Ouabain and substrate affinities of human Na+-K+-ATPase alpha 1beta 1, alpha 2beta 1, and alpha 3beta 1 when expressed separately in yeast cells

Jochen Müller-Ehmsen1,3, Padmaja Juvvadi1, Curtis B. Thompson1, Lusine Tumyan1, Michelle Croyle2, Jerry B. Lingrel2, Robert H. G. Schwinger3, Alicia A. McDonough1, and Robert A. Farley1

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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Human Na+-K+-ATPase alpha 1beta 1, alpha 2beta 1, and alpha 3beta 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 alpha 1beta 1 and alpha 3beta 1 at 37°C and 32 nM for alpha 2beta 1, indicating that the human alpha -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: alpha 2 (6.3 ± 2.4 mM) > alpha 3 (1.6 ± 0.5 mM) approx  alpha 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 alpha 1beta 1 (6,652 min-1) was about twice as high as that by alpha 3beta 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 alpha 2beta 1 complex in yeast membranes was significantly less stable than alpha 1beta 1 or alpha 3beta 1, resulting in a lower functional expression level. The alpha 2beta 1 complex was also more easily denatured by SDS than was the alpha 1beta 1 or the alpha 3beta 1 complex.

sodium pump; cardiac glycosides; heterologous expression


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SODIUM-POTASSIUM-ADENOSINETRIPHOSPHATASE is a heterodimeric protein consisting of alpha - and beta -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 alpha -subunit contains the amino acids involved in catalytic function and the ion, nucleotide, and cardiac glycoside binding sites, the function of the beta -subunit is not completely understood. The beta -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 alpha -subunits (alpha 1, alpha 2, alpha 3, and alpha 4) and beta -subunits (beta 1, beta 2, and beta 3) (2). Although the alpha 1-isoform is expressed in most tissues, alpha 2 is predominant in skeletal muscle and can be detected in the brain (34) and heart (28), alpha 3 is found in excitable tissues (34), and alpha 4 is found in testis (29). Similarly, the beta 1-isoform is fairly ubiquitous, whereas beta 2 and beta 3 are mostly found in skeletal muscle, neural tissues, lung, and liver (1, 2). In human heart, only alpha 1beta 1, alpha 2beta 1, and alpha 3beta 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 alpha 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 alpha 1-isoform is resistant to the binding and pharmacological effects of ouabain, whereas most other mammalian alpha 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 alpha -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 alpha 1-3- and beta 1-3-isoforms were expressed in Xenopus oocytes, and the properties of the expressed pumps were compared (3). All the alpha -subunit isoforms expressed in Xenopus oocytes with the beta 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 alpha 2- and alpha 3-isoforms are usually expressed in human tissues together with the alpha 1-isoform.

In the present study, human Na+-K+-ATPase alpha 1beta 1, alpha 2beta 1, and alpha 3beta 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 alpha beta complex for Na+, K+, and ATP were obtained from ouabain binding measurements, and the enzymatic turnover of ATP was measured for the alpha 1beta 1 and alpha 3beta 1 complexes. The results of these measurements were in good agreement with those obtained for human Na+-K+-ATPase expressed in Xenopus oocytes. The alpha 2beta 1 complex expressed in yeast was much less stable than the alpha 1beta 1 and alpha 3beta 1 complexes, however, possibly indicating that the lipid environment of the protein is less than optimal in these cells.


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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 (alpha 1 and alpha 3) and pKC4 (alpha 2 and beta 1).

Construction of yeast expression plasmids YhNalpha 1, YhNalpha 2, YhNalpha 3, and GhNbeta 1. Human alpha 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 alpha 1 was confirmed by automated sequencing (Biochemical Core Laboratory, University of Southern California). The resulting yeast expression plasmid in which human Na+-K+-ATPase alpha 1-isoform is expressed under the control of yeast PGK promoter was designated YhNalpha 1.

Human alpha 2 cDNA (3,063 bp) was released from its original vector (pKC4) and subcloned into the BamHI site of pBluescript II KS(+) (Stratagene). PCR-mediated mutagenesis was performed to introduce EcoRI sites directly upstream of the initiating ATG and downstream of the termination codon. The alpha 2 fragment was then released using EcoRI (resulting in 1 untranslated bp at the 5' end and 2 bp at the 3' end) and ligated into the EcoRI-digested yeast expression plasmid YEp1PT. The correct orientation of the insert was confirmed by restriction analysis, and the sequence of the coding region of the new plasmid, YhNalpha 2, was confirmed as described above.

The coding region of alpha 3 (3,042 bp) was constructed from two different fragments. One was released from a pRcCMV plasmid, ligated into pBluescript II KS(+), and used for the 5' part of the final clone (nucleotides 1-1143). A new EcoRI site was inserted upstream of the coding region by PCR. The 3' part of the alpha 3 coding region (nucleotide 1144 to STOP) was released from a pBluescript II SK plasmid using AflIII and ligated with the 5' part of alpha 3 at the AflIII site. The complete alpha 3 cDNA was then released by EcoRI digestion, which resulted in a fragment containing one untranslated nucleotide at the 5' end and 163 nucleotides at the 3' end of the coding sequence. This fragment was ligated into the EcoRI-digested yeast expression plasmid YEp1PT. The correct orientation of the insert in the new plasmid, YhNalpha 3, was confirmed by restriction analysis, and the sequence of the alpha 3 coding region was determined by DNA sequencing.

Human beta 1 cDNA (912 bp) was released from the pKC4 vector by digestion with BstXI/BclI, the 5' end was cut back, and the 3' end was filled in by treatment with T4 DNA polymerase, resulting in a blunt-ended fragment with no untranslated base pairs at the 5' end and two untranslated base pairs at the 3' end of the coding sequence. The electroeluted fragment was then ligated into the BamHI-digested and nuclease-treated yeast expression vector pG1T, in which it is expressed under the control of the inducible GAL1 promoter (5). The correct orientation of the insert was confirmed by restriction analysis, and the coding region of the yeast expression plasmid (GhNbeta 1) was sequenced.

Transformation of S. cerevisiae and yeast membrane preparation. Standard yeast media were used throughout the study. The yeast strain 30-4 (MAT alpha , trp1, ura3, Vn2, GAL+) was used for the coexpression of the human Na+-K+-ATPase alpha - and beta -isoforms. Yeast cells were cotransformed with YhNalpha and GhNbeta 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 beta 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 alpha beta 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.

The affinity of the human Na+-K+-ATPase alpha beta complexes for ouabain was determined by equilibrium binding of [3H]ouabain. Total ouabain concentrations were varied by increasing the concentrations of unlabeled ouabain while maintaining a constant level of radiolabeled ouabain (0.2-2 nM). The binding conditions and experimental procedures were identical to those described above, unspecific binding was determined in the presence of excess unlabeled ouabain (1 mM), and specific binding was normalized to the amount of recovered protein (mock assay). The data were fitted to the following equation, as previously described by Johnson et al. (14)
bound<IT>=</IT><FR><NU>B<SUB>max</SUB><FENCE><FR><NU>[<SUP>3</SUP>H]ouabain</NU><DE><IT>K</IT><SUB>d</SUB></DE></FR></FENCE></NU><DE>1<IT>+</IT><FENCE><FR><NU>[<SUP>3</SUP>H]ouabain</NU><DE><IT>K</IT><SUB>d</SUB></DE></FR></FENCE><IT>+</IT><FENCE><FR><NU>ouabain</NU><DE><IT>K</IT><SUB>d</SUB></DE></FR></FENCE></DE></FR>
where Bmax is maximum binding capacity.

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 alpha 1beta 1 and alpha 3beta 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 alpha -isoform and the beta 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 alpha 1 (1:100; obtained from M. Kashgarian, Yale University) (15), McB2 against alpha 2 (1:100; obtained from K. Sweadner, Harvard University) (32), anti-TED against alpha 3 (1:200; obtained from T. Pressley, Texas Tech) (26), or anti-human beta 1 (obtained from P. Martin-Vassallo, Tenerife, Spain) (36). Blots of alpha 1, alpha 3, and beta 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 alpha 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 mM-1 (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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Amino acid sequences of human Na+-K+-ATPase alpha 1-, alpha 2-, alpha 3-, and beta 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 alpha 1, alpha 2, and beta 1, the results were identical to the sequences that have been previously published (16, 17, 30, 31). In contrast, the alpha 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 alpha 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 alpha 1 and alpha 2, whereas valine, which has been previously reported to be at this position of human alpha 3, was found only in yeast transporters. Amino acid 555 has been reported to be a tyrosine in human alpha 3, chicken alpha 3, and human gastric H+-K+-ATPase, but it is a phenylalanine in our human Na+-K+-ATPase alpha 3 clone, in rat alpha 3 and pig alpha 3, and in human, rat, pig, and chicken alpha 1 and alpha 2. Amino acid 557 was reported to be a glutamine only for human Na+-K+-ATPase alpha 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 alpha 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 alpha 3-isoform, a glycine was reported in this position only for dog alpha 1. All other sequences and the sequence we have found for human Na+-K+-ATPase alpha 3 contain an aspartic acid at this position. From these comparisons we conclude that the human alpha 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 alpha beta 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 alpha -subunit isoform and beta 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 alpha 2-subunit was expressed at significantly lower levels in the yeast than the alpha 1- or alpha 3-subunit, and it was difficult to visualize the alpha 2-subunit on blots using 125I-protein A, which was used to visualize the alpha 1-, alpha 3-, and beta 1-subunits. In Fig. 1, alpha 2 was visualized using ECL. Consistent with the low expression level, the amount of ouabain bound by yeast clones expressing human Na+-K+-ATPase alpha 2beta 1 was ~10-fold lower than the amount bound by clones expressing the alpha 1beta 1- or alpha 3beta 1-subunits. The clones for each individual isoform with the highest amount of ouabain bound were selected for further analysis.


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Fig. 1.   Western blot of Na+-K+-ATPase (NKA) isoforms expressed in yeast. Yeast membrane protein (100 µg) from cells expressing human Na+-K+-ATPase alpha 1beta 1, alpha 2beta 1, or alpha 3beta 1 were separated by SDS-PAGE and transferred to Immobilon-P membranes. Four different clones from stably transformed cells expressing each isoform (1-4) were assayed. Membranes from human brain (HB), rat brain (RB), dog kidney (DK), and untransformed yeast cells (U) were included as controls. The alpha - and beta -subunits were detected with isoform-specific antibodies (see METHODS).

Ouabain affinity of human Na+-K+-ATPase alpha 1beta 1, alpha 2beta 1, and alpha 3beta 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 alpha beta complexes. As predicted by the screening experiments, the Bmax of ouabain was ~10-fold lower for yeast expressing alpha 2beta 1 than for cells expressing alpha 1beta 1 or alpha 3beta 1. A comparison of the Kd values of the human Na+-K+-ATPase isoforms showed that the alpha 1beta 1 and alpha 3beta 1 complexes bind ouabain with about twofold higher affinity than alpha 2beta 1; the relatively lower affinity of alpha 2beta 1 for ouabain was also observed in oocytes expressing human alpha -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 alpha beta 1-isoforms expressed in Xenopus oocytes (3).


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Fig. 2.   Equilibrium ouabain binding to Na+-K+-ATPase isoforms expressed in yeast. Yeast membrane protein (0.5-1 mg) from cells expressing human Na+-K+-ATPase alpha 1beta 1 (A), alpha 2beta 1 (B), or alpha 3beta 1 (C) were incubated for 60 min at 37°C with 0.2-2 nM [3H]ouabain and increasing concentrations of nonradioactive (cold) ouabain. Bound ouabain was determined as previously described (7). The amount of bound ouabain increased as the total ouabain concentration increased, but the bound disintegrations per minute (dpm) decreased as the specific activity of the radiolabeled ouabain was diluted with cold ouabain. Representative experiments are shown for each alpha beta complex. Lines through data are the best fit of a self-competition model (14) to the data.


                              
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Table 1.   Ouabain binding and substrate interactions in yeast microsomal membranes expressing human Na+-K+-ATPase isoforms

Na+, K+, and ATP affinity of human Na+-K+- ATPase alpha 1beta 1, alpha 2beta 1, and alpha 3beta 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
E2·K<IT>↔</IT>K<SUP><IT>+</IT></SUP><IT>+</IT>E2<IT>+</IT>P<SUB>i</SUB> ↔ E–P (1)

<IT>+</IT>ouabain ↔ E–P(ouabain)
Similarly, Na+ ions antagonize ouabain binding by binding to E1 and reducing the amount of E2 available for phosphoenzyme formation
E1·Na<SUP>+</SUP> ↔ Na<SUP>+</SUP><IT>+</IT>E1 ↔ E2<IT>+</IT>P<SUB>i</SUB> ↔ E–P (2)

<IT>+</IT>ouabain ↔ E–P(ouabain)
Under conditions where phosphorylation or ouabain is not limiting, the affinity of E2 for K+ is approximated by titration of reaction 1 with K+. The reaction with Na+ is more complex, involving equilibration between E1 and E2 forms of the enzyme in addition to the affinity of E1 for Na+. Typical data for these experiments are shown in Fig. 3, A and B. Hill coefficients (mean ± SD) for Na+ antagonism of ouabain binding were 1.7 ± 0.3 (alpha 1), 1.7 (alpha 2, n = 1), and 1.9 ± 0.1 (alpha 3). For K+ antagonism of ouabain binding, the Hill coefficients were 1.2 ± 0.3 (alpha 1), 2.0 ± 0.4 (alpha 2), and 1.5 ± 0.1 (alpha 3). Only the Hill coefficient for K+ antagonism of ouabain binding to alpha 2beta 1 was significantly different (P < 0.01) from values obtained for the other isoforms. Values of K0.5, the concentration of ligand that yields half-maximal response, were obtained from the fits and are shown in Table 1. Apparent affinity of the pumps for Na+ was also estimated from the Na+ dependence of ouabain binding to phosphoenzyme formed from ATP, shown in reaction 3, called the "front-door" reaction
E1<IT>+</IT>Na<SUP><IT>+</IT></SUP><IT>+</IT>ATP ↔ E–P (3)

<IT>+</IT>ouabain ↔ E–P(ouabain)
In this assay (Fig. 3C), the amount of ouabain bound after a 3-min incubation increased as the concentration of Na+ increased. For alpha 1beta 1 and alpha 3beta 1, the data were fit by an equation for a single population of binding sites; however, the alpha 2beta 1 data were best fit by a model with two populations of sites each having different K0.5 values for Na+. These values are listed in Table 1. The K0.5 values of each human alpha beta 1 complex for ATP were determined from the ATP concentration dependence of reaction 3 (data not shown), and the values obtained from these measurements are also shown in Table 1.


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Fig. 3.   Effect of KCl and NaCl on ouabain binding. A: 0.5-1 mg of yeast membrane protein from cells expressing human Na+-K+-ATPase alpha 1beta 1, alpha 2beta 1, or alpha 3beta 1 was incubated with 20 nM [3H]ouabain and KCl concentrations shown on the abscissa. After incubation for 60 min at 37°C, the amount of bound ouabain was determined as described in METHODS. Lines through data are best fits of a modified Hill equation (13) to the data. B and C: ouabain binding conducted as described in A, except NaCl was used instead of KCl.

Instability of human alpha 2beta 1 heterodimers expressed in yeast cells. The reduced Bmax of ouabain by alpha 2beta 1 complexes compared with alpha 1beta 1 and alpha 3beta 1 indicated that fewer functional alpha 2beta 1 pumps than other complexes were present in the steady state. The fact that it was necessary to use ECL to clearly visualize the alpha 2 protein is consistent with this observation. A time course of ouabain binding to alpha 2beta 1 was measured, and the results showed that the amount of ouabain bound by alpha 2beta 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 alpha 2beta 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 alpha 2beta 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 alpha 2beta 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 alpha 2beta 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 alpha 2beta 1 complexes was obtained by SDS extraction of yeast membranes. For membranes containing alpha 1beta 1 or alpha 3beta 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 alpha 2beta 1 (data not shown).


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Fig. 4.   Time course of ouabain binding to alpha 1beta 1 and alpha 2beta 1. Yeast membrane protein (0.5 mg) containing alpha 1beta 1 or alpha 2beta 1 was incubated with 20 nM [3H]ouabain at 37°C, and aliquots were withdrawn at 1.5, 2.5, 3.5, 4.5, 6.5, and 24 h for measurement of bound ouabain. Reactions containing alpha 2beta 1 were done in the presence () or absence () of 5 mM KCl. Lines were drawn through data points by eye.

ATPase activity. Ouabain-sensitive ATP hydrolysis was measured in yeast membranes containing human alpha 1beta 1 or alpha 3beta 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 alpha 2beta 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 alpha 1beta 1. The molecular turnover for ATP hydrolysis by alpha 1beta 1 was calculated to be about twice as high as that for ATP hydrolysis by alpha 3beta 1 (Table 1), consistent with the observation by Crambert et al. (3) that the number of charges per second per molecule transported by alpha 1beta 1 expressed in Xenopus oocytes is about four times as large as that transported by alpha 3beta 1.


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Fig. 5.   ATP hydrolysis by human Na+-K+-ATPase alpha 1beta 1. SDS-extracted yeast membrane protein (10-50 µg) was used for each determination of the rate of ATP hydrolysis at different ATP concentrations (see METHODS). Rate of ouabain-sensitive ATP hydrolysis is plotted on the ordinate, and ATP concentrations ([ATP]) are shown on the abscissa. Line through data points is the nonlinear least-squares fit of the Michaelis-Menten equation to the data. Maximum velocity for this reaction was 0.162 µmol · mg-1 · min-1, and the Michaelis-Menten constant for ATP was 208 µM.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Human Na+-K+-ATPase alpha 1beta 1, alpha 2beta 1, and alpha 3beta 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 alpha beta complexes in yeast provides a convenient system in which to study the properties of individual alpha -subunit isoforms. This is not possible in human tissues, where alpha 2 and alpha 3 are expressed with each other or with alpha 1.

The results of equilibrium ouabain binding measurements indicated that the affinity of the alpha 1beta 1 and alpha 3beta 1 complexes for ouabain is similar and is slightly higher than the affinity of the alpha 2beta 1 complex (P < 0.01). The twofold difference in Kd values between alpha 1/alpha 3 and alpha 2 complexes with beta 1 would not be apparent in equilibrium binding measurements in tissues containing alpha 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 alpha 1beta 1 and alpha 3beta 1 complexes but was significantly lower for the alpha 2beta 1 complex. This result is probably due to an instability of the alpha 2beta 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 alpha -subunit isoforms were expressed from the same expression plasmid, it seems likely that the instability of alpha 2beta 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 alpha 2beta 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 alpha beta complex indicates that the alpha 1- and alpha 3-isoforms have a similar affinity for K+ (0.9 ± 0.6 and 1.3 ± 0.1 mM for alpha 1 and alpha 3, respectively) but that the affinity of alpha 2 for K+ is significantly lower (6.3 ± 2.4 mM, P < 0.001). The values for alpha 1beta 1 and alpha 3beta 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 alpha 2beta 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 alpha 1beta 1- and alpha 2beta 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 alpha 3beta 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 alpha 1- and alpha 3-isoforms were found to have K0.5 values of 1.5 and 2.8 mM, respectively. Na+-dependent ouabain binding to the alpha 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 alpha 2beta 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 alpha -subunit exhibited a similar K0.5 for ATP in the same assay.

Both alpha 1beta 1 and alpha 3beta 1 expressed in yeast are capable of hydrolyzing ATP. The molecular turnover of alpha 1beta 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 alpha 1beta 1 expressed in insect cells (12). This value is approximately twofold higher than that of alpha 3beta 1 expressed in yeast. No independent value is available for ATPase activity of human alpha 3beta 1, but in human alpha 3beta 1 expressed in Xenopus oocytes, turnover was ~25% of that in alpha 1beta 1 (3). Because alpha 3beta 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 alpha 1-, alpha 2-, and alpha 3-isoforms of Na+-K+-ATPase have been expressed in functional form in yeast cells with the human beta 1-subunit, and the biochemical characteristics of each alpha beta -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.


    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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