the currents averaged from 3 applications of the
voltage-clamp protocol. Superfusate contained 0 mM Na+ and
15 mM K+, and pipette solution contained 80 mM
Na+ and 80 mM K+. Membrane capacitance of the
myocyte was 120 pF.
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The voltage dependence of steady-state membrane currents determined
with the K+-containing filling solutions is summarized in
Fig. 2. We also performed experiments
with a pipette filling solution containing 70 mM Cs+. To
allow comparison with a previous study (7), the
superfusate contained 1.5 mM Na+ and 5.6 mM K+.
A summary of the currents is included in Fig. 2. Currents of myocytes
patch-clamped with Cs+-containing pipettes were shifted in
the outward direction relative to the currents of myocytes
patch-clamped with K+-containing pipettes. This may be
accounted for by an inhibitory effect of the 80 mM K+ in
pipette solutions on Ip that was greater than
the inhibitory effect of 70 mM Cs+ (11). The
slope conductance, however, was similar for experiments performed with
K+- and Cs+-containing pipette solutions
(1.84 ± 0.15 vs. 1.85 ± 0.14 pA/mV). The conductance was
also virtually identical to that found previously in guinea pig
myocytes patch-clamped with Cs+-containing pipette
solutions (estimated from Refs. 7 and 23). We conclude
that nonpump membrane currents in experiments performed with
K+-containing pipettes are similar to the currents one can
expect with the Cs+-containing pipette solutions commonly
used when studying pump currents in cardiac myocytes.

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Fig. 2.
Effect of Cs+ and K+ on the slope
conductance summarized from 8 and 10 myocytes, respectively. Pipette
solutions contained 70 mM Cs+ ( ) or 80 mM
K+ ( ). Vm, membrane
voltage. Iwhole cell, holding current before
exposure of myocytes to ouabain.
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Effect of pipette K+ on
Ip-Vm relationships.
To examine the effect of intracellular K+ on the voltage
dependence of Ip, we determined the
Ip-Vm relationship for
myocytes patch-clamped with [K]pip ranging from 0 to 80 mM. An example of membrane currents before and after exposure to
ouabain in an experiment using a [K]pip of 80 mM is shown
in Fig. 1. A summary of
Ip-Vm relationships
determined with all levels of [K]pip is shown in Fig.
3A. A Mann-Whitney test
indicated that the mean value of Ip pooled for
measurements at all test potentials with pipette solutions containing
only 2.5 mM K+ was significantly less than the
corresponding mean value of Ip measured with
K+-free solutions.

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Fig. 3.
A: effect of pipette K+
concentration ([K]pip) on Na+-K+
pump current (Ip)-Vm
relationships. Superfusate was Na+ free and contained 15 mM
K+, and pipette solutions contained 80 mM Na+
and variable [K]pip. n, No. of myocytes in
each group. B: normalized
Ip-Vm relationships for a
[K]pip of 0 or 10 mM.
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Figure 3A shows that there is a
[K]pip-dependent decrease in Ip.
Ip does not appear to decrease by the same
factor at all test potentials. This can be further examined by
normalizing Ip to the Ip
recorded at a fixed membrane potential. If there is a proportional,
voltage-independent change in Ip with a change in pipette cation concentration, the normalized
Ip-Vm relationships should overlap (7). We normalized
Ip to the Ip recorded at 0 mV for experiments performed with a [K]pip of 0 or 10 mM. The normalized Ip-Vm
relationships are shown in Fig. 3B. The slopes of the
normalized relationships at test potentials more positive than
40 mV
were compared by linear regression. The slope determined with a
[K]pip of 10 mM was significantly more negative than the slope determined with K+-free patch pipette solutions.
There was also a significant difference when the normalized
Ip-Vm relationships were
compared with a Mann-Whitney test. These findings indicate that
[K]pip-induced Na+-K+ pump
inhibition is voltage dependent.
Figure 3A indicates that within the
[K]pip range we could study, K+ inhibition of
the pump current is not complete, i.e., saturating concentrations of
K+ do not reduce Ip to zero. The
incomplete inhibition induced by K+ suggests that cells may
contain K+-sensitive and relatively
K+-insensitive subpopulations of pumps (see
DISCUSSION). To highlight the incomplete inhibition, we
plotted Ip as a function of [K]pip in Fig. 4A at one of the test
potentials (0 mV). To further characterize the inhibitory effect of
[K]pip on Ip
(Ii), we subtracted the mean
Ip measured at each [K]pip from
the mean Ip measured when pipette solutions were
K+ free. This subtraction was performed at each
Vm. A
[K]pip-Ii relationship derived
from data obtained at 0 mV is shown in Fig. 4B. The Hill
equation has been fitted to the data.

