©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Substitutions of Glutamate 781 in the Na,K-ATPase Subunit Demonstrate Reduced Cation Selectivity and an Increased Affinity for ATP (*)

(Received for publication, July 28, 1995; and in revised form, November 20, 1995)

Joseph C. Koster Gustavo Blanco Paul B. Mills Robert W. Mercer (§)

From the Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The intramembrane Glu residue of the Na,K-ATPase alpha subunit has been postulated to have a role in the binding and/or occlusion of cations. To ascertain the role of Glu, the residue was substituted with an aspartate, alanine, or lysine residue and the mutant Na,K-ATPases were coexpressed with the native beta1 subunit in Sf9 insect cells using the baculovirus expression system. All alpha mutants are able to efficiently assemble with the beta1 subunit and produce catalytically competent Na,K-ATPase molecules with hydrolytic activities comparable to that of the wild-type enzyme. Analysis of the kinetic properties of the mutated enzymes showed a decrease in apparent affinity for K compared to wild-type Na,K-ATPase, with the lysine and alanine substitutions displaying the greatest reduction. All Na,K-ATPase mutants demonstrated a significant increase in apparent affinity for ATP compared to wild-type Na,K-ATPase, while the sensitivity to the cardiotonic inhibitor, ouabain, was unchanged. The dependence on Na, however, differs among the mutant enzymes with both the Glu Asp and Glu Ala mutants displaying a decrease in the apparent affinity for the cation, while the Glu Lys mutant exhibits a modest increase. Furthermore, in the absence of K, the Glu Ala mutant displays a Na-ATPase activity and a cellular Na influx suggesting that Na is substituting for K at the extracellular binding sites. The observation that trypsin digestion of the Glu Ala mutant in Na medium produces a K-stabilized tryptic fragment also intimates a decreased capacity of the mutant to discriminate between Na and K at the extracellular loading sites. All together, these data implicate Glu of the Na,K-ATPase alpha subunit as an important coordinate of cation selectivity and activation, although the modest effect of Glu Lys substitution seemingly precludes direct involvement of the residue in the cation binding process. In addition, the fifth membrane segment is proposed to represent an important communicative link between the extramembraneous ATP binding domain and the cation transport regions of the Na,K-ATPase.


INTRODUCTION

The Na,K-ATPase, or sodium pump, is a membrane-spanning, heterodimeric protein that uses the energy from the hydrolysis of ATP to maintain the low intracellular sodium concentration and high intracellular potassium concentration common to most animal cells. During enzyme turnover, three intracellular sodium ions and two extracellular potassium ions are countertransported across the plasma membrane for each molecule of ATP hydrolyzed. The ion-motive enzyme consists of a 110-kDa catalytic alpha subunit and a smaller noncovalently associated beta subunit. The catalytic alpha subunit contains the binding sites for ATP and the cardiotonic inhibitor, ouabain; it is phosphorylated by ATP, and undergoes the cation-dependent E(1) E(2) conformational transitions associated with cation transport. The exact function of the glycosylated beta subunit remains unknown, although, it has been implicated in the K-binding process of the enzyme (Lutsenko and Kaplan, 1993).

Understanding the molecular events associated with the Na,K-ATPase catalytic cycle requires a knowledge of the individual amino acids that coordinate the binding of the enzyme ligands. While data from site-directed mutagenesis and chemical modification studies have implicated specific residues within the alpha subunit in ATP binding and ouabain sensitivity (for review, see Lingrel and Kuntzweiler(1994)), comparatively little is known concerning the intramembrane amino acids that comprise the monovalent cation binding and occlusion sites. It is generally believed that movement of sodium and potassium through the low dielectric medium of the plasma membrane involves the neutralization and stabilization of the positively charged ions through coordination with carboxyl-bearing residues within or near the membrane. The use of hydrophobic, carboxyl-selective reagents to chemically modify and inactivate cation binding of the Na,K-ATPase has proven useful in identifying intramembrane residues in the alpha subunit that may be involved in cation transport function (Goldshleger et al., 1992; Argüello and Kaplan, 1994).

Recently, a fluorescent, carboxyl-reactive derivative of diazomethane, 4-(diazomethyl)-7-(diethylamino)coumarin (DEAC)(^1), has been shown to inactivate cation occlusion of the Na,K-ATPase in a K and Na preventable manner by covalently labeling Glu, a residue in the fifth transmembrane segment (Argüello and Kaplan, 1994). DEAC inhibition appears to be selective for the cation binding site as the modified enzyme is still able to undergo the E(1) E(2) conformational transitions and displays normal levels of ATP/ADP binding. The importance of this residue in Na,K-ATPase function is further evidenced by the findings that (i) substitution of Glu with a leucine or aspartate residue inhibits Na,K-ATPase-dependent cell growth in transfected HeLa cells (Jewell-Motz and Lingrel, 1993; Feng and Lingrel, 1995), (ii) replacement of the corresponding glutamate residue, Glu, in the sarco(endo)plasmic reticulum Ca-ATPase blocks Ca-dependent functions of the enzyme (Clarke et al., 1989), and (iii) the glutamate residue is highly conserved among members of the P-type family of ATPases (Argüello and Kaplan, 1994). More recently, expression of Glu mutations in transfected mammalian cells have shown that this residue, although not required for activity, has an important role in cation interactions or selectivity (Vilsen, 1995; Feng and Lingrel, 1995).

