1 Department of Biological
Sciences, A competition assay of
86Rb+
uptake in HeLa cells transfected with ouabain-resistant
Na+-K+-ATPase
mutants revealed a stimulation of
86Rb+
uptake at low external concentrations (1 mM) of competitor
(K+). Of the models that were
tested, those that require that two K+ be bound before transport
occurs gave the worst fits. Random and ordered binding schemes
described the data equally well. General models in which both binding
and transport were allowed to be cooperative yielded parameter errors
larger than the parameters themselves and could not be utilized. Models
that assumed noncooperative transport always showed positive
cooperativity in binding. E327Q and E327L mutated forms of rat
sodium pump; mutagenesis; transport; potassium flux; mechanistic
modeling
THE ROLE OF
Na+-K+-ATPase,
present in most eukaryotic cells, is to maintain the ionic gradients of
Na+ and
K+ (14, 27, 38). It is generally
agreed that the stoichiometry (i.e., the number of internal-to-external
transport sites) is three internal
(Na+) to two external
(K+) to one phosphorylation
site. Unlike the stoichiometry, which is fixed by the number of sites,
the coupling ratio (i.e., the ratio of
Na+ extruded to
K+ taken up) can vary depending on
ionic conditions (25). Under normal physiological conditions, the
coupling ratio is three Na+ pumped
out to two K+ pumped into the
cells.
The
Na+-K+-ATPase
shows a high degree of homology to other P-type ATPases, which include
the sarcoplasmic reticulum
Ca2+-ATPase and the
H+-K+-ATPase,
found in stomach and colon (17). Site-directed mutagenesis studies of
the sarcoplasmic reticulum
Ca2+-ATPase (7) have suggested
that six polar, oxygen-containing residues, predicted to reside within
the transmembrane domains, appear to comprise the high-affinity binding
site for Ca2+. Four of these
residues are conserved in the
Na+-K+-ATPase,
where they correspond in the rat
Both chemical modification (15) and site-directed mutagenesis (27) have
been used to elucidate the mechanism by which Na+ and
K+ are transported against their
concentration gradients and to identify and locate specific amino acids
involved in this transport process.
N, N'-dicyclohexylcarbodiimide
has been used to modify the carboxyl side chains of Glu and Asp. The
results of these studies suggested that the conserved Glu,
corresponding to residues 953 and 327 of rat
Lingrel and co-workers (35, 40) have developed a strategy to study
site-directed mutations in
Na+-K+-ATPase.
This consists of introducing substitutions into a cDNA for a
ouabain-insensitive enzyme, followed by transfection of the cDNA into
HeLa cells, which have an endogenous enzyme that is ouabain sensitive
(35). Cell growth in the presence of 1 µM ouabain is then used as an
indication that the transfected enzyme is capable of functioning as a
pump. Using site-directed mutagenesis, Van Huysse and Lingrel (40)
showed that Glu-955 and Glu-956 of the rat
Materials.
86Rb+
was purchased from DuPont-NEN. The specific radioactivity varied from
~2 to 10 mCi/mg. Ouabain and furosemide were from Calbiochem or
Sigma. Scintillation fluid (Scintiverse BD) was from Fisher. Cell
culture supplies were purchased from GIBCO, Life Technologies, Sigma,
and Fisher. All other reagents [NaCl, KCl,
tris(hydroxymethyl)aminomethane (Tris) base, HCl, phosphoric acid, and
MgCl2] were from Fisher.
Mutagenesis and cloning.
The mutated rat Culture of HeLa cells.
Wild-type HeLa cells and HeLa cells transfected with the rat
Rb+ uptake
assay and ouabain dose response.
Native HeLa cells transfected with rat
Rb+ uptake
competition assay.
For routine experiments, all solutions contained 1 µM ouabain and 1 mM furosemide. Cells were preincubated in PBS containing ouabain and
furosemide for 30 min at 37°C. The cells were rinsed with
K+-free PBS (in mM: 135 NaCl, 0.5 CaCl2, 0.5 MgCl2, 5 glucose, 6.5 Na2HPO4,
and 1.5 NaH2PO4)
or K+-free
low-Na+ PBS (in mM: 135 choline
chloride, 0.5 CaCl2, 0.5 MgCl2, 5 glucose, 6.5 Na2HPO4,
and 1.5 NaH2PO4).
The cells were then changed to prewarmed PBS or
low-Na+ PBS varying in
[K+] as indicated.
86Rb+
was immediately added and incubated for various times up to 30 min.
Because these rates of uptake were always linear up to 15 min for all
combinations of external ion concentrations, the majority of the
experiments were carried out for a single time of 10 min. Cells were
rinsed and extracted as described in the previous paragraph. In some
experiments, 4 mM monensin, a Na+
ionophore, was used during the K+
competition assays to stabilize the internal
[Na+] during the
course of the incubation.
Measurement of internal
[Na+].
To determine the amount of internal
Na+, cells were grown on 100-mm
dishes and treated exactly as above, but without
86Rb+.
After 10 min, the cells were washed with an ice-cold solution containing (in mM) 135 choline chloride, 5 glucose, 0.5 MgCl2, 0.5 CaCl2, and 15 Tris phosphate (pH
7.4). The cells were then scraped from the plates and suspended in
water. The [Na+] of
the lysates was measured using an International Laboratories IL353
double-beam atomic absorption spectrophotometer. The concentrations were normalized to protein levels determined by the method of Lowry et
al. (28).
Data analysis.
Data were plotted and curve fits obtained using KaleidaGraph by
Abelbeck Software. The KaleidaGraph program uses the
Levenberg-Marquardt algorithm for nonlinear curve fitting. Parameter
values were constrained to be nonnegative. Starting values for the
parameters were determined from examination of the plots and
systematically varied. The initial estimates did not affect the
parameter values obtained on convergence. Equations for the curve fits
are given in the APPENDIX. Nonspecific uptake is defined as the amount of
86Rb+
associated with the cells in the presence of excess unlabeled competing
ligand (100 mM KCl) and was obtained in some cases by direct
measurement or, in the case in which
K+ was varied over a wide range,
by fits to the models. The use of "constant percent" error is
well established for weighting radioligand binding data (29). The data
of the present study were therefore weighted using the reciprocals of
the weighting factors (w) calculated
from the equation
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
Appendix
References
2 had lower apparent affinities for the first K+ bound than did
wild-type rat
2 modified to be
ouabain resistant. The mutations did not affect the apparent affinity
of the second K+ bound. Models
that assumed noncooperativity in binding always showed positively
cooperative transport, i.e., enzymes with two K+ bound had a higher flux than
those with one K+ bound. Increases
in external Na+ decreased the
apparent affinity for K+ for all
models and decreased the ratio of the apparent influx rate constants
for E327L.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
Appendix
References
2-isoform to Glu-327 and
Glu-778, Thr-806, and Asp-807.
2-isoform, are sites of
modification by
N, N'-dicyclohexylcarbodiimide
(15) and thus potential cation binding sites. Another
carboxyl-modifying reagent, 4-(diazomethyl)-7-(diethylamino)coumarine, identified Glu-779 (corresponding to residue 778 in rat
2) as a possible cation
binding site (2).