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Fig. 4.
A: Pump current remaining after inhibition by
[K]pip. Ip at a test potential of
0 mV has been plotted for different [K]pip values.
B: [K]pip dependence of inhibition by
[K]pip on Ip
(Ii) at Vm = 0 mV.
The Hill equation has been fitted (solid line) to experimental data
( ). Half-maximal inhibitory [K]pip
(KK1/2) was 7.2 mM, and the Hill coefficient was
1.0. There was a statistically significant increase in the Hill
coefficients with an increase in Vm from 0.7 to
1.5 over the range of Vm studied. C:
simulated inhibitory effect of cytosolic K+ on pump rate.
Pump rate was simulated for cytosolic K+ corresponding to
the [K]pip values used experimentally. The Hill equation
has been fitted to the simulated values. KK1/2
derived from the fit was 8.0 mM, and the Hill coefficient was 1.5. The
maximum turnover number at zero [K]pip used for the
simulations was 10 s 1 (at 24°C). D: voltage
dependence of KK1/2 derived from
[K]pip-Ii relationships at test
potentials from 100 to +60 mV ( ). Solid line
represents the best fit of a Boltzmann distribution to the
Vm-KK1/2 relationship.
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A K+-induced acceleration of the backward reaction
E1 + 2K+
E2(K+)2 is a possible mechanism for
the [K]pip-dependent Ii.
If fast competitive binding of K+ and Na+ at
two sites on the E1 conformation and selective binding of Na+ at a third site are in equilibrium, the following
equation can be derived to describe the dependence of the observed rate
constant, kobs, on cytosolic Na+ and
K+ concentrations
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(1)
|
where kmax represents the maximum rate for
the E1 + 2K+
E2(K+)2 transition when two sites
on E1 are fully occupied by K+ ions.
KN and KK represent the
microscopic association constants for Na+ and
K+ ions for the two nonspecific binding sites on
E1, and K'N represents the
association constant for the third binding site specific for Na+. A kmax of 550 s
1
(4), a KK of 100 M
1
(31), a KN of 1.2 × 102 M
1 (16), and a
K'N of 3.9 × 102
M
1 (16) were obtained from the literature.
It is apparent from the equation that K+-induced activation
of the backward reaction should slow down the overall forward reaction.
To test this, we carried out numerical simulations by adapting
differential rate Eqs. 8-11 of Lüpfert et al.
(20) for saturating substrate conditions to nonsaturating conditions, and we extended the equations by including Eq. 1
given above to take into account the E1 + 2K+
E2(K+)2
backward reaction and Na+/K+ competition for
E1 binding sites. The simulated inhibitory effect of
cytosolic K+ shown in Fig. 4C was consistent
with the inhibition of the K+-sensitive
Na+-K+ pump subpopulation by
[K]pip shown in Fig. 4B.
To describe the effect of Vm on the
[K]pip-induced pump inhibition, we fitted the Hill
equation to the [K]pip-Ii
relationships at all test potentials. The [K]pip values
for half-maximal inhibitory effect (KK1/2)
derived from the fits are shown in Fig. 4D.
KK1/2 appears to be voltage dependent. A
dielectric coefficient of 0.15 was derived from a fit of the Boltzmann
equation to the values for KK1/2.
Interaction of cytosolic K+ with the pump within an access
channel is one of the possible mechanisms for voltage dependence of
KK1/2. Such interaction may give rise to
ouabain-sensitive transient currents that can be detected in response
to sudden changes in Vm when experimental
conditions allow only a limited set of partial reactions of the pump
cycle (22, 26). In an attempt to demonstrate transients,
we used Na+-free superfusates containing 15 mM
K+ and Na+-free patch pipette solutions
containing 10-25 mM K+. We could not detect
ouabain-sensitive current transients, even when reducing the
[ATP]pip to 50-100 µM and the temperature to 22°C to prolong their duration. Although our experimental system may
not have allowed the detection of transients of short duration or low
amplitude, these experiments do not support the existence of an access channel.
Effect of pipette Cs+ on
Ip-Vm relationships.