To further examine the role of the glutamate in Na,K-ATPase function, site-directed mutagenesis was used to replace Glu (the equivalent residue in the rat Na,K-ATPase) with an alanine, aspartate, or lysine residue, and the mutant Na,K-ATPase polypeptides were expressed in Spodoptera frugiperda (Sf9) cells using the baculovirus expression system. All mutant sodium pumps are catalytically active, but display altered affinities for Na, K, and ATP. In addition, the Glu Ala mutant displays an increased affinity for Cs, a congener of K in the reaction cycle. In the absence of potassium ions, the Glu Ala mutant mediates a Na-dependent ATPase activity and a cellular Na influx suggesting that Na is acting at the extracellular binding sites as a surrogate for K. An identical Na-dependent ATPase activity was recently reported for the Glu Ala mutant of the alpha subunit in transfected COS cells (Vilsen, 1995), but not in HeLa cells (Feng and Lingrel, 1995). The cause of this difference is unclear. In addition, trypsin digestion of the Glu Ala mutant demonstrates that Na alone is sufficient to stabilize the E(2)(K) conformational state of the enzyme. All together, these results suggest that in the Glu Ala mutant, the Na is acting at extracellular binding sites as a replacement for K in the catalytic cycle. Moreover, these results suggest that Glu is an important coordinate of cation selectivity and activation, and may represent a link between the extramembraneous ATP binding domain and the cation transport regions of the Na,K-ATPase.


MATERIALS AND METHODS

DNA and Viral Constructions

The cDNAs corresponding to the rat Na,K-ATPase alpha1 (Schneider et al., 1985) and beta1 (Mercer et al., 1986) subunits were subcloned into the dual promoter baculovirus transfer vector p2Bac (Invitrogen Corp., San Diego, CA) to allow for simultaneous expression in the same vector. Transfection of linearized Autographica californica multiple nuclear polyhedrosis virus genomic DNA, and recombinant baculovirus preparation and selection were performed following the manufacturer's procedures (Invitrogen Corp.). Amino acid substitutions in the alpha1 subunit were introduced using oligonucleotide-directed mutagenesis (Amersham Corp.). Prior to subcloning, mutant cassettes were sequenced to ensure that the desired mutation had been incorporated and that no other mutations were introduced. Mutant cassettes of the alpha1 subunit were subcloned as PflmI/MscI restriction fragments into the wild-type alpha1beta1 p2Bac transfer vector.

Viral Infections and Membrane Preparation

Sf9 cells were grown in suspension cultures (from 50 ml to 2 liters) in TNM:FH medium (defined in O'Reilly et al.(1992); JRH Biosciences, Lenexa, KS), supplemented with 10% (v/v) fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml fungizone (complete medium). Infections were performed in serum-free medium for 1 h using a viral multiplicity of infection ranging from 5 to 10. After addition of complete medium, cultures were maintained for 72 h. All experiments were performed using membrane preparations from the insect cells. For isolation of membranes, Sf9 cells were centrifuged (5,000 times g) for 10 min and resuspended at a concentration of 5 times 10^6 cells/ml in 0.1 mM EDTA, 30 mM imidazole HCl, pH 7.4. The cells were homogenized on ice using a glass homogenizer and the lysate was centrifuged for 10 min at 1,000 times g. The supernatant was removed, centrifuged for an additional 10 min at 12,000 times g, and the final pellet was suspended in 250 mM sucrose, 0.1 mM EGTA, and 25 mM imidazole HCl, pH 7.4.

PAGE and Immunoblot Analysis

The expressed proteins were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Proteins were separated by SDS-PAGE (Laemmli, 1970), transferred to nitrocellulose (Hybond C, Amersham Corp.), and immunoblotted as described previously (Blanco et al., 1995). The alpha1 and beta1 isoforms were identified with a polyclonal antibody (poly(alphaA)) that recognizes both the rat alpha1 and beta1 subunits (DeTomaso et al., 1993). In the immunoprecipitation experiments, the beta1 subunit was detected using an anti-beta1 antiserum raised against purified beta1 subunit from dog kidney (provided by Dr. Amir Askari, Medical College of Ohio, Toledo, OH).