1-subunit (corresponding to
Glu-952 and Glu-953 of rat
2-subunit) are not essential
for enzyme function and that substitution of Gln or Asp at these
positions had very little effect on the cation dependence of ATPase
activity. Substitution in rat
2
modified to be ouabain resistant (rat
2*; see
Mutagenesis and cloning below) of
Glu-778 and Asp-807 with Leu and of Asp-803 with Leu, Asn, or Glu
caused inactivation of the enzyme (19). Substitution of Asp-925 with
Leu or Asn yielded functional enzymes. The Leu substitution caused a
twofold increase in the concentration of K+ that stimulated
enzymatic activity 50% (K1/2), but the Asn substitution
decreased the
K1/2 for
K+. Substitution of Glu 327 with
Ala or Asp failed to yield functional enzymes, whereas
substitution of this position with Gln or Leu yielded functional
enzymes with lower apparent affinities for both
Na+ and
K+ (9, 19, 41). Thus Glu-327 and
Glu-778 and Asp-803, Asp-807, and Asp-925 of rat
2 might comprise at least a
portion of the cation binding site. The purpose of the present study
was to determine the effects of mutations at Glu-327 on the ability of
the mutated enzyme to pump Rb+
into intact cells.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
Appendix
References
2-isoform
referred to as rat
2* has been
modified by the substitution of Arg for Leu at residue 111 and Asp for
Asn at residue 122 (using the rat
2 numbering system) at the
borders of the first intracellular domain (34). This makes rat
2* resistant to ouabain
[concentration for 50% inhibition (IC50) is ~50 µM].
Site-directed mutagenesis was used to make further mutations at
position 327 as previously described (19). E327Q signifies that Glu-327
(using the rat
2 numbering
system) has been replaced by Gln. E327L signifies that Glu-327 has been replaced by Leu.
2* mutants were maintained in
Dulbecco's modified Eagle's medium with 10% calf serum, 100 U/ml
penicillin, 0.1 mg/ml streptomycin, and 250 ng/ml amphotericin B,
incubated at 37°C in a 5% CO2
atmosphere. Unless otherwise indicated, transfected cells were
maintained in 1 µM ouabain.
2* were plated at 3 × 104 cells/ml in 24-well tissue
culture plates (1 ml/well). The effect of ouabain on
86Rb+
uptake by cells transfected with rat
2* was determined. Cells were
incubated until about 80% confluent (both in the absence and presence
of 1 µM ouabain) and then rinsed with phosphate-buffered saline (PBS;
in mM: 135 NaCl, 3.5 KCl, 0.5 CaCl2, 0.5 MgCl2, 5 glucose, 6.5 Na2HPO4,
and 1.5 KH2PO4)
and incubated with PBS containing the indicated ouabain concentrations
for 30 min at 37°C.
86Rb+
was then added at ~2 µCi/ml, and cells were incubated for 10 min at
37°C. The concentration of
86Rb+
([86Rb+])
typically ranged from 2 to 15 µM. The incubation was stopped by
submerging the plate in an ice-cold solution of 0.9% NaCl and 5 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (pH 7.4). The wells were then rinsed eight times in this solution.
Total rinse time was <1 min. Cells were extracted with 0.5 ml of 0.2 N NaOH for 1 h and then neutralized with HCl before counting. Samples
were counted in a Packard Tricarb liquid scintillation analyzer (model
2000CA), which has an efficiency for
86Rb+
of 97%. Each data point represents the average of the radioactivity present in four separate wells. Protein concentration was determined by
the method of Lowry et al. (28).
where
CPM is the mean amount of radioactivity taken up per well for
quadruplicate determinations at each
[K+]. Suspected
outliers (<1% of total replications) among the quadruplicates were
discarded on the basis of a Q-test
(16). The coefficient C of
Eq. 1 was determined by an error analysis of several months of experiments.
From a total of 885 measurements of
Rb+ uptake, each determined in
quadruplicate, the square of the SD for these measurements was plotted
against the square of the mean CPM bound. A fit to
Eq. 1 gave the value 0.00928 for C
(equivalent to a constant percent error of 9.63%). The goodness of fit
was assessed by comparing the data to the curve predicted by the model. It was evaluated in terms of the size of the parameter errors, the
(1)
2 value, and the number of
runs, where a run is the change of sign of the residual. The greater
the number of runs, the better the fit. The probability that the
distribution of the runs was random was determined using published
tables (8). Student's t-test was used
to determine that parameter values were statistically different.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The experiments reported here assessed the effect of external K+ on the steady-state net uptake of 86Rb+ by HeLa cells. The term "uptake" is used to describe the radioactivity [in counts/min (cpm) of 86Rb+] accumulated by the cells in 10 min. Because the apparent affinities for activation of ATPase activity by the two ions are virtually identical (4), it has been assumed that K+ is a congener of Rb+. If it is assumed that the apparent binding constants for transport and the influx rate constants for Rb+ and K+ are also identical, one can estimate the steady-state unidirectional influx of the sum of 86Rb+ and unlabeled K+, referred to in this article as "total influx." To calculate the unidirectional influx of K+ plus 86Rb+, it is necessary to calculate the specific radioactivity at each [K+]. The assumption here is that adding K+ is equivalent to adding unlabeled Rb+. The specific radioactivity as a function of [K+] is equal to the specific radioactivity of carrier-free 86Rb+ multiplied times the factor [86Rb+]/([86Rb+]+[K]). The uptake of 86Rb+ can be converted to total influx of K+ plus 86Rb+ by dividing the uptake (units of cpm/10 min) by the specific radioactivity (units of cpm/pmol) at each [K+]. If contaminating K+, which might be present in the water and reagents (estimated to be, at the highest, 5 µM), were comparable to the [86Rb+] (2-30 µM), it could affect this calculation. We emphasize that this calculated total influx is not the net rate of K+ transport, since it does not take efflux of K+ or 86Rb+ into account. The efflux of 86Rb+ will be negligible during the assay period because of the high [K+] in the cell. There may be, however, some K+ efflux, as the result of K+-K+ exchange. Influx rate constants, used in the expressions for uptake (U), are assumed to include all steps beyond the binding of the ion to the external site, i.e., occlusion and deocclusion.
|
The rat 2*-isoform is a
modified form of
Na+-K+-ATPase,
designed to be insensitive to ouabain so that functional activity of the endogenous HeLa enzyme (which has a high affinity for ouabain) can
be eliminated by use of low concentrations of ouabain. Preliminary experiments with the parental HeLa line were carried out to determine a
concentration of ouabain that would eliminate
86Rb+
uptake due to the endogenous HeLa
Na+-K+-ATPase.
Uptake of
86Rb+
is significantly inhibited by increasing concentrations of ouabain, with an IC50 of ~140 nM (Fig.
1). At saturating
concentrations of ouabain, however, there is still significant
86Rb+
uptake. A transport system that could be responsible for this ouabain-independent
86Rb+
uptake is the
Na+-K+-Cl
cotransporter (24). This cotransporter is inhibited by bumetanide or
furosemide. Figure 1 shows that, in the presence of 1 mM furosemide, essentially all
86Rb+
uptake in parental HeLa cells was blocked by the addition of 1 µM
ouabain (IC50 for ouabain is 244 nM). Approximately 40% of the
86Rb+
uptake is sensitive to furosemide but insensitive to ouabain. The
IC50 for inhibition of
86Rb+
uptake by ouabain in these experiments, 140-240 nM, is consistent with the known sensitivity of the human
1-isoform present in HeLa cells. Subsequent experiments were carried out in the presence of
1 mM furosemide plus 1 µM ouabain to eliminate (>95%) uptake by
endogenous HeLa
Na+-K+-ATPase.
Figure 2 shows a ouabain dose-response
curve for HeLa cells transfected with the rat
2* cDNA and cultured in the
absence or presence of 1 µM ouabain. Uptake of
86Rb+
was followed in the presence of 1 mM furosemide and 5 mM
K+. The dose-response curve for
the cells grown in the absence of ouabain is clearly biphasic, with
IC50 values for the two components of 70 ± 2 nM and 240 ± 60 µM. The higher affinity
component is the endogenous HeLa
Na+-K+-ATPase,
since its IC50 is close to that
for HeLa cells alone (cf. Fig. 1). For the cells grown in the presence
of 1 µM ouabain, uptake by the endogenous HeLa
Na+-K+-ATPase
is inhibited, and therefore only one component, with an IC50 of 150 ± 30 µM, is detected, consistent with it being the transfected
ouabain-insensitive rat
2*
Na+-K+-ATPase.
The magnitudes of the components are approximately the same, suggesting
that the amount, or at least the activity, of the transfected enzyme is
equal to the endogenous HeLa
Na+-K+-ATPase.
Thus the
86Rb+
uptake observed in the presence of 1 mM furosemide and 1 µM ouabain represents uptake by the rat
2*
(transfected) enzyme.
|
The effect of varying external
[Na+] and
[K+] on uptake
kinetics was examined over a 20-min period. Figure
3 indicates that in the presence of 15 mM
external Na+ (low-Na PBS), the
time course of
86Rb+
uptake into HeLa cells was linear for at least 15 min at all [K+] tested. Figure 3
shows the rate of uptake of
86Rb+
at three different external
[K+]. The highest rate
of uptake is observed at 0.5 mM
K+, with lower rates observed both
at 0.01 mM and at 4 mM. The intercepts of the plots were not different
from those of nonspecific uptake determined in the presence of 100 mM
K+, suggesting that there is
neither a lag time nor an initial burst in the uptake. The rate of
uptake in the presence of 150 mM external Na+ was also linear. The cells
used in the experiments shown were transfected with E327Q; likewise,
cells transfected with either rat
2* or E327L (in 15 and 150 mM
Na+) also showed this linear
relationship at a variety of external [K+] (data not shown).
Having demonstrated the linearity of the uptake over the range of
external [Na+] and
[K+] employed,
subsequent experiments were done at a single time point of 10 min.
|
Figure 4 shows uptake of trace amounts of 86Rb+ in the presence of increasing amounts of K+ (K+ competition curve). The data clearly do not fit to a conventional Hill equation, since the competition curve predicted by the Hill equation is monophasic. As K+ is increased from the lowest concentrations, the amount of 86Rb+ uptake in 10 min increases. As the concentration of competitor continues to increase, there is a decrease in 86Rb+ uptake. This is consistent with Fig. 3, which shows that, at the lowest external [K+] shown, there was a relatively low rate of 86Rb+ uptake. At 0.5 mM external K+, the peak of stimulation under the conditions of this experiment (15 mM Na+), the highest rate of 86Rb+ uptake is observed.
|
Changing the external K+ solution to anything other than 5 mM could have caused an immediate change in membrane potential. Although the degree of change is not expected to have a significant effect on the activity of the pump (36), any change in membrane potential could, in theory, be followed by a slower increase in internal [Na+]. To test the possibility that the internal [Na+] was changing over the time course of our experiments, the effect of external K+ on internal Na+ was monitored using atomic absorption spectroscopy. The [Na+] (normalized to protein concentration) does not vary significantly over the range of [K+] in which the peak of stimulation of uptake is observed (Fig. 5). The results shown are for E327Q and are analogous to those obtained for all cell lines investigated. Thus the observed stimulation of uptake cannot be explained by changes in internal [Na+]. Further evidence for this conclusion was obtained by conducting a K+ competition experiment in the presence of 4 µM monensin (data not shown). The degree of stimulation of uptake in the presence of monensin and 15 mM external Na+ was comparable to the stimulation in the absence of monensin. This suggests that stimulation of uptake was not a result of changes in internal [Na+]. In addition, the linearity of Fig. 3 is consistent with the concept that we have maintained steady state over the 10-min time period of the experiments. If the uptake were increasing over a 10-min period, one would expect the curve to be parabolic.
|
To determine whether the nature of the external competitor makes a difference in the apparent stimulation of Rb+ uptake, the competing K+ was replaced by unlabeled Rb+. The dissociation constants determined with unlabeled Rb+ were within 1 SD of those determined with K+ as the competitor (data not shown), suggesting that Rb+ and K+ are interacting with the binding site with similar affinities.
In all the experiments so far described, the procedure for varying the [K+] was that a K+-free PBS solution was made. Various amounts of a 1 M KCl solution were added to this to achieve the specific [K+]; thus ionic strength varied somewhat in different samples. To test whether these variations in ionic strength had any effect on the dissociation constants, an experiment was carried out in which ionic strength was maintained constant by addition of choline chloride to the incubation solutions (choline chloride + KCl = 50 mM). The values for the dissociation constants determined in this experiment were no different from those seen in the routine experimental design (data not shown).
To test whether the stimulation of 86Rb+ uptake by K+ is due to the activity of the Na+-K+-ATPase, the effect of ouabain on the system was determined (Fig. 6). When the effect of K+ on 86Rb+ uptake was determined in the presence of 1 µM ouabain, the standard conditions for these experiments, stimulation was observed. In the presence of 10 mM ouabain, the 86Rb+ uptake is reduced almost to background, indicating that the stimulating component is due to activity of the Na+-K+-ATPase.
|
The degree of stimulation of
86Rb+
uptake by K+ is greater with the
E327Q mutation than with rat 2*
(Figs. 5 and 7 vs. Figs. 4 and 6). We
therefore attempted to fit the data to models that predict both
stimulatory and inhibitory phases. We considered 11 models, which are
described in detail in the APPENDIX.
Two models (see DISCUSSION) were
eliminated because they did not show stimulation of uptake. Fits to the
remaining nine models were applied to data obtained from eight separate
experiments with mutant E327Q (external Na+ = 150 mM) and are shown in
Table 1. The
2 values and the number of runs
are also shown in Table 1. The fact that the
2 values for our fits were
approximately equal to the degrees of freedom (Table 1) indicates that
the weighting scheme employed is appropriate (33). The data set used in
Fig. 7 is experiment 6 (Table 1).
|
|
Figure 7A shows fits to the three
models in which the binding of K+
is described in terms of a single dissociation constant
(K1). The
K1 for E327Q
usually had a value of 1-2 mM in the presence of 150 mM
Na+. These three models assume
either infinitely cooperative binding (model
III) or infinitely cooperative
transport with noncooperative binding
(models
IC2 and
IIC2). For all three models,
transport by definition occurs only when two cations
(K+ or
Rb+) are bound. The shapes of
the fits are similar to the actual data, and the errors in the
parameters are reasonable. The
2 values are, however,
unacceptably high (>100), along with a very low number of runs (Table
1), and the models were therefore rejected on this basis.
When the possibility of infinitely cooperative transport with
cooperative binding was allowed
(models
IC1 and
IIC1), the unrestrained weighted
fits indicated negative cooperativity, i.e., the binding of the second
K+
(K3 = 40-70
mM) had a much lower apparent affinity than the first (K1 = 0.01-0.02 mM). The shape of the curve was very different from the
data points (Fig. 7B). With the
exceptions of experiments 4 and
8 (Table 1), these fits had very large
2 values and a low number of runs compared with the
models that assume that enzyme with either one or two
K+ bound can transport. With some
data sets, under some conditions (e.g., nonweighted fits, exclusion of
nonspecific binding from the fits), convergence could also be obtained
in which the binding was positively cooperative. These fits, however,
also had large
2 values and
unacceptable errors in the parameters. These infinitely cooperative
transport models were therefore rejected.
Figure 7C shows fits to random and
ordered models (models
IA1 and
IIA1) in which transport is
noncooperative. On the basis of criteria of parameter errors,
2, and runs, these two models
clearly fit well to all the data. For these two models, the binding is
always positively cooperative. Figure
7D shows fits to cooperative transport
models (models
IB1 and
IIB1;
f1
f2;
f = f2/f1;
where f1 is the
flux constant for the singly occupied species and
f2 is the flux
constant for the doubly occupied species, with units of
time
1, and
f is the ratio of influx rate
constants) in which the binding of
K+ is assumed to be noncooperative
(K1 = K3). The
f is always >1. That is, doubly
occupied enzyme transports faster than singly occupied enzyme. Table
2 summarizes the analyses of the wild type
and E327 mutants in terms of binding constants or in terms of varying
ratios of influx rate constants.
|
Of the models that we tested, whether cooperative binding or cooperative transport, the only models that fit the data were models that allow transport with one or two K+ bound (variable coupling models). In all of the variable coupling models, the numerator for the equation for transport contains both [K+] and [K+]2 terms. In all of the fixed ratio models (models that allow transport only when 2 K+ are bound), the numerator contains only [K+]2 terms. Thus, at [K+] much less than the dissociation constant the total influx is linearly dependent on [K+], whereas in the fixed ratio models the total influx is linearly dependent on [K+]2. Data from uptake experiments were therefore transformed to total influx and plotted against [K+] and [K+]2. Figure 8 shows that uptake is a linear function of K+, consistent with variable ratio models. Identical results were obtained with the other mutants.
|
The effect of external Na+ on 86Rb+ uptake was determined. Experiments were carried out at an external [Na+] of 15 mM rather than the 150 mM present in PBS. Figure 9 shows the results of lowering external [Na+] on 86Rb+ uptake by the E327Q mutant: a higher level of 86Rb+ uptake was observed at the lower [Na+]. Whether the data were fit to the cooperative binding model (model IIA1) or cooperative transport model (model IIB1), the apparent affinity for K+ was increased at the lower [Na+]. These results are consistent with the fact that external Na+ competes with external K+ for transport. Similar effects were seen with the other transfectants tested. In all cases, lowering the external Na+ decreased the apparent binding constant(s) for K+ (Table 2).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The 86Rb+ uptake methodology used for these experiments has a very good signal-to-noise ratio; in the presence of furosemide and ouabain, 86Rb+ uptake is essentially completely inhibited to the nonspecific level (Figs. 1 and 2). In addition, the utilization of intact cells simplifies interpretation of the results, since, unlike fragmented membrane preparations, the concentrations of ions on the extracellular side can be varied independently of the concentrations on the intracellular side. This enables testing of more complex models (cf. APPENDIX) of transport than has previously been done with mutated Na+-K+-ATPase. The primary experimental result that had to be rationalized was the stimulation of 86Rb+ uptake at low concentrations of unlabeled competitor (Figs. 4-7). The peak of stimulation of 86Rb+ uptake occurs at ~1 mM external K+. The subsequent decrease of 86Rb+ taken up occurs because the two sites become occupied by nonradioactive competing ligand.
A portion of the observed stimulation of uptake of
86Rb+
might have resulted from changes in the membrane potential as a result of the variations in external K+.
Such a change could have two consequences, a direct effect of the
membrane potential change on the activity of the pump and/or an
indirect effect on pump activity due to changes in intracellular [Na+]. The latter
possibility was tested by monitoring the internal [Na+] in different
external [K+]. As
shown in Fig. 5, the
[Na+] (normalized to
protein concentration) did not vary significantly over the range of
[K+] in which the peak
of stimulation of uptake is observed. It has been shown that changes in
resting membrane potential independent of changes in internal
Na+ can affect the pump turnover
number. For example, at constant external
K+, depolarization of resting
membrane potential caused a slight monotonic increase in steady-state
pump current (3). On the basis of previous reports (1, 22), we have
estimated the expected membrane potentials at various
[K+] and the expected
changes in current that would result. For rat 2* cells,
86Rb+
uptake is relatively flat up to 0.1 mM and reaches a peak at ~1 mM.
As the external K+ is changed from
0.1 to 1 mM, the change in resting membrane potential would be
approximately
79.4 to
76.4 mV (3.8%). This would, by itself, directly increase the pump current by 7.6% (3). The uptake of
86Rb+
increases by 43% over this same change in
[K+], and the
unidirectional influx increases 16-fold. Therefore, although the change
in membrane potential does directly influence the pump turnover rate,
the effect is small compared with the effect of
K+ on unidirectional flux. Changes
in membrane potential cannot account for the majority of the
stimulation of
86Rb+
uptake that we observe.
A number of models were tested to determine how well they fit the data. In general, the models that we tested describe 86Rb+ uptake in terms of binding constants and influx rate constants. It should be emphasized that the models that were tested are no doubt simplistic compared with the actual transport model. For example, occlusion and/or deocclusion steps could not be included, since the more complex models incorporating these steps contain too many parameters for fitting. The term "noncooperative transport" refers to models in which transport can occur with one or two cations bound to the extracellular site and the influx rate constants are assumed to be the same for doubly and singly occupied enzyme. In these models, the binding can be either cooperative or noncooperative. The term "cooperative transport" refers to models in which either doubly or singly occupied enzyme can transport and the influx rate constants differ; i.e., transport can occur at different rates for singly or doubly occupied enzyme. In addition, the binding can be cooperative or noncooperative. "Infinitely cooperative transport" refers to models in which the flux for singly occupied enzyme is zero. That is, transport takes place only when two K+ are bound to the extracellular side of the pump. We make the assumption that the rate of uptake of K+ is proportional to the amount of K+ bound in a rapid equilibrium manner. This will be true even if the rapid binding of K+ is followed by a slower occlusion step, as suggested by Forbush (10). It should be pointed out that the binding and flux constants of our schemes are not necessarily intrinsic binding constants and may have embedded in them other rate constants of the transport cycle. They should therefore be considered apparent constants.
Uptake is also a function of the actual rate of movement of the bound ions across the membrane, which, in turn, is determined by any rate-limiting step or steps (e.g., occlusion of K+) that follow the binding of K+. Because the influx rate constant for the singly occupied species could be different from that of the doubly occupied species, the uptake rate is a function of the two influx rate constants and the amount of singly occupied and doubly occupied enzyme (E): transport = f1[EK] + f2[KEK]. Because there is a flux term in both the denominator and numerator of the equation used for fitting, one cannot derive absolute values for the rate constants, but only ratios.
Of the 11 models tested, 2 were eliminated because they
could not show stimulation of uptake. These were random and ordered models in which neither transport nor binding was cooperative. Of the
remaining nine models (shown in Table 1), five can be eliminated on the
basis of statistical criteria for goodness of fit, including
2 and the runs tested.
The simplest models that allow stimulation of
86Rb+
uptake are infinitely cooperative transport or infinitely cooperative
binding models in which the binding of
K+ is described by a single
effective dissociation constant (Fig. 7A). For example, early studies (12)
of
86Rb+
uptake were analyzed using either 1)
models that assume the presence of two infinitely cooperative binding
sites, sometimes called simultaneous models, or
2) cooperative transport models
consisting of two identical noninteracting, noncooperative binding
sites. Implicit in the three models above
(models
III,
IC2, and
IIC2; see
APPENDIX) is the concept that the
transport process is infinitely cooperative; i.e., only enzyme that has
two K+ (or
Rb+) bound to external sites can
undergo the transport process. In the infinitely cooperative binding
model, this is because the apparent affinity of the enzyme for a second
K+ is so high that there are
effectively no singly occupied enzyme molecules present. For the
infinitely cooperative transport models, the singly occupied species
influx rate constant equals zero, reflecting no ion movement by singly
occupied enzyme. These cooperative transport models, in which the
binding of K+ is described by a
single effective dissociation constant, did not fit our data (Fig.
7A and Table 1), although the apparent dissociation constant obtained from these fits (0.06-0.10 mM for rat 2* in the presence of 15 mM
external Na+) is not very
different from those obtained from previous studies (0.14-0.40 mM
in the absence of external Na+)
in erythrocytes (13, 31, 32, 42). More recent
86Rb+
uptake studies of rat
2*
reported an apparent binding constant of 0.20 mM in the presence of 10 mM external Na+ (30). Infinitely
cooperative transport in which the binding is cooperative
(models
IC1 and
IIC1) also does not fit our data very well (Fig. 7B). Thus, of the
models that we tested, those that require that external sites of the
enzyme be filled with two K+ (or
Rb+) before transport can occur
(infinitely cooperative transport) do not fit our data, whether the
binding of K+ is cooperative or
noncooperative.
The four remaining models that do adequately fit the data describe the stimulation of 86Rb+ uptake by external K+ either in terms of cooperativity of binding (Fig. 7C) or cooperativity of transport (Fig. 7D). For the binding models described in the APPENDIX, two different possibilities were considered, random or ordered models. In these two binding models, transport can occur whether one or two K+ are bound to the external site. In support of this concept, at limiting [K+], total influx is dependent on [K+] not [K+]2 (Fig. 8). In physical terms, the random model would describe an enzyme with two separate binding sites for K+. The ordered model would suggest a binding pocket in which the two K+ bind in sequence. The random binding model contains one more parameter (an extra binding constant for K+) than the ordered model. It was always necessary to make the simplifying assumption that the two binding constants for K+ had the same apparent affinities to obtain convergence. In this case, the equations for the two binding models are almost identical. Therefore, the random and ordered models gave fits that were almost superimposable, and we cannot distinguish between the two binding models. The remainder of the discussion is therefore limited to the less complex ordered binding model
With the ordered binding model, it is possible to get stimulation of
uptake at low concentration of unlabeled competitor by either positive
cooperativity of binding, positive cooperativity of transport, or a
combination of both. Because we were unable to obtain satisfactory fits
with the most general case (APPENDIX, scheme
2;
K1 K3,
f1
f2), we were
forced to consider the two extreme cases: cooperative binding,
noncooperative transport
(K1
K3,
f1 = f2)
or noncooperative binding, cooperative transport (K1 = K3,
f1
f2). When the
results are described in terms of cooperative binding (Table 2), the
only models that fit the data show positive cooperativity. This
suggests that stimulation of 86Rb+
uptake by external K+ could be a
result of the fact that the binding of the first
K+ (or
Rb+) increases the apparent
affinity of the enzyme for the second ion. Enzyme with either one or
two K+ can transport, and as
external [K+]
increases there will be more molecules capable of transport. This will
cause an increase in the amount of
86Rb+
taken up. In contrast, describing the results in terms of cooperativity of transport (Table 2) suggests that the influx rate constant of the
doubly occupied enzyme is greater than that of the singly occupied
enzyme (f > 1). As the external
[K+] increases, there
are more doubly occupied enzyme molecules capable of this higher rate
of transport; thus there is an increase in 86Rb+
uptake. The stimulation, of course, could also be caused by a combination of cooperativity in both transport and binding.
Data for K+ activation of ATPase
activity (26) were fit best by a model that had two nonequivalent
K+ sites with dissociation
constants (extrapolated to 0 Na+) of 0.2 and 0.1 mM. Recent
studies by Buhler and Apell (5) reject a simultaneous binding of two
K+ to the extracellular face.
Their data allowed a quantitative distinction between the binding of
the first and second K+. Studies
of K+-induced quench of
fluorescein isothiocyanate-labeled enzyme (39) suggested that two
K+ are bound before the
conformational change can occur. The data (39) could then be fit either
by an ordered model in which the binding was negatively cooperative or
by a random binding model in which the two sites were independent and
identical. Our data, in contrast, suggested that transport would occur
with only one external site occupied and that the cooperativity was
positive (K1/K3 = 4 for rat 2*).
Cation transport is a complex process consisting of a series of steps such as binding, occlusion, and actual ion movement. The existence of half-occluded states, i.e., occlusion when only a single ion is bound, has been suggested by Forbush (11) in the development of his flickering gate model. Sachs (37) concluded earlier from steady-state kinetic analysis that movement of a lone 86Rb+ was possible in the absence of external Na+ when external Rb+ was limited. These findings are consistent with the concept that, unlike the stoichiometry, which is fixed by the number of sites, the coupling ratio (i.e., the actual flux of Na+ extrusion and K+ uptake) can vary depending on ionic conditions (25).
Analysis of our 86Rb+ uptake data did not allow us to distinguish between ordered or random binding. A study of inhibition by K+ of [3H]ouabain binding to purified sheep enzyme indicated, however, that an ordered binding model fit the data better than a random binding model (21). Other lines of investigation that distinguish between random and ordered binding of K+ include deocclusion experiments. Forbush (10) has suggested, on the basis of transient measurement of both 22Na+ efflux and 86Rb+ uptake within a single turnover cycle of the enzyme, that Na+-K+-ATPase has two distinct cation sites. He proposed a slow site and a fast site from which Rb+ is released. The release of the occluded ions is slow because a conformational change must occur before Rb+ can be released inside the cell. Because Rb+ in the fast site can exchange with Rb+ in the slow sites, this is consistent with a model in which the two external transport sites are located in the same region of the enzyme in a sort of "binding pocket."
A steady-state model for transport consistent with the findings of Forbush (11) is the ordered model (APPENDIX, model II). One can envision that the external cation transport site consists of a binding pocket into which two K+ (or Rb+) can fit. The first K+ binds with an apparent affinity K1. Binding of the first K+ so alters the force field in the pocket that the second K+ binds with a different apparent affinity, K3. For stimulation by external K+ to be observed in Rb+ uptake, K3 must be lower than K1, i.e., the binding must be positively cooperative. The degree of stimulation is dependent on the ratio of K1 to K3 (Table 2). Alternatively, the stimulation could be due to an increased rate of ion movement that occurs after the second K+ is bound. Finally, stimulation can be explained as some combination of cooperativity of binding and transport.
On the basis of chemical modification studies (15), it had been
hypothesized that Glu-327 participates in coordinating
K+. Site-directed mutagenesis
studies (9, 19, 41) have shown that substitution of Glu-327 of the
2-isoform with either Ala or
Asp yielded enzyme that could not support cell viability (18). In
contrast, substitution with either Gln or Leu yielded a mutated enzyme
that was able to transport cations and maintain cell viability. From
these results, it was concluded that neither the negative charge nor
the carboxyl side chain at position 327 was absolutely essential for
overall functioning of the enzyme. By examination of the cation
activation of ATPase activity, it was shown that, under conditions of
the experiments (30 mM Na+),
mutant E327Q had a twofold lower apparent affinity for
Na+ and a three- to sixfold lower
apparent affinity for K+
(19). Mutant E327L had a fourfold lower apparent affinity for Na+ and a twofold lower apparent
affinity for K+. It was suggested
that the length of the side chain at position 327 might be
important for enzyme function.
The effects of mutation at amino acid 327 as analyzed from 86Rb+ uptake data are consistent with those described previously. Both of the mutants (E327L and E327Q) have a reduced apparent affinity for the first K+. Table 2 provides the values for apparent binding constants that were calculated using the ordered, noncooperative transport model (model IIA1). Although all three cell lines show stimulation of 86Rb+ uptake by external K+, the two mutants show greater stimulation than the wild type, as reflected in higher ratios of K1 to K3. All three lines show positive cooperativity (K3 < K1). Table 2 provides the values of f calculated from the same data using the cooperative transport, noncooperative binding model (model IIB1). Again, compared with wild type, the mutants have a reduced apparent affinity for K+, as reflected in the increase in the average binding constant determined from the fit. In all three lines, the apparent influx rate constant for the doubly occupied enzyme, f2, is greater than that for the singly occupied enzyme, f1 (or f > 1). The reduced apparent affinity for K+ in activation of 86Rb+ uptake in mutants E327L and E327Q is in qualitative agreement with earlier studies of K+ activation of ATPase activity. In addition, our present studies, using intact cells, demonstrate altered apparent cation affinities, specifically for an external cation transport site. It should be emphasized that the binding constants and f are apparent and may have embedded in them other rate constants of the turnover cycle.
The degree of stimulation and the apparent affinity of the enzyme for
K+ are functions of the specific
enzyme (wild type or mutant) and of the external
[Na+] (Table 2). With
the assumption that Na+ is purely
competitive with K+, the apparent
intrinsic affinity of rat 2*
for K+ (extrapolated to 0 external
Na+) would be 0.20 mM for the
cooperative transport model. The apparent intrinsic affinity for
Na+ would be 32 mM. Similar
analyses for mutants E327Q and E327L yielded apparent intrinsic
affinities of 0.32 and 0.4 mM for
K+ and 18 and 8.4 mM for
Na+, respectively. These values
are not very different from each other and are consistent with previous
studies in erythrocytes (13, 31, 32, 42) and fragmented enzyme
preparations (26).
With the assumption that Na+ is a
purely competitive inhibitor of K+
binding, there should be no effect of external
Na+ on
f for the two enzyme species capable
of transport. Although the external
[Na+] does not affect
f for rat
2* and E327Q, there was a
threefold increase in f of E327L on
lowering the [Na+] to
15 mM. This suggests that Na+ is
not simply competing with K+ for
the transport sites in the mutant but plays a more complex role.
Cavieres and Ellory (6), as well as Sachs (37), have suggested the
existence of an allosteric Na+
site that affects
86Rb+
uptake.
Although substitutions E327A and E327D expressed in HeLa cells produce nonfunctional Na+-K+-ATPase (19), the same mutants when expressed in NIH/3T3 cells are able to support [3H]ouabain binding (23), indicating that these mutations are able to insert into the membrane correctly. [3H]ouabain-binding experiments indicate that K+ interacts with the E327A mutant expressed in 3T3 cells with an affinity not very different from wild type (20, 23). K+ also interacts with the E327D mutant as probed by [3H]ouabain binding, although there is an activation rather than an inhibition. It was concluded from these studies that Glu-327 was important for stabilizing a K+-induced conformation within the catalytic cycle of the enzymatic cycle, which is normally not rate limiting. Removal of the charge alters the rate-limiting step. Interestingly, Vilsen (41) has suggested that the E327Q mutation has a large effect on the E2(K)-to-E1 transition.
Our 86Rb+ studies are consistent with these previous studies that suggest that, although Glu-327 is not essential for cation binding, it plays an important role. Although we cannot conclude that Glu-327 directly interacts with K+, it is clear that mutation to Gln or Leu alters the apparent affinity of the enzyme for the external transport site for K+. In addition, the present study has been able to test more complex models of enzyme mechanism than can be tested with isolated membrane preparations. Of the models that were tested, those that require that transport occur only when two K+ are bound gave the worst fits. Models tested that allowed transport to occur with either one or two K+ bound and that, in addition, showed positive cooperativity in either binding or transport gave reasonable fits.
![]() |
APPENDIX |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our data, consistent with previously published data, require models that involve two K+ (Rb+) sites per enzyme molecule. We have considered two binding schemes, random and ordered, as well as three transport schemes, cooperative, noncooperative, and infinitely cooperative. In schemes 1-3 and Eq. A1-A5 below, E represents the enzyme, Rb+ the labeled ligand, and K+ the competing unlabeled ligand. It is assumed that the dissociation constants (Kx) for Rb+ and K+ are identical (e.g., Kx for K+ is equal to Kx for Rb+). For all the models considered, it is assumed that 86Rb+ uptake is proportional to the amount of enzyme that has 86Rb+ bound.
Random Binding Models
The most general binding model, which under certain conditions allows for stimulation of uptake, is the random binding model (model I) shown in scheme 1
![]() |
If one assumes that all cation-containing species are capable of transport of the cation(s), then uptake of 86Rb+ radioactivity (U) is a function of all 86Rb+-containing forms. Mathematically, U = f1[ERb] + f2[RbE] + f3(2[RbERb] + [KERb] + [RbEK]), where f1, f2, and f3 are the first-order kinetic influx rate constants for transport through the membrane.
The maximal amount of uptake
(Umax) is defined to be the
amount of uptake that would occur if all of the enzyme present
(Etotal) contained two
86Rb+.
Mathematically, Umax = 2f3[Etotal].
U and Umax have the units cpm per
10 min. Nonspecific uptake is defined as the constant NS multiplied by
[86Rb+].
Thus the term NS[Rb+]
represents the amount of radioactivity (CPM) associated with the cells
in the presence of a vast excess of specific competitor (K+). With these definitions,
the general equation below (Eq.
A1) represents the radioactive
uptake under steady-state conditions, as described by a random binding
model For model IA1,
K1 = K2 K3 and f1 = f2 = f3; for model
IB1, K1 = K2 = K3 and
f1 = f2
f3.
Case A: noncooperative transport
(f1 = f2 = f3 ).
Equation.
A1 contains eight parameters to be
fit, which proved too many for convergence. To reduce the number of
parameters, simplifying assumptions had to be made. One such assumption
is that f1 = f2 = f3, in which case
the influx rate constants drop out of the equation describing the
uptake. Note that it is assumed transport can occur whether the enzyme
has one or two cations bound. Attempts to fit the data to the general
case (K1 K2) yielded
parameter errors that were much larger than the parameters themselves,
and convergence was obtained only if the initial estimates of
K1 and
K2 were similar.
We therefore made a further simplification that
K1 = K2. Whether one
assumes that K1 = K2 or
K1
K2,
model I predicts stimulation of
Rb+ uptake by low concentrations
of unlabeled cation (K+) only if
K3 is less than
both K1 and
K2 (i.e.,
positively cooperative binding). If it is assumed that
K1 = K2, the amount of
stimulation is dependent on the ratio of
K3 to
K1. The results
of a fit of model
IA1 to an experiment with mutant E327Q
in the presence of 150 mM external
Na+ is shown in Fig.
7C. Binding
model
I with
K1 = K2 = K3 and
f1 = f2 = f3 cannot show
stimulation, does not fit our data, and was not considered further.
Case B: cooperative transport
(f1 = f2 f3 ).
The assumption of this model is that transport can occur when the
enzyme has either one or two cations bound. To reduce the number of
parameters to be fit, it was assumed that
f1 = f2 and that
K1 = K2.
Equation A1 is still the equation used to fit
the data but unlike in case
A yields a ratio of influx rate
constants (f = f3/f1)
as well as binding constants. Attempts to fit the data to the case
K1 = K2
K3 yielded
parameter errors that were much larger than the parameters themselves,
and convergence was obtained only if the initial estimates of
K1 and
K3 were similar. With the assumption that
K1 = K2 = K3, this model
predicts stimulation of Rb+ uptake
by low concentrations of unlabeled cation
(K+) only if
f is >1 (i.e., positively
cooperative transport; Fig 7D).
Case C: infinitely cooperative transport
(f1 = f2 = 0, f3 > 0).
A version of the cooperative transport model assumes that only doubly
occupied forms of the enzyme ([RbERb], [KERb],
[RbEK], and [KEK]) are capable of transport
(infinitely cooperative transport). This is equivalent to assuming that
f1 and
f2 are zero.
Mathematically, U = f3(2[RbERb] + [KERb] + [RbEK]).
Equation A1 can, therefore, be simplified
to the equation used for the curve fitting of
the uptake data. For model
IC1,
K1 = K2 K3; for
model
IC2,
K1 = K2 = K3.
Ordered Binding Models
A simplification of the random model illustrated in model I is an ordered binding model (model II) shown as scheme 2
![]() |
U is a function of all 86Rb+-containing forms. Mathematically, U = f1[ERb] + f2([EKRb] + 2[ERb2]) and Umax = 2f2[Etotal]. With these definitions, the radioactive uptake under steady-state conditions is represented by the general equation
![]() |
![]() |
(A3) |
Case A: noncooperative transport
(f1 = f2 ).
Equation A3 contains six parameters to be fit,
which proved too many for convergence. To reduce the number of
parameters, simplifying assumptions had to be made. One such assumption
is that f1 = f2, in
which case, the influx rate constants drop out of the equation describing the uptake. Again, it is assumed transport can occur whether
the enzyme has one or two cations bound. Stimulation of uptake at low
concentrations of unlabeled cation
(K+) will occur only if
K3 < K1
(model
IIA1). The amount of stimulation will depend on the ratio of
K3 to
K1. The results
of fits to model IIA1 using mutant rat
2* and E327Q in the presence of
150 mM external Na+ are shown in
Figs. 4 and 7C, respectively. Binding
model
II with K1 = K3 cannot show
stimulation, does not fit our data, and was not considered further.
Case B: cooperative transport
(f1 f2 ).
Stimulation of transport can occur when the enzyme has either one or
two cations bound, if
f1 is assumed to
be unequal to f2.
Equation A3 is still the equation used to fit
the data but unlike in case
A yields an
f (=
f2/f1)
as well as binding constants. Attempts to fit the data to the general
case, K1
K3, yielded parameter errors that were larger than the parameters themselves, and
convergence was obtained only if the initial estimates of K1 and
K3 were similar.
If K1 is assumed
to be equal to
K3,
model II predicts stimulation of
Rb+ uptake by low concentrations
of unlabeled cation (K+) only if
f is >1 (i.e., positively
cooperative transport). See Fig 7D.
Case C: infinitely cooperative transport (f1 = 0). It is assumed in this model that only doubly occupied forms of the enzyme (ERb2 and EKRb) are capable of transport (infinitely cooperative transport; f1 = 0). Mathematically, U = f2(2[ERb2] + [EKRb]) and Umax = 2f2[Etotal]. Equation A3 therefore reduces to
![]() |
![]() |
(A4) |
Simultaneous or Infinitely Cooperative Binding
Finally, we considered an infinitely cooperative (or simultaneous) binding model in which the affinity of the second K+ (or Rb+) is so high that no significant amount of the EK or ERb form exists (model III). Model III describes the affinity of K+ in terms of a single effective dissociation constant, K1, as shown in scheme 3
![]() |
![]() |
(A5) |
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Theresa Kuntzweiler, Dr. Terry Kirley, and David Balshaw for helpful discussions and Dr. J. S. Lee for assisting with uptake experiments.
![]() |
FOOTNOTES |
---|
Address for reprint requests: K. Tepperman, Dept. of Biological Sciences, McMicken College of Arts and Sciences, University of Cincinnati, Cincinnati, OH 45221-0006.
Received 12 July 1995; accepted in final form 31 July 1997.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Amorino, G. P.,
and
M. H. Fox.
Heat-induced changes in intracellular sodium and membrane potential: lack of a role in cell killing and thermotolerance.
Rad. Res.
146:
283-292,
1996[Medline].
2.
Arguello, J. M.,
and
J. H. Kaplan.
Glutamate 779, an intramembrane carboxyl, is essential for monovalent cation binding by the Na,K-ATPase.
J. Biol. Chem.
269:
6892-6899,
1994
3.
Bahinski, A.,
M. Nakao,
and
D. Gadsby.
Potassium translocation by the Na+/K+ is voltage insensitive.
Proc. Natl. Acad. Sci. USA
85:
3412-3416,
1988[Abstract].
4.
Bell, M. V.,
F. Tonduer,
and
J. R. Sargent.
The activation of sodium-plus-potassium ion-dependent adenosine triphosphatase from marine teleost gills by univalent cations.
Biochem. J.
163:
185-187,
1977[Medline].
5.
Buhler, R.,
and
H.-J. Apell.
Sequential potassium binding at the extracellular side of the Na,K-pump.
J. Membr. Biol.
145:
165-173,
1995[Medline].
6.
Cavieres, J. D.,
and
J. C. Ellory.
Allosteric inhibition of the sodium pump by external sodium.
Nature
255:
338-340,
1975[Medline].
7.
Clarke, D. M.,
T. W. Loo,
and
D. H. MacLennan.
Functional consequences of alterations to polar amino acids located in the transmembrane domain of the Ca2(+)-ATPase of sarcoplasmic reticulum.
J. Biol. Chem.
265:
6262-6267,
1990
8.
Draper, N. R.,
and
H. Smith.
Applied Regression Analysis. New York: Wiley, 1966.
9.
Feng, J.,
and
J. B. Lingrel.
Analysis of amino acid residues in the H5-H6 transmembrane and extracellular domains of Na+,K+-ATPase subunit identifies threonine 797 as a determinant of ouabain sensitivity.
Biochemistry
33:
4218-4224,
1994[Medline].
10.
Forbush, B.
Na+, K+, and Rb+ movements in a single turnover of the Na/K pump.
Curr. Top. Membr. Transp.
28:
19-39,
1987.
11.
Forbush, B.
Rapid release of 42K or 86Rb from two distinct transport sites on the Na,K-pump in the presence of Pi or vanadate.
J. Biol. Chem.
262:
11116-11127,
1987
12.
Garay, R. P.,
and
P. J. Garrahan.
The interaction of sodium and potassium with the sodium pump in red cells.
J. Physiol. (Lond.)
231:
297-325,
1973[Medline].
13.
Garrahan, P. J.,
and
I. M. Glynn.
The sensitivity of the sodium pump to external sodium.
J. Physiol. (Lond.)
192:
175-188,
1967[Medline].
14.
Glynn, I. M.,
and
S. J. D. Karlish.
Occluded cations in active transport.
Annu. Rev. Biochem.
59:
171-205,
1990[Medline].
15.
Goldshleger, R.,
D. M. Tal,
J. Moorman,
W. D. Stein,
and
S. J. D. Karlish.
Chemical modification of Glu-953 of the chain of Na+,K+-ATPase associated with inactivation of cation occlusion.
Proc. Natl. Acad. Sci. USA
89:
6911-6915,
1992[Abstract].
16.
Gordon, A. J.,
and
R. A. Ford.
The Chemist's Companion. New York: Wiley, 1972.
17.
Horisberger, J.-D.
The Na,K,-ATPase: Structure-Function Relationship. Boca Raton, FL: CRC, 1994.
18.
Jewell, E. A.,
and
J. B. Lingrel.
Comparison of the substrate dependence properties of the rat Na,K-ATPase 1,
2, and
3 isoforms expressed in HeLa cells.
J. Biol. Chem.
266:
16925-16930,
1991
19.
Jewell-Motz, E. A.,
and
J. B. Lingrel.
Site-directed mutagenesis of the Na,K-ATPase: consequences of substitutions of negatively-charged amino acids localized in the transmembrane domains.
Biochemistry
32:
13523-13530,
1993[Medline].
20.
Johnson, C. L.,
T. A. Kuntzweiler,
J. B. Lingrel,
C. G. Johnson,
and
E. T. Wallick.
Glutamic acid 327 in the sheep 1 isoform of Na+,K+-ATPase is a pivotal residue for cation-induced conformational changes.
Biochem. J.
309:
187-194,
1995[Medline].
21.
Johnson, C. L.,
P. J. Schultheis,
J. B. Lingrel,
C. G. Johnson,
and
E. T. Wallick.
Comparison of the effects of potassium on ouabain binding to native and site-directed mutants of Na,K-ATPase.
Arch. Biochem. Biophys.
317:
133-141,
1995[Medline].
22.
Kort, J. J.,
and
G. Koch.
The Na+, K+, 2Cl-cotransport system in HeLa cells and HeLa cell mutants exhibiting and altered efflux pathway.
J. Cell. Physiol.
141:
181-190,
1989[Medline].
23.
Kuntzweiler, T. A.,
E. T. Wallick,
C. L. Johnson,
and
J. B. Lingrel.
Glutamic acid 327 in the sheep 1 isoform of Na+,K+-ATPase stabilizes a K+-induced conformational change.
J. Biol. Chem.
270:
2993-3000,
1995
24.
Lauf, P. K.,
J. Bauer,
N. C. Adragna,
A. Fujise,
M. M. Zade-Oppen,
K. H. Ryu,
and
E. Delpire.
Erythrocyte K-Cl cotransport: properties and regulation.
Am. J. Physiol.
263 (Cell Physiol. 32):
C917-C932,
1992
25.
Lauger, P.
Electrogenic Ion Pumps. Sunderland, MA: Sinauer, 1991.
26.
Lindenmayer, G. E.,
A. Schwartz,
and
H. K. Thompson, Jr.
A kinetic description for sodium and potassium effects on (Na+ + K+)-adenosine triphosphatase: a model for a two-nonequivalent site potassium activation and an analysis of multiequivalent site models for sodium activation.
J. Physiol. (Lond.)
236:
1-28,
1974[Medline].
27.
Lingrel, J. B.,
and
T. Kuntzweiler.
Na+,K+-ATPase.
J. Biol. Chem.
269:
19659-19662,
1994
28.
Lowry, O. H.,
N. J. Rosebrough,
A. L. Farr,
and
R. J. Randall.
Protein measurement with the Folin phenol reagent.
J. Biol. Chem.
193:
265-275,
1951
29.
Munson, P. J.,
and
D. Rodbard.
Ligand: a versatile computerized approach for characterization of ligand-binding systems.
Anal. Biochem.
107:
220-239,
1980[Medline].
30.
Munzer, J. S.,
S. E. Daly,
E. A. Jewell-Motz,
J. B. Lingrel,
and
R. Blostein.
Tissue- and isoform-specific kinetic behavior of the Na,K-ATPase.
J. Biol. Chem.
269:
16668-16676,
1994
31.
Post, R. L.,
C. R. Merritt,
C. R. Kinsolving,
and
C. D. Albright.
Membrane adenosinetriphosphatase as a participant in the active transport of sodium and potassium in human erythrocyte.
J. Biol. Chem.
235:
1796-1802,
1960[Medline].
32.
Preistland, R. N.,
and
R. Whittam.
The influence of external sodium ions on the sodium pump in erythrocytes.
Biochem. J.
109:
369-374,
1968[Medline].
33.
Press, W. H.,
S. A. Teukolsky,
W. T. Vetterling,
and
B. P. Flannery.
Numerical Recipes. Cambridge, UK: Cambridge University Press, 1992.
34.
Price, E. M.,
and
J. B. Lingrel.
Structure-function relationships in the Na,K-ATPase subunit: site-directed mutagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates a ouabain-resistant enzyme.
Biochemistry
27:
8400-8408,
1988[Medline].
35.
Price, E. M.,
D. A. Rice,
and
J. B. Lingrel.
Site-directed mutagenesis of the border residues from the H1-H2 extracellular domain of the subunit.
J. Biol. Chem.
265:
6638-6641,
1990
36.
Rakowski, R. F.,
D. C. Gadsby,
and
P. De Weer.
Voltage dependence of the Na/K pump.
J. Membr. Biol.
155:
105-112,
1997[Medline].
37.
Sachs, J. R.
Inhibition of the Na-K pump by external sodium.
J. Physiol. (Lond.)
264:
449-470,
1977[Abstract].
38.
Skou, J. C.,
and
M. Esmann.
The Na,K-ATPase.
J. Bioenerg. Biomembr.
24:
249-261,
1992[Medline].
39.
Smirnova, I. N.,
and
L. D. Faller.
Mechanism of K+ interaction with fluorescein 5'-isothiocyanate-modified Na+,K+-ATPase.
J. Biol. Chem.
268:
16120-16123,
1993
40.
Van Huysse, J. W.,
and
J. B. Lingrel.
Nonpolar amino acid substitutions of potential cation binding residues Glu-955 and Glu-956 of the rat 1 isoform of Na+,K+-ATPase.
Cell. Mol. Biol. Res.
39:
497-507,
1993[Medline].
41.
Vilsen, B.
Glutamate 329 located in the fourth transmembrane segment of the -subunit of the rat kidney Na+,K+-ATPase is not an essential residue for active transport of sodium and potassium ions.
Biochemistry
32:
13340-13349,
1993[Medline].
42.
Whittam, R.,
and
M. E. Ager.
Vectorial aspects of adenosine-triphosphatase in relation to active cation transport.
Biochem. J.
93:
337-348,
1964[Medline].