Because Cs+ can act as a K+ congener by
inhibiting the pump at intracellular sites (11), we
examined the effect of Cs+ in pipette solutions on the
Ip-Vm relationship. We
determined Ip-Vm
relationships in superfusates containing 1.5 mM Na+ and 5.6 mM K+ (adopted from Ref. 7). Pipette solutions
were K+ and Cs+ free or contained 70 mM
Cs+. The
Ip-Vm relationships are
summarized in Fig. 5A. We
compared the mean of the Ip values at all test
potentials with Cs+-containing or Cs+-free
pipette solutions by using the Mann-Whitney rank-sum test. Mean
Ip of myocytes patch-clamped with
Cs+-containing pipette solutions was significantly less
than mean Ip of myocytes patch-clamped with
Cs+-free solutions. To examine whether the inhibitory
effect of Cs+ on Ip is voltage
dependent, we normalized the
Ip-Vm relationships shown
in Fig. 5A to the Ip measured at 0 mV. The normalized relationships are shown in Fig. 5B. We
fitted a regression line to the
Ip-Vm relationships
determined with and without Cs+ in pipette solutions at
test potentials more negative than
20 mV and a regression line to the
relationships at potentials of
20 mV or more positive. The slopes
determined with and without pipette Cs+ were significantly
different in both voltage ranges. To avoid any assumption of linearity,
we also compared Ip-Vm
relationships in Fig. 5B with a Mann-Whitney rank-sum test.
There was a significant difference between the normalized relationships
in both voltage ranges compared. We conclude that cytosolic
Cs+ inhibits Ip in a
voltage-dependent fashion, i.e., such inhibition is not specific for
K+.

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Fig. 5.
A: effect of pipette Cs+
concentration ([Cs]pip) on the
Ip-Vm relationship. The
pipette solutions contained 80 mM Na+ and Cs+
as indicated. n, No. of myocytes in each group.
B: data from A normalized to the
Ip recorded at 0 mV to illustrate the effect of
pipette Cs+ on the slopes of the
Ip-Vm relationship.
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Effect of intracellular Na+ on
Ip-Vm relationships.
To examine the effect of cytosolic Na+ on the voltage
dependence of the Na+-K+ pump, we performed
experiments with different levels of [Na]pip. Patch
pipettes contained K+ in a first series of experiments. We
used a constant [K]pip of 80 mM to maintain the
inhibitory effect of intracellular K+ on
Ip constant. [Na]pip ranged from 5 to 80 mM. The superfusate was Na+ free and contained 15 mM
K+. The Ip-Vm
relationships determined with the different levels of
[Na]pip are shown in Fig.
6A. There is a monotonic
increase in Ip with an increase in
[Na]pip at all test potentials. However, Ip does not appear to increase by the same
factor at all test potentials. To further examine this, we normalized
Ip-Vm relationships determined with a [Na]pip of 10 or 80 mM to the
Ip recorded at 0 mV. The normalized
relationships are shown in Fig. 6B. Linear regression
demonstrated a statistically significant difference between the slopes.
A Mann-Whitney rank-sum test also indicated a significant difference
between the normalized
Ip-Vm relationships.

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Fig. 6.
Effect of [Na]pip on
Ip-Vm relationships.
Pipette solutions contained 80 mM K+ and variable
[Na]pip. n, No. of myocytes in each group.
B: data from A normalized to
Ip recorded at 0 mV when [Na]pip
was 10 and 80 mM.
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To further describe the voltage dependence of the interaction of
cytosolic Na+ with the pump, we transformed the
Ip-Vm relationships shown
in Fig. 6A to
[Na]pip-Ip relationships at
different test potentials. The
[Na]pip-Ip relationship at one
test potential (0 mV) is shown in Fig.
7A. The Hill equation has been
fitted to the data. The [Na]pip values for half-maximal
stimulation (KNa1/2) derived from Hill equations
fitted to [Na]pip-Ip relationships
at all test potentials are shown in Fig. 7B. A dielectric
coefficient of 0.15 was derived from a fit of the Boltzmann equation.

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Fig. 7.
A: [Na]pip dependence of
Ip at Vm = 0 mV. The
Hill equation has been fitted (solid line) to experimental data
( ). [K]pip was 80 mM. There was a
statistically significant increase in the Hill coefficients with an
increase in Vm from 1.5-2.0 over the range
of Vm studied. B: voltage dependence
of the [Na]pip inducing half-maximal
Ip (KNa1/2) derived from
[Na]pip-Ip relationships at test
potentials from 100 to +60 mV ( ). Solid line
represents the best fit of a Boltzmann distribution to the
Vm-KNa1/2
relationship.
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In a second series of experiments we examined the effect of
[Na]pip on the
Ip-Vm relationship with
K+-free patch pipette solutions. We wished to detect
[Na]pip-dependent activation of the
Na+-K+ pump with high resolution at low levels
of [Na]pip as well as to measure
Ip with nearly maximal pump activation. We
therefore clustered the [Na]pip values at relatively low
levels and at high levels with a range from 0.1 to 100 mM. The
superfusate was Na+ free and contained 15 mM
K+. The Ip-Vm
relationships determined with the different levels of
[Na]pip are shown in Fig.
8A. There was a monotonic
increase in Ip with an increase in
[Na]pip at all test potentials. To examine whether the
[Na]pip-dependent increase in Ip
is voltage dependent, we normalized
Ip-Vm relationships
determined with a [Na]pip of 5 or 100 mM to the
Ip recorded at 0 mV. The normalized
relationships are shown in Fig. 8B. Linear regression
demonstrated a statistically significant difference between the slopes.
A Mann-Whitney rank-sum test also indicated a significant difference
between the normalized Ip-Vm relationships.

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Fig. 8.
A: effect of [Na]pip on
Ip-Vm relationships.
Pipette solutions were K+ and Cs+ free and
contained a variable [Na]pip. n, No. of
myocytes in each group. B: data from A normalized
to Ip recorded at 0 mV when
[Na]pip was 5 or 100 mM.
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We transformed the Ip-Vm
relationships shown in Fig. 8A to
[Na]pip-Ip relationships at
different test potentials. The
[Na]pip-Ip relationship at one
test potential (0 mV) is shown in Fig.
9A. The Hill equation has been
fitted to the data. The KNa1/2 values derived
from Hill equations fitted to
[Na]pip-Ip relationships at all
test potentials are shown in Fig. 9B. A dielectric
coefficient of 0.06 was derived from a fit of the Boltzmann equation.

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Fig. 9.
A: [Na]pip dependence of
Ip at Vm = 0 mV. The
Hill equation has been fittted (solid line) to experimental data
( ). [K]pip was 0 mM. Hill coefficients
varied from 1.5 to 1.4 with an increase in Vm
over the range studied. The variation of the Hill coefficient with
voltage was not statistically significant. B: voltage
dependence of KNa1/2 derived from
[Na]pip-Ip relationships at test
potentials from 100 to +60 mV ( ). Solid line
represents the best fit of a Boltzmann distribution to the
Vm-KNa1/2
relationship.
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DISCUSSION |
Specificity of ouabain-induced changes in membrane currents.
The interaction of Na+ (13, 14, 22) and
K+ (26, 28) with the
Na+-K+ pump at extracellular sites is voltage
dependent. Figure 3 indicates that a negative slope of the
Ip-Vm relationship can
occur under conditions expected to eliminate this voltage dependence.
We identified Ip as the shift in membrane
current induced by ouabain. Although ouabain is a highly specific
inhibitor of the Na+-K+ pump, the inhibition
can give rise to changes in membrane current not arising directly from
electrogenic Na+/K+ exchange.
Pump inhibition reduces cellular consumption of ATP. This, in turn,
might inactivate ATP-sensitive K+ channels
(15). However, at test potentials positive to the equilibrium potential for K+ (EK),
ouabain-induced shifts in currents arising from ATP-sensitive K+ channels and the Na+-K+ pump are
in the same direction. This would result in an overestimation of pump
currents and hence a positive slope of the
Ip-Vm relationship. The
slope was negative at test potentials positive to the
EK calculated from K+ concentrations
in pipette solutions and superfusate according to the Nernst equation
(Fig. 3). In addition, inclusion of 20 mM TEA-Cl in all pipette
solutions should have blocked ATP-sensitive K+ channels
(15).
Ouabain might also affect Na+-activated K+
channels reported to exist in cardiac myocytes of some species
(18) if pump inhibition results in an increase in the
Na+ concentration at cytosolic sites despite perfusion of
the intracellular compartment. However, a role of
Na+-activated K+ channels in the mechanism
generating the negative slope of the Ip-Vm relationship is
unlikely because the slope was reproduced when Cs+ replaced
K+ in pipette solutions (Fig. 5) and cytosolic
Cs+ blocks the Na+-activated K+
channel (18).
Inhibition of Ip by cytosolic
K+ and Cs+.
Figure 4, A and B, suggests that
K+-induced inhibition saturates at a level where inhibition
is incomplete. The mechanism for the incomplete inhibition is not
identified. We speculate that the inhibition of
Ip that is identified is attributable to a
subpopulation of pump units. The measured Ip is
generated by all isoforms of the pump in all locations. It is
conceivable there is a difference in the affinity of cytosolic
K+ for different isoforms of the pump or that the same
isoform in different microdomains of the sarcolemma have
different functional properties. It is possible, for example, that
pumps in the cardiac myocyte sarcolemmal caveolae (21) are
affected by the lipid-rich milieu and/or proximity to kinases
characterizing these important microdomains. Experimental constraints
did not allow us to examine whether additional inhibition of
Ip occurs at [K]pip >80 mM.
K+-induced inhibition of Ip might be
due to an effect of K+ on a rate-limiting E2
E1 transition. The transition is probably rate limiting
even under physiological circumstances (20). It seems
highly likely that the transition was rate limiting under the
conditions of our experiments because an allosteric effect of
extracellular Na+ causes the rate of transition to decrease
with a decrease in the extracellular Na+ concentration
(6, 17, 33). Our experiments were performed with
Na+-free superfusates.
Inhibition of the E2
E1 transition could be
due to binding of cytosolic K+ to the E1 or
E2 conformation. Inhibition by binding directly to the
E2 conformation is unlikely because such inhibition is only
quantitatively significant at cytosolic ATP concentrations much lower
than used in this study (33). Inhibition by binding to the E1 conformation could be due to inhibition of the
forward reaction or acceleration of the backward reaction.
Inhibition of the forward reaction could in principle occur if
K+ binds to cytosolic Na+ binding sites and
inhibits the ATP phosphorylation reaction (29). At the
cytosolic concentrations of Na+ and ATP we used, the rate
constant (at 24°C) for the phosphorylation reaction in the absence of
K+ can be estimated from Kane et al. (16) to
be ~175 s
1. If Eq. A8 of Kane et al.
(16) is modified to include competition with
K+ at two cytosolic Na+ binding sites and if
one assumes that the microscopic binding constant for K+ is
100 M
1 (31), the rate constant for the
phosphorylation reaction at 80 mM K+ decreases by 7% only.
This is much less than the K+-induced decrease in
Ip we observed.
We used a model to examine the hypothesis that cytosolic K+
inhibits the overall forward pump rate by binding to the E1
conformation and accelerating the backward reaction. The model was
based on kinetics reported under conditions similar to those we used in our experiments. It was consistent with the K+-induced pump
inhibition we observed, even at the lowest [K]pip, and
there was good agreement between the
[K]pip-Ii relationship and the
simulated K+-induced pump inhibition (Fig. 4) in the
concentration range we could examine experimentally. The similarity in
the effects of 10 mM [K]pip and 70 mM
[Cs]pip on
Ip-Vm relationships (see
Figs. 3 and 5) is also consistent with the hypothesis that pump
inhibition is due to binding of K+ or the K+
congener to the E1 conformation of the pump because the
affinity of the E1 conformation for K+ is
~10-fold higher than its affinity for Cs+
(29).
[K]pip-induced inhibition of Ip
increased with a shift in membrane potential toward positive values
(Fig. 4D). Inspection of Eq. 1 indicates this
could be due to a voltage-dependent increase in the association
constant of K+ for the E1 conformation of the
pump (KK) or to an increase in the rate constant
for the conformational transition
E1(K+)2
E2(K+)2
(kmax). Previous studies do not support the
existence of such voltage dependence in isolated
Na+-K+-ATPase (1).
The Na+-K+ pump is a membrane-embedded
molecule, and membrane constituents make an important contribution to
pump function. It is pertinent to the present study that the membrane
environment is a particularly important determinant of cytosolic
K+/Na+ antagonism (32) and that
such antagonism is a prominent feature of
Na+-K+-ATPase in the heart (31).
To our knowledge, voltage dependence of
Na+-K+- ATPase has not been reported for
enzyme isolated from the heart and might be difficult to study. Unless
a recovery equally representative of all isoforms and microdomains can
be ensured, it is unlikely that Na+-K+-ATPase
in isolated membrane fragments reflects properties of all
Na+-K+ pumps in an intact cell. Pumps embedded
in lipid-rich membrane microdomains may be of particular concern in
this regard because in situ membrane lipids, believed to be important
in the regulation of the Na+-K+ pump
(32), are easily altered in isolation procedures. It is not known whether they contribute to voltage-dependent regulation. The
sarcolemma and the T tubular system of myocytes provide a highly
inhomogeneous environment and a complexity that might be avoided in
studies on cells with a less complex membrane structure. However, a
detailed study on the effect of cytosolic K+ on the voltage
dependence of the pump in such cells has not been reported.
Figure 3 suggests that the voltage-dependent component of
K+-induced pump inhibition diminishes at a high
[K]pip, and voltage dependence of the inhibition may be
of little importance at the even higher physiological levels of
cytosolic K+. The main practical implication is that our
study may explain findings in previous studies. The effect of variable
concentrations of the K+ congener Cs+ in
pipette solutions (23) on
Ip-Vm relationships may
have prevented the detection of voltage-dependent interaction of
Na+ with cytosolic sites.
Voltage-dependent activation of pump by cytosolic
Na+.
Previous studies reported that
Ip-Vm relationships of
ventricular myocytes, normalized to the Ip
recorded at a test potential of 20 mV (23) or a
[Na]pip of 85 mM (30), have similar slopes for different [Na]pip values. This suggests that pump
activation by intracellular Na+ is independent of
Vm. However, a decrease in [Cs]pip
was used to maintain osmotic balance with increases in
[Na]pip. Our study suggests that a decrease in
[Cs]pip shifts the slope of the
Ip-Vm relationship in a
positive direction (Fig. 5) and offsets effects of increases in
[Na]pip on the slope in the opposite direction. Without
such effects of Cs+,
Ip-Vm relationships in
the previous studies (23, 30) might have been similar to
the Ip-Vm relationships
shown in Figs. 6 and 8 in the present study. A change in the normalized
relationships with a change in [Na]pip is consistent with
voltage-dependent binding of Na+ at a pump site facing the
cytoplasmic side of the Na+-K+ pump.
Barmashenko et al. (3) also reported findings attributed
to voltage-dependent binding of cytosolic Na+ to the
Na+-K+ pump in intact cells. They measured
Ip of voltage-clamped guinea pig ventricular
myocytes suspended in Na+-free superfusates and perfused
with K+- and K+ congener-free pipette
solutions. A dielectric coefficient of ~0.08 was derived for the
voltage dependence of the interaction of Na+ with cytosolic
sites. We derived similar dielectric coefficients. However,
discrepancies in important details between the two studies should be noted.
The Ip measured by Barmashenko et al.
(3) at a test potential of 0 mV saturated at ~0.75
pA/pF, whereas we measured a much higher Ip of
~4.5 pA/pF at the same test potential (Fig. 8). Barmashenko et al.
(3) attributed the low maximal Ip
to the use of Cs+ rather than K+ to activate
the pump at extracellular sites. Their patch pipettes had initial
resistances of 2-4 M
. The access resistance is expected to have
been approximately twofold higher. In our hands a resistance at such
levels rather than the composition of solutions limits maximal pump
rates. Regardless of the mechanism, any rate limitation not determined
by the composition of pipette solutions must influence values for
KNa1/2 of
[Na]pip-Ip relationships and the
dielectric coefficients that are derived.
We derived the dielectric coefficients from fits of Hill equations to
the relationships between [Na]pip and
Ip. The values we report were based on a fitting
procedure that allowed the Hill coefficient to float. The dielectric
coefficients we derived were sensitive to details of the fitting
procedure. When we used Hill coefficients fixed at estimated mean
values, we obtained different numerical values for the dielectric
coefficients. However, they were within the 0.06-0.15 range of
those derived with a floating Hill coefficient.
The numerical values of our dielectric coefficients are less than the
dielectric coefficient of ~0.25 derived from measurements of charge
movement of the electrogenic step with the pump restricted to a partial
reaction in isolated membrane fragments (12). Our analysis
is based on the overall forward pump rate and reflects interaction of
Na+ with all three cytosolic sites, including the two
believed to be located outside the electrical field of the membrane.
Such analysis does not allow a distinction between the sites
(25) and may have underestimated the dielectric
coefficient of the electrogenic partial reaction. Our values are in
reasonable agreement with those derived from Hill plots of pump rates
measured in noncellular systems (5, 9, 25) and support the
qualitative conclusion that the interaction of cytosolic
Na+ with the Na+-K+ pump is voltage
dependent. Our study does not justify conclusions about quantitative
details of this interaction.
Kinetic equivalence of changes in substrate concentration and
Vm in an access channel predicts a shift of
Ip-Vm relationships along
the voltage axis without a change in maximal current or shape of the
relationship with a change in [Na]pip (28).
Such a shift was not observed in this study (Figs. 5 and 6) and may be
difficult to demonstrate for a shallow access channel. In addition, the
conformational change from E1P to E2P is
voltage dependent and we cannot rule out a contribution from it to the
shape of the Ip-Vm
relationships and hence a deviation from the prediction of an access
channel model. The rate constant in one study is such that the
E1P to E2P conformational change may be rate
limiting (34), whereas much higher rate constants in two
other studies (2, 8) suggest that it is unlikely that the
conformational change contributes to the shape of the
Ip-Vm relationship.
Lu et al. (19) found no charge movements related to
binding of Na+ at cytoplasmic sites in giant membrane
patches excised from cardiac myocytes. This implies that binding is
independent of voltage or that voltage dependence is not a constant
feature necessarily preserved in the excised membrane patches. A recent
study (10) suggests the latter is the case. Exposure of
cardiac myocytes to insulin shifted the
Ip-Vm relationships in a
manner consistent with elimination of voltage dependence of cytosolic
Na+ binding. Effects of pharmacological blockers indicated
involvement of cellular messengers and suggested effects on voltage
dependence were mediated by dephosphorylation (10). The
experiments of Lu et al. (19) were performed without ATP
in cytosolic solutions. This may have facilitated dephosphorylation and
hence reduced or eliminated voltage dependence of Na+
binding. These speculations are consistent with suggestions that voltage dependence of cytosolic Na+ binding under control
of cellular messengers may be important in the regulation of
Na+-K+ pump function (27).
 |
ACKNOWLEDGEMENTS |
This study was supported by the North Shore Heart Research
Foundation. P. S. Hansen received a Postgraduate Medical Research Scholarship from the National Heart Foundation of Australia, K. A. Buhagiar was supported by National Heart Foundation of Australia Grant
G96S4589, and D. F. Gray received a National Health and Medical
Research Council Postgraduate Research Scholarship. R. J. Clarke
acknowledges support from the Australian Research Council.
 |
FOOTNOTES |
Address for reprint requests and other correspondence:
H. H. Rasmussen, Dept. of Cardiology, Royal North
Shore Hospital, Pacific Highway, St. Leonards, NSW 2065, Australia
(E-mail: helger{at}med.usyd.edu.au).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpcell.01343.2000
Received 19 December 2000; accepted in final form 24 June 2002.
 |
REFERENCES |
1.
Apell, HJ,
and
Karlish SJ.
Functional properties of Na,K-ATPase, and their structural implications, as detected with biophysical techniques.
J Membr Biol
180:
1-9,
2001[ISI][Medline].
2.
Babes, A,
and
Fendler K.
Na+ transport and the E1P-E2P conformational transition of the Na+/K+-ATPase.
Biophys J
79:
2557-2571,
2000[Abstract/Free Full Text].
3.
Barmashenko, G,
Kockskamper J,
and
Glitsch HG.
Depolarisation increases the apparent affinity of the Na+-K+ pump to cytoplasmic Na+ in isolated guinea-pig ventricular myocytes.
J Physiol
517:
691-698,
1999[Abstract/Free Full Text].
4.
Bugnon, P,
Doludda M,
Grell E,
and
Merbach AE.
High pressure stopped-flow spectrometer for kinetic studies of fast bioinorganic reactions by absorbance and fluorescence detection.
In: High Pressure Research in the Biosciences and Biotechnology, edited by Heremans K.. Leuven, Belgium: Leuven Univ. Press, 1997, p. 143-146.
5.
Cornelius, F.
Hydrophobic ion interaction on Na+ activation and dephosphorylation of reconstituted Na+,K+-ATPase.
Biochim Biophys Acta
1235:
183-196,
1995[ISI][Medline].
6.
Cornelius, F,
and
Skou JC.
The sided action of Na+ on reconstituted shark Na+/K+-ATPase engaged in Na+-Na+ exchange accompanied by ATP hydrolysis. II. Transmembrane allosteric effects on Na+ affinity.
Biochim Biophys Acta
983:
142-152,
1989[ISI][Medline].
7.
Gadsby, DC,
and
Nakao M.
Steady-state current-voltage relationship of the Na/K pump in guinea pig ventricular myocytes.
J Gen Physiol
94:
511-537,
1989[Abstract].
8.
Ganea, C,
Babes A,
Lüpfert C,
Grell E,
Fendler K,
and
Clarke RJ.
Hofmeister effects of anions on the kinetics of partial reactions of the Na+,K+-ATPase.
Biophys J
77:
267-281,
1999[Abstract/Free Full Text].
9.
Goldschlegger, R,
Karlish SJD,
Rephaeli A,
and
Stein WD.
The effect of membrane potential on the mammalian sodium-potassium pump reconstituted into phospholipid vesicles.
J Physiol
387:
331-355,
1987[Abstract].
10.
Hansen, PS,
Buhagiar KA,
Gray DF,
and
Rasmussen HH.
Voltage-dependent stimulation of the Na+-K+ pump by insulin in rabbit cardiac myocytes.
Am J Physiol Cell Physiol
278:
C546-C553,
2000[Abstract/Free Full Text].
11.
Hemsworth, PD,
Whalley DW,
and
Rasmussen HH.
Electrogenic Li+/Li+ exchange mediated by the Na+-K+ pump in rabbit cardiac myocytes.
Am J Physiol Cell Physiol
272:
C1186-C1192,
1997[Abstract/Free Full Text].
12.
Heyse, S,
Wuddel I,
Apell HJ,
and
Stürmer W.
Partial reactions of the Na,K-ATPase: determination of rate constants.
J Gen Physiol
104:
197-240,
1994[Abstract].
13.
Hilgemann, DW.
Channel-like function of the Na,K pump probed at microsecond resolution in giant membrane patches.
Science
263:
1429-1432,
1994[ISI][Medline].
14.
Holmgren, M,
Wagg J,
Bezanilla F,
Rakowski RF,
De Weer P,
and
Gadsby DC.
Three distinct and sequential steps in the release of sodium ions by the Na+/K+-ATPase.
Nature
403:
898-901,
2000[ISI][Medline].
15.
Kabakov, AY.
Activation of KATP channels by Na/K pump in isolated cardiac myocytes and giant membrane patches.
Biophys J
75:
2858-2867,
1998[Abstract/Free Full Text].
16.
Kane, DJ,
Fendler K,
Grell E,
Bamberg E,
Taniguchi K,
Froehlich JP,
and
Clarke RJ.
Stopped-flow kinetic investigations of conformational changes of pig kidney Na+,K+-ATPase.
Biochemistry
36:
13406-13420,
1997[ISI][Medline].
17.
Karlish, SJD,
and
Stein WD.
Cation activation of the pig kidney sodium pump: transmembrane allosteric effects of sodium.
J Physiol
359:
119-149,
1985[Abstract].
18.
Lawrence, C,
and
Rodrigo GC.
A Na+-activated K+ current (IK,Na) is present in guinea-pig but not rat ventricular myocytes.
Pflügers Arch
437:
831-838,
1999[ISI][Medline].
19.
Lu, CC,
Kabakov A, A,
Markin VS,
Mager S,
Frazier GA,
and
Hilgemann DW.
Membrane transport mechanisms probed by capacitance measurements with megahertz voltage clamp.
Proc Natl Acad Sci USA
92:
11220-11224,
1995[Abstract].
20.
Lüpfert, C,
Grell E,
Pintschovius V,
Apell HJ,
Cornelius F,
and
Clarke RJ.
Rate limitation of the Na+,K+-ATPase pump cycle.
Biophys J
81:
2069-2081,
2001[Abstract/Free Full Text].
21.
Mohammadi, K,
Liu L,
Aynafshar BB,
Wang H,
Tian J,
Ivanov A,
Xie Z,
and
Askari A.
Na+/K+-ATPase is present in caveolae microdomains of the plasma membrane (Abstract).
Biophys J
82:
265a,
2002.
22.
Nakao, M,
and
Gadsby DC.
Voltage dependence of Na translocation by the Na/K pump.
Nature
323:
628-630,
1986[ISI][Medline].
23.
Nakao, M,
and
Gadsby DC.
[Na] and [K] dependence of the Na/K pump current-voltage relationship in guinea pig ventricular myocytes.
J Gen Physiol
94:
539-565,
1989[Abstract].
24.
Neher, E.
Correction for liquid junction potentials in patch clamp experiments.
Methods Enzymol
207:
123-131,
1992[ISI][Medline].
25.
Or, E,
Goldschlegger R,
and
Karlish SJD
An effect of voltage on binding of Na+ at the cytoplasmic surface of the Na+-K+ pump.
J Biol Chem
271:
2470-2477,
1996[Abstract/Free Full Text].
26.
Peluffo, RD,
and
Berlin JR.
Electrogenic K+ transport by the Na+-K+ pump in rat cardiac ventricular myocytes.
J Physiol
501:
33-40,
1997[Abstract].
27.
Rakowski, RF,
Gadsby DC,
and
De Weer P.
Voltage dependence of the Na/K pump.
J Membr Biol
155:
105-112,
1997[ISI][Medline].
28.
Sagar, A,
and
Rakowski RF.
Access channel model for the voltage dependence of the forward running Na+/K+ pump.
J Gen Physiol
103:
869-894,
1994[Abstract].
29.
Schneeberger, A,
and
Apell HJ.
Ion selectivity of the cytoplasmic binding sites of the Na,K-ATPase. II. Competition of various cations.
J Membr Biol
179:
263-273,
2001[ISI][Medline].
30.
Stimers, JR,
Liu S,
and
Kinard TA.
Effect of Nai on activity and voltage dependence of the Na/K pump in adult rat cardiac myocytes.
J Membr Biol
135:
39-47,
1993[ISI][Medline].
31.
Therien, AG,
and
Blostein R.
K+/Na+ antagonism at cytoplasmic sites of Na+-K+-ATPase: a tissue-specific mechanism of sodium pump regulation.
Am J Physiol Cell Physiol
277:
C891-C898,
1999[Abstract/Free Full Text].
32.
Therien, AG,
and
Blostein R.
Mechanisms of sodium pump regulation.
Am J Physiol Cell Physiol
279:
C541-C566,
2000[Abstract/Free Full Text].
33.
Van der Hijden, HTWM,
and
de Pont JJHHM
Cation sidedness in the phosphorylation step of Na+/K+-ATPase.
Biochim Biophys Acta
983:
142-152,
1989[ISI][Medline].
34.
Wuddel, I,
and
Apell HJ.
Electrogenicity of the sodium transport pathway in the Na,K-ATPase probed by charge-pulse experiments.
Biophys J
69:
909-921,
1996[Abstract].
Am J Physiol Cell Physiol 283(5):C1511-C1521
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