Immunoprecipitations

Uninfected and 48 h infected Sf9 cells grown in 6-well tissue culture plates were lysed with 1% CHAPS in 150 mM NaCl, 25 mM HEPES, pH 7.4. After removal of the insoluble material (10 min; 15,000 times g), samples were subjected to immunoprecipitation. To precipitate the alpha1 isoform, 50 µl of a monoclonal antibody hybridoma supernatant that is specific for the alpha1 subunit (C464-6B, provided by Dr. Michael Caplan, Yale University) and 100 µl (1 mg/ml) of goat anti-mouse coated magnetic beads (BioMag; PerSeptive Diagnostics, Inc., Cambridge, MA) were used. After overnight incubation on a rocking table at 4 °C, beads were isolated by holding the microcentrifuge tube to a magnet and aspirating the supernatant. The beads were washed 3 times in the lysis buffer. The precipitated protein was eluted by resuspending the beads in sample buffer (100 mM Tris-HCl, pH 6.8, 2% SDS, 33% glycerol, 100 mM dithiothreitol) and incubating for 15 min at 65 °C. Eluted proteins were separated by SDS-PAGE (7.5% gel), transferred to nitrocellulose, and probed with the anti-beta1 specific antiserum.

Trypsin Digestion

Membrane proteins (200 µg) from alpha1beta1 or alpha1(Glu Ala)beta1 infected Sf9 cells were preincubated in a medium containing 1 mM EDTA, 0.05% CHAPS, and 25 mM imidazole, pH 7.5, in the absence or presence of 150 mM NaCl. Membranes were incubated for 20 min at 37 °C in the respective medium, then L-1-tosylamido-2-phenylethyl chloromethyl ketone-trypsin (Sigma) was added at a protein/trypsin ratio (w/w) of 1:10,000. After 20 min, digestions were stopped by the addition of soybean inhibitor (2-3-fold weight excess over trypsin) and incubated on ice for 5 min. Proteins were precipitated in 10% trichloroacetic acid, centrifuged (10 min, 15,000 times g), and the pellet resuspended in the sample buffer described above. Proteins were resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with an anti-alpha antiserum (anti-NASE; provided by Dr. Thomas Pressley, University of Texas, Houston, TX), that recognizes an amino acid sequence (KNPNASEPKHLL) common to both the 77- and 58-kDa tryptic fragments of the alpha subunit.

Biochemical Assays

Protein assays were performed using the bicinchoninic acid/copper sulfate solution as described by the supplier (Pierce Chemical Co.).

Na,K-ATPase activity was assayed through determination of the initial rate of release of P(i) from [-P]ATP as described previously (Beaugé and Campos, 1983). Samples of 50-100 µg of total protein were assayed in a final volume of 0.25 ml. Following a 30-min incubation period at 37 °C, the reaction was terminated by the addition of 5.5% trichloroacetic acid (final) and incubating on ice for 5 min. Released P(i)-P(i) was then converted to phosphomolybdate and extracted with isobutyl alcohol. Finally, 0.15 ml of the organic phase were taken and radioactivity measured by liquid scintillation counting. The ATP hydrolyzed never exceeded 15% of the total ATP present in the sample and hydrolysis was linear over the incubation time. Na,K-ATPase activity was determined as the difference in ATP hydrolysis in the absence and presence of 1 mM ouabain. For maximal Na,K-ATPase activity, the medium contained 120 mM NaCl, 30 mM KCl, 3 mM MgCl(2), 3 mM ATP, 0.2 mM EGTA, 2.5 mM sodium azide, 30 mM Tris-HCl, pH 7.4, ± 1 mM ouabain. For the cations, incubation media were the same as above except that for Na dependence, Na concentration was varied from 2.5 to 122.5 mM. For K and Cs stimulations, the respective ion concentrations were varied from 0 to 30 mM. In medium depleted of K, flame photometry demonstrated that the concentration of free K was approximately 30 µM. Choline chloride was added so that the final concentration of Na or K plus choline totaled 150 mM. The ATP dependence was determined under saturating concentrations of all cations (120 mM Na, 30 mM K, and 3 mM Mg). To determine the effect of different ouabain concentrations on Na,K-ATPase activity, samples were preincubated with the indicated concentrations of ouabain for 30 min at 37 °C in the reaction medium; the reaction was started by the addition of ATP.

For Na transport assays, infected Sf9 cells were harvested at 48 or 72 h post-infection. Cells were centrifuged (2,000 times g) and resuspended at a density of 1 times 10^6 cells/ml in preincubation medium (150 mM NaCl, 100 µM bumetanide, 25 mM HEPES, and 2.5 mM MgCