1 Department of Physiology and Biophysics, Keck School of Medicine, University of Southern California, Los Angeles, California 90033; 2 Department of Molecular Genetics, Biochemistry, and Microbiology, College of Medicine, University of Cincinnati, Cincinnati, Ohio 45267-0524; and 3 Laboratory of Muscle Research and Molecular Cardiology, Clinic III of Internal Medicine, University of Cologne, 50924 Cologne, Germany
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ABSTRACT |
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Human
Na+-K+-ATPase
1
1,
2
1, and
3
1
heterodimers were expressed individually in yeast, and ouabain
binding and ATP hydrolysis were measured in membrane fractions. The
ouabain equilibrium dissociation constant was 13-17 nM for
1
1 and
3
1
at 37°C and 32 nM for
2
1, indicating
that the human
-subunit isoforms have a similar high affinity for
cardiac glycosides. K0.5 values for antagonism of ouabain binding by K+ were ranked in order as follows:
2 (6.3 ± 2.4 mM) >
3
(1.6 ± 0.5 mM)
1 (0.9 ± 0.6 mM),
and K0.5 values for Na+ antagonism
of ouabain binding to all heterodimers were 9.5-13.8 mM. The
molecular turnover for ATP hydrolysis by
1
1 (6,652 min
1) was about
twice as high as that by
3
1 (3,145 min
1). These properties of the human heterodimers
expressed in yeast are in good agreement with properties of the human
Na+-K+-ATPase expressed in Xenopus
oocytes (G Crambert, U Hasler, AT Beggah, C Yu, NN Modyanov, J-D
Horisberger, L Lelievie, and K Geering. J Biol Chem
275: 1976-1986, 2000). In contrast to Na+ pumps
expressed in Xenopus oocytes, the
2
1 complex in yeast membranes was
significantly less stable than
1
1 or
3
1, resulting in a lower functional
expression level. The
2
1 complex was also more easily denatured by SDS than was the
1
1 or the
3
1 complex.
sodium pump; cardiac glycosides; heterologous expression
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INTRODUCTION |
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SODIUM-POTASSIUM-ADENOSINETRIPHOSPHATASE
is a heterodimeric protein consisting of - and
-subunits.
Na+-K+-ATPase is also called the
Na+ pump and is a ubiquitous protein responsible for
establishing and maintaining the electrochemical gradient for
Na+ and K+ across the plasma membrane
of mammalian cells. The Na+-K+-ATPase
is also the therapeutic target for cardiac glycosides. Although the
-subunit contains the amino acids involved in catalytic function and
the ion, nucleotide, and cardiac glycoside binding sites, the function
of the
-subunit is not completely understood. The
-subunit is
essential, however, for the normal activity of the enzyme
(21) and is involved in the transport of the functional Na+-K+-ATPase to the plasma membrane. Several
isoforms of the Na+-K+-ATPase have been
identified for
-subunits (
1,
2,
3, and
4) and
-subunits
(
1,
2, and
3)
(2). Although the
1-isoform is expressed in
most tissues,
2 is predominant in skeletal muscle and
can be detected in the brain (34) and heart
(28),
3 is found in excitable tissues
(34), and
4 is found in testis
(29). Similarly, the
1-isoform is fairly
ubiquitous, whereas
2 and
3 are mostly
found in skeletal muscle, neural tissues, lung, and liver (1,
2). In human heart, only
1
1,
2
1, and
3
1 have been found, implicating these heterodimers in the actions of
cardiac glycosides (27).
Besides the tissue-specific expression pattern of Na+ pump
isoforms in physiological conditions, isoform expression is
specifically altered in a tissue-specific manner in diseases such as
hyper- and hypothyroidism, hypokalemia, hypertension, and heart failure (18, 20, 22, 27). Although the functional relevance of tissue- and disease-specific regulation of
Na+-K+-ATPase isoform expression is not always
obvious, the physiological importance of multiple isoforms may be
underscored by the maintenance of multiple isoforms throughout
evolution (2). For example, in heart failure,
Na+ pump expression falls, which may be an adaptive
compensation analogous to inhibiting the pump with cardiac glycosides
(23, 27). In K+-depleted animals, skeletal
muscle 2-isoform expression is severely depressed,
allowing net exit of K+ from the muscle to buffer the fall
in plasma K+ (33). Differences in the
subcellular localization of some isoforms may also indicate specific
functional roles for the different isoforms in certain cells
(24).
Cardiac glycosides are inotropic drugs used to treat heart failure and
atrial fibrillation. They bind specifically to the Na+-K+-ATPase and inhibit its activity. In
cardiomyocytes treated with cardiac glycosides, the resultant increase
in cytoplasmic Na+ results in a reduction in
Ca2+ extrusion by the Na+/Ca2+
exchanger and an increase in sarcoplasmic reticulum Ca2+
storage. During subsequent action potentials in the myocytes, more
Ca2+ is released from the sarcoplasmic reticulum than in
the absence of the drug, leading to a greater contractile force. In
rodents, it is well established that the 1-isoform
is resistant to the binding and pharmacological effects of ouabain,
whereas most other mammalian
1-isoforms bind ouabain
with high affinity. Data for ouabain binding to human heart have been
inconsistent, with some reports concluding that all isoforms of the
-subunit have a similar affinity for the drugs (Ref.
27; Wang J, Velotta JB, McDonough AA, and Farley RA,
unpublished observation) and other reports showing evidence for
multiple ouabain receptors with different affinities (4,
9). Recently, nine combinations of human Na+-K+-ATPase
1-3- and
1-3-isoforms were expressed in Xenopus
oocytes, and the properties of the expressed pumps were compared
(3). All the
-subunit isoforms expressed in
Xenopus oocytes with the
1-subunit had
ouabain dissociation constants (Kd) of
4.5-22 nM. These Kd values are similar to
the value of 18 ± 6 (SD) nM that we measured for ouabain binding
to several human tissues and human cell lines (Wang et al., unpublished
observations), indicating that differences in chemical composition
between human and amphibian cell membranes may not affect the
properties of the pump. Nevertheless, a comparison of biochemical
properties of the pump isoforms expressed in different expression
systems would be useful to validate the results obtained in the
Xenopus oocytes, since human
2- and
3-isoforms are usually expressed in human tissues
together with the
1-isoform.
In the present study, human Na+-K+-ATPase
1
1,
2
1, and
3
1 have been expressed individually in
the yeast Saccharomyces cerevisiae. Yeast cells do not
contain endogenous ouabain-sensitive Na+ pumps, which
allows the assessment of Na+-K+-ATPase
properties without any background activity of host enzyme. Equilibrium
ouabain binding was measured, the apparent affinities of each
complex for Na+, K+, and ATP were obtained from
ouabain binding measurements, and the enzymatic turnover of ATP was
measured for the
1
1 and
3
1 complexes. The results of these
measurements were in good agreement with those obtained for human
Na+-K+-ATPase expressed in Xenopus
oocytes. The
2
1 complex expressed in
yeast was much less stable than the
1
1
and
3
1 complexes, however, possibly
indicating that the lipid environment of the protein is less than
optimal in these cells.
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METHODS |
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Cloning of cDNA of human
Na+-K+-ATPase
isoforms.
Total RNA was prepared from human tissue (heart, brain, and kidney)
samples obtained from the Cooperative Human Tissue Network (Columbus,
OH). First-strand synthesis was primed by oligo(dT) and extended with
Superscript (Life Technology) reverse transcriptase in PCR buffer (50 mM KCl, 10 mM Tris · HCl, 2.5 mM MgCl2, and 0.2 mM
dNTPs). The first strand was then amplified with isoform-specific primers to generate overlapping cDNA products. The PCR products were
subcloned into pBluescript II SK and sequenced with Sequenase 2.0 (US
Biochemical) according to the manufacturer's specification. Mutagenesis reactions were performed using the Quick Change procedure (Stratagene, La Jolla, CA) to correct errors introduced by the PCR.
Unique restriction sites were then utilized to subclone the complete
cDNA into the plasmids pRcCMV (1 and
3)
and pKC4 (
2 and
1).
Construction of yeast expression plasmids YhN1, YhN
2,
YhN
3, and GhN
1.
Human
1 cDNA (3,072 bp) was released from pRcCMV by
restriction digestion with NcoI/DraIII. After
filling in the 5' end and exonuclease treatment of the 3' end (T4 DNA
polymerase) and electroelution, a blunt-ended fragment was obtained
(1-bp untranslated sequence at the 5' end and 15-bp sequence at the 3'
end) that was then ligated (T4 ligase) into the EcoRI
digested yeast expression plasmid YEp1PT (11). The correct
orientation of the insert was confirmed by restriction analysis, and
the coding sequence of
1 was confirmed by automated
sequencing (Biochemical Core Laboratory, University of Southern
California). The resulting yeast expression plasmid in which human
Na+-K+-ATPase
1-isoform is
expressed under the control of yeast PGK promoter was designated
YhN
1.
Transformation of S. cerevisiae and yeast membrane preparation.
Standard yeast media were used throughout the study. The yeast strain
30-4 (MAT , trp1, ura3, Vn2, GAL+) was used for the
coexpression of the human Na+-K+-ATPase
-
and
-isoforms. Yeast cells were cotransformed with YhN
and
GhN
1 plasmids using the lithium acetate procedure as previously described (8). After transfection, yeast cells
were grown at 30°C on selective minimal medium in which galactose was used as the carbohydrate source to induce the expression of
1. Up to eight colonies for each human Na+
pump heterodimer were further propagated in minimal medium to identify
the clones with the highest expression levels of the enzyme by ouabain
binding (see below). Frozen glycerol stocks of the transformants were
stored at
80°C. A microsomal fraction of yeast cell membranes was
prepared as previously described (7). Yeast microsomal
membranes were extracted with 0.1% (wt/vol) SDS as described
previously (6). Protein concentrations were determined by
the method of Lowry et al. (19).
Ouabain binding experiments in yeast membranes.
Initially, the expression levels of the human
Na+-K+-ATPase in yeast were investigated by
[3H]ouabain binding experiments with all clones for each
combination. Between 0.25 and 1 mg of membrane protein were
incubated with 20 nM [3H]ouabain per assay. The reaction
conditions were chosen as previously described (7); the
buffer consisted of 4 mM H3PO4, 4 mM
MgCl2, and 50 mM Tris · HCl (pH 7.4). The assays
were incubated at 37°C for 1 h, chilled on ice/H2O
for 15 min, and pelleted in a microcentrifuge for 15 min at 4°C. The
pellets were rinsed briefly with 0.5 ml of ice-cold water, and pellets
were dissolved in 1% SDS before scintillation counting. Nonspecific
binding was determined by the addition of 1 mM nonradioactive ouabain
and was subtracted from assay values. Mock assays were performed
without [3H]ouabain, and the pellets were solubilized in
1% SDS and assayed for protein recovery. Membranes from untransformed
yeast or yeast transformed with vectors alone showed
[3H]ouabain binding equal to nonspecific binding,
typically ~20 fmol [3H]ouabain/mg microsomal protein.
Clones with the highest specific ouabain binding were used for
subsequent experiments.
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Inhibition of ouabain binding by K+. To estimate the K+ affinity of the human Na+-K+-ATPase isoforms, ouabain binding was performed as described above with the following modifications. The concentration of [3H]ouabain was 5-20 nM, and 0-100 mM KCl was added to the assay. Ionic strength was maintained constant in some experiments with choline chloride; however, at monovalent cation concentrations <100 mM, the addition of choline chloride had no noticeable effect on the results. The concentration of KCl in stock solutions was determined by flame photometry, and the values obtained for the KCl concentration were used for calculations. The binding conditions and experimental procedures were identical to those described above.
Ouabain binding as a function of
Na+ concentration.
Microsomal membranes from yeast expressing human
Na+-K+-ATPase isoforms were washed free of
Na+ by diluting membranes ~25-fold in
Na+-free buffer [25 mM imidazole-HCl and 1 mM EDTA (free
acid), pH 7.4] and pelleting them for 60 min at 100,000 g
at 4°C. The pellet was suspended in Na+-free buffer and
pelleted again before being suspended in a small volume of buffer.
Final concentrations for the reaction were 20 nM
[3H]ouabain, 3 mM MgCl2, 25 mM imidazole-HCl,
pH 7.4, 3 mM ATP-Tris (Sigma), and 0-100 mM NaCl. Ionic strength
was adjusted by complementary concentrations of choline chloride in
some experiments. Microsomal membranes and reaction tubes were
prewarmed for 10 min at 37°C, and the reaction was initiated by the
addition of membranes. Reactions were incubated for 3 min at 37°C and
stopped by transfer to ice/H2O. Bound
[3H]ouabain was separated from free
[3H]ouabain by centrifugation for 15 min at 4°C in a
microcentrifuge and aspiration of the supernatant. The reaction tubes
were rinsed with 0.5 ml of ice-cold distilled water, and pellets were
dissolved in 200 µl of 1% SDS before scintillation counting. Stock
solutions of 10× concentrations of all reagents and washed membranes
were assayed by flame photometry for Na+. In all cases, the
reagents failed to register a measurable amount of Na+,
with a sensitivity of the instrument being 1 mM Na+ minimum.
Ouabain binding as a function of ATP concentration. Ouabain binding as a function of the ATP concentration was performed to obtain a measure for the ATP affinity of the different isoforms. Microsomal membranes from yeast (250 µg) expressing human Na+-K+-ATPase isoforms were incubated in 50 mM imidazole-HCl, pH 7.4, and 5 mM NaN3 with [3H]ouabain (40 nM) and 0-50 µM Na2ATP at 37°C for 5 min. Reactions were stopped by transfer to ice/H2O. Bound [3H]ouabain was separated from free [3H]ouabain by centrifugation for 15 min at 4°C in a microcentrifuge and aspiration of the supernatant. The reaction tubes were rinsed with 1 ml of ice-cold 25 mM imidazole-HCl, pH 7.4, and 1 mM Na2EDTA. Then pellets were dissolved in 200 µl of 1% SDS before scintillation counting. Protein recovery was determined in mock assays without radiolabeled ouabain. Assays were done in duplicate, and specific binding was determined by the subtraction of nonspecific binding (addition of 1 mM nonradioactive ouabain) from total binding.
Coupled assay to determine ATPase activity.
Na+-K+-ATPase activity was measured on
SDS-extracted yeast membranes containing expressed human
Na+-K+-ATPase isoforms using an NADH-coupled
enzyme assay at 37°C. Between 10 and 50 µg of membrane protein were
used for each sample, and the ATPase activity that was inhibited by 10 µM ouabain was identified as Na+-K+-ATPase
activity. Turnover values for 1
1 and
3
1 were obtained by dividing the maximum
ATPase activity by the ouabain binding stoichiometry.
Western blots.
Immunoblot analysis was conducted on samples of microsomal fractions of
yeast membranes from cells that were transformed with the yeast
expression plasmids for one -isoform and the
1-isoform. Membrane fractions from human and rat brain
and dog kidney were prepared as previously described (36)
and were included to verify isoform specificity of antibodies.
Membranes from untransformed cells were included as a negative control.
Yeast membrane protein (100 µg/sample) was resolved by SDS-PAGE and
electrophoretically blotted onto Immobilon P membrane. Blots were
probed with one of the following antibodies: 464.6 against
1 (1:100; obtained from M. Kashgarian, Yale University)
(15), McB2 against
2 (1:100; obtained from
K. Sweadner, Harvard University) (32), anti-TED against
3 (1:200; obtained from T. Pressley, Texas Tech)
(26), or anti-human
1 (obtained from P. Martin-Vassallo, Tenerife, Spain) (36). Blots of
1,
3, and
1 were prepared
and processed as described previously (20) using
125I-labeled protein A and autoradiography for quantitation
of the antibody-antigen complexes. Because of the low expression level of
2, enhanced chemiluminescence (ECL) was used to
visualize this isoform. Exposure times were chosen to optimize signal
detection and were different for each antibody.
Materials.
All enzymes used for molecular cloning were obtained from New England
Biolabs (Beverly, MA) or Boehringer Mannheim and were used as
recommended by the suppliers. Pfu polymerase was obtained from Stratagene. Unlabeled ouabain was purchased from Sigma-Aldrich, and the concentration of ouabain in solution was determined by spectrophotometry using an extinction coefficient of 18.8 mM1 (35). [3H]ouabain was
obtained from NEN Life Science Products (Boston, MA). The specific
activity was determined as described by Wallick and Schwartz
(35) using 5 µg of purified dog kidney membrane protein
for the binding experiment.
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RESULTS |
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Amino acid sequences of human
Na+-K+-ATPase
1-,
2-,
3-, and
1-isoforms.
The cDNA sequences of the Na+-K+-ATPase
isoforms in the yeast expression plasmids were determined to ensure
that no unwanted mutations of the cDNA had occurred during the
subcloning. For
1,
2, and
1, the results were identical to the sequences that have
been previously published (16, 17, 30, 31). In contrast, the
3 cDNA used in this study, which was assembled from
expressed sequence tags 3-5 and 3-6, contains the following
differences in the amino acid sequence compared with the published
human
3 sequence (25): V336L, Y555F, Q557K,
and G583D. The nucleotide sequence differences were found in the cDNA
used as well as in two independent expressed sequence tags, indicating
that there may be a polymorphism of these amino acids. In amino acid
sequence alignments with related proteins, we found that amino acid 336 is a leucine in all mammalian Na+-K+-ATPase
isoforms including human
1 and
2, whereas
valine, which has been previously reported to be at this position of
human
3, was found only in yeast transporters. Amino
acid 555 has been reported to be a tyrosine in human
3,
chicken
3, and human gastric H+-K+-ATPase, but it is a phenylalanine in our
human Na+-K+-ATPase
3 clone, in
rat
3 and pig
3, and in human, rat, pig, and chicken
1 and
2. Amino acid 557 was
reported to be a glutamine only for human
Na+-K+-ATPase
3-isoform and rat
nongastric H+-K+-ATPase, whereas in our clones
we found a lysine, which has also been found in the homologous position
of rat and chicken Na+-K+-ATPase
3-isoform. At this position the amino acid residue is highly variable, with other frequent amino acids being arginine, glutamic acid, and proline. Amino acid 583 is part of the DPPR and
homologous sequences, which have been shown to be highly conserved in
Na+-K+-ATPase and other P-type ATPases
(10). We found that, among the 70 closest related proteins
to human Na+-K+-ATPase
3-isoform, a glycine was reported in this position only for dog
1. All other sequences and the sequence we have
found for human Na+-K+-ATPase
3
contain an aspartic acid at this position. From these comparisons we
conclude that the human
3 clone used in this study is correct.
Expression of human
Na+-K+-ATPase
isoforms in yeast cells.
Untransformed yeast does not bind ouabain; thus acquisition of
high-affinity ouabain binding can be used as an indicator of functional
expression of the human Na+-K+-ATPase in these
cells. Microsomal membranes from up to eight clones for each
combination were screened using 20 nM [3H]ouabain.
Western blotting was used to confirm isoform expression. Results shown
in Fig. 1 demonstrate expression of each
-subunit isoform and
1 in the appropriate yeast
clones. No immunoreactivity was observed in membranes from
untransformed yeast, and isoform specificity of the antibodies was
verified using membranes from kidney and brain. The
2-subunit was expressed at significantly lower levels in
the yeast than the
1- or
3-subunit, and
it was difficult to visualize the
2-subunit on blots
using 125I-protein A, which was used to visualize the
1-,
3-, and
1-subunits. In
Fig. 1,
2 was visualized using ECL. Consistent with the
low expression level, the amount of ouabain bound by yeast clones expressing human Na+-K+-ATPase
2
1 was ~10-fold lower than the amount
bound by clones expressing the
1
1- or
3
1-subunits. The clones for each
individual isoform with the highest amount of ouabain bound were
selected for further analysis.
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Ouabain affinity of human
Na+-K+-ATPase
1
1,
2
1, and
3
1 when separately expressed in yeast
cells.
Equilibrium ouabain binding was performed using microsomal membrane
preparations in the presence of Mg2+ and inorganic
phosphate (Pi). The conditions were chosen so that the
Na+-K+-ATPase was largely in its phosphorylated
state, to which ouabain binds with high affinity. Low concentrations
(0.5-5 nM) of radiolabeled ouabain were used, and the
concentration of unlabeled ouabain was varied. Increasing
concentrations of unlabeled ouabain compete with the radiolabeled
ouabain for binding to the pumps, and the data were fit with a
self-competition model (14). A typical experiment is shown
in Fig. 2, and Table
1 summarizes the equilibrium binding
(Bmax) and Kd values of the three
human
complexes. As predicted by the screening experiments, the
Bmax of ouabain was ~10-fold lower for yeast expressing
2
1 than for cells expressing
1
1 or
3
1. A
comparison of the Kd values of the human
Na+-K+-ATPase isoforms showed that the
1
1 and
3
1 complexes
bind ouabain with about twofold higher affinity than
2
1; the relatively lower affinity of
2
1 for ouabain was also observed in
oocytes expressing human
-subunit isoforms (3). The
Kd values are in agreement with recent
measurements in our laboratory of ouabain binding to human tissues and
cultured human cells expressing different combinations of
Na+-K+-ATPase isoforms (Wang et al.,
unpublished observation) and are 1.5- to 3.6-fold higher than the
Kd values reported for human Na+-K+-ATPase
1-isoforms
expressed in Xenopus oocytes (3).
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Na+,
K+, and ATP affinity of human
Na+-K+- ATPase
1
1,
2
1, and
3
1.
The effects of Na+ and K+ on equilibrium
ouabain binding were measured to estimate the affinity of the pumps for
these cations. Ouabain binding is antagonized by K+ ions,
which compete for E2 with phosphoenzyme formed from Pi according to the following scheme, called the "back-door" reaction. Mg2+ is an essential metal ion cofactor in the reaction
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(1) |
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(2) |
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(3) |
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Instability of human 2
1 heterodimers
expressed in yeast cells.
The reduced Bmax of ouabain by
2
1 complexes compared with
1
1 and
3
1
indicated that fewer functional
2
1 pumps
than other complexes were present in the steady state. The fact that it
was necessary to use ECL to clearly visualize the
2
protein is consistent with this observation. A time course of ouabain binding to
2
1 was measured, and the
results showed that the amount of ouabain bound by
2
1 decreased with incubation time (Fig.
4, squares). In this
experiment, membranes were incubated with 20 nM
[3H]ouabain for up to 6 h before ouabain binding was
measured. Including 5 mM KCl in the buffer (Fig. 4, circles)
reduced the amount of ouabain bound by
2
1
by ~50% after 1.5 h, as expected from the competition between
phosphoenzyme formation and the binding of K+. The amount
of ouabain bound by the
2
1 complex
increased slightly in the presence of KCl between 1.5 and 3.5 h to
a maximum and was constant for
2.5 h thereafter. The effect of KCl on
ouabain binding by
2
1 can be explained by
two mechanisms: 1) KCl antagonizes ouabain binding according
to reaction 1, accounting for the initial reduction in
binding observed at short times compared with the reaction conducted in
the absence of KCl. 2) KCl seems to stabilize the
2
1 complex for
6 h, thereby slowing the
rate of loss of ouabain binding capacity that is seen in the absence of
KCl. Additional evidence for the instability of
2
1 complexes was obtained by SDS
extraction of yeast membranes. For membranes containing
1
1 or
3
1,
the amount of ouabain bound per milligram of membrane protein increased
after extraction with 0.1% SDS, whereas all ouabain binding was lost
after SDS extraction of membranes containing
2
1 (data not shown).
|
ATPase activity.
Ouabain-sensitive ATP hydrolysis was measured in yeast membranes
containing human 1
1 or
3
1 complexes. Because the endogenous yeast ATPase activity is more sensitive to denaturation by SDS than the
Na+-K+-ATPase, the membranes were extracted
with 0.1% SDS to increase the specific
Na+-K+-ATPase activity. It was not possible to
measure ATPase activity for the
2
1
complex, because this complex is denatured by SDS extraction of the
yeast membranes. Figure 5 shows the ATP
concentration dependence of ATPase activity for
1
1. The molecular turnover for ATP
hydrolysis by
1
1 was calculated to be
about twice as high as that for ATP hydrolysis by
3
1 (Table 1), consistent with the
observation by Crambert et al. (3) that the number of
charges per second per molecule transported by
1
1 expressed in Xenopus
oocytes is about four times as large as that transported by
3
1.
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![]() |
DISCUSSION |
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Human Na+-K+-ATPase
1
1,
2
1, and
3
1 were expressed individually in yeast
cells in this study, and biochemical characteristics of each complex
were measured. Because yeast cells do not contain any
ouabain-sensitive ATPase activity or high-affinity receptors for
ouabain, the properties of the expressed
Na+-K+- ATPase could be measured in the
absence of any endogenous background. The heterologous expression of
the different
complexes in yeast provides a convenient system in
which to study the properties of individual
-subunit isoforms. This
is not possible in human tissues, where
2 and
3 are expressed with each other or with
1.
The results of equilibrium ouabain binding measurements indicated that
the affinity of the 1
1 and
3
1 complexes for ouabain is similar and
is slightly higher than the affinity of the
2
1 complex (P < 0.01).
The twofold difference in Kd values between
1/
3 and
2 complexes with
1 would not be apparent in equilibrium binding
measurements in tissues containing
2 with the other
isoforms and is consistent with results from a panel of different human tissues and human cell lines in which only a single population of
ouabain binding sites was detected (Wang et al., unpublished observation). These findings make it unlikely that the difference in
sensitivity to cardiac glycosides observed between patients with
congestive heart failure and normal individuals is due to differences
in the affinity of the Na+-K+-ATPase isoforms
for the drugs. Instead, the increased sensitivity of heart failure
patients is likely to be due to a reduction in the total number of
pumps in the hearts of these patients. A reduction in pump abundance in
failing human hearts compared with nonfailing hearts was recently
documented by ouabain binding and immunoblots (27). This
reduction may itself be a compensatory mechanism designed to increase
Ca2+ loading in the sarcoplasmic reticulum.
The steady-state abundance of pumps expressed by yeast cells was
similar for the 1
1 and
3
1 complexes but was significantly lower
for the
2
1 complex. This result is
probably due to an instability of the
2
1
complex that was manifest as a loss of ouabain binding capacity during
prolonged incubation at 37°C (Fig. 4). The reasons for this
instability are not known; however, because all the
-subunit
isoforms were expressed from the same expression plasmid, it seems
likely that the instability of
2
1 is
related to protein-protein or protein-lipid interactions, rather than differences in transcription or translation. Possibly the yeast membrane does not provide a stable environment for this particular heterodimer, or other protein factors that normally interact with Na+-K+-ATPase in mammalian cells are absent
from yeast and their absence destabilizes the
2
1 complex.
Ouabain binding to the phosphoenzyme formed from Mg2+, and
Pi is competitive with K+. The
K0.5 for K+ antagonism of ouabain
binding to each complex indicates that the
1- and
3-isoforms have a similar affinity for K+
(0.9 ± 0.6 and 1.3 ± 0.1 mM for
1 and
3, respectively) but that the affinity of
2 for K+ is significantly lower (6.3 ± 2.4 mM, P < 0.001). The values for
1
1 and
3
1
are similar to those previously found by Crambert et al.
(3) to antagonize ouabain binding to human
Na+-K+-ATPase expressed in Xenopus
oocytes. The K0.5 for
2
1, however, is about twice as high for
the pump expressed in yeast. The binding of ouabain to
Na+-K+-ATPase is also antagonized by
Na+, and the K0.5 for
Na+ inhibition of ouabain binding to the human
1
1- and
2
1-isoforms expressed in yeast is similar
to the values obtained for Na+ activation of pump current
for these complexes expressed in Xenopus oocytes. For
3
1, the K0.5
obtained for Na+ antagonism of ouabain binding to yeast
membranes is about one-half of that found to activate pump current in
oocytes. The ability of Na+ to support ATP-dependent
phosphorylation of the Na+-K+-ATPase can also
be measured by ouabain binding, and in this assay the
1-
and
3-isoforms were found to have
K0.5 values of 1.5 and 2.8 mM, respectively.
Na+-dependent ouabain binding to the
2-isoform was more complex, however, and was best fit by
the sum of two populations of sites with K0.5 of
0.6 ± 0.1 and 18.6 ± 8.1 mM. These values may reflect the
presence of two conformations of the protein, the normal wild-type and
partially unfolded pumps, consistent with the observed instability of
2
1 in yeast membranes. That the
interaction of Na+ is more sensitive to conformational
differences than the interaction of K+ may be because of
the same structural constraints that distinguish the almost absolute
specificity of Na+-K+-ATPase for
Na+ compared with the relatively weaker selectivity of the
enzyme for K+. The ability of several ions to replace
K+ in Na+-K+-ATPase activity assays
is well known, whereas no other ion can substitute efficiently for
Na+. Consistent with a unique sensitivity of
Na+ interactions to conformational perturbations was the
observation that all isoforms of the
Na+-K+-ATPase
-subunit exhibited a similar
K0.5 for ATP in the same assay.
Both 1
1 and
3
1 expressed in yeast are capable of
hydrolyzing ATP. The molecular turnover of
1
1 expressed in yeast is similar to that
calculated for Na+-K+-ATPase purified from dog
renal medulla in our laboratory and for sheep
Na+-K+-ATPase
1
1
expressed in insect cells (12). This value is
approximately twofold higher than that of
3
1 expressed in yeast. No independent value is available for ATPase activity of human
3
1, but in human
3
1 expressed in Xenopus
oocytes, turnover was ~25% of that in
1
1 (3). Because
3
1 is found in brain (Peng L,
Martin-Vasallo P, and Sweadner KJ, unpublished observation) and heart
(36), the difference in turnover may be physiologically important.
In summary, the human 1-,
2-, and
3-isoforms of Na+-K+-ATPase have
been expressed in functional form in yeast cells with the human
1-subunit, and the biochemical characteristics of each
-subunit were found to agree well with those found for these pumps expressed in Xenopus oocytes. The agreement between
the results obtained from these different expression systems makes it
likely that the properties of the human isoforms in human tissues are
also similar. The ability to study individual isoforms in the absence
of endogenous Na+-K+-ATPase activity makes the
expression of Na+ pumps in yeast an attractive system for
many experiments that cannot be accomplished in human tissues that
express more than one isoform.
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ACKNOWLEDGEMENTS |
---|
The authors are grateful to Dr. Chinh Tran, Dr. David Kane, and Robert Ahlstrom for advice and helpful discussions and to Hoan Tang and Inken Neu for excellent technical assistance.
![]() |
FOOTNOTES |
---|
This work was supported by National Institutes of Health Grants GM-28673 (R. A. Farley) and P01 HL-41496-12 and 5 R01 HL-28573-17 (J. B. Lingrel), an American Heart Association grant-in-aid from the Western States Affiliate (A. A. McDonough), Deutsche Forschungsgemeinschaft Grant Mu 1469/1-1 (J. Müller-Ehmsen), and Köln Fortune (R. H. G. Schwinger).
Address for reprint requests and other correspondence: R. A. Farley, Dept. of Physiology and Biophysics, USC Keck School of Medicine, 1333 San Pablo St., MMR 250, Los Angeles, CA 90033 (E-mail: rfarley{at}hsc.usc.edu).
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.
Received 17 January 2001; accepted in final form 22 May 2001.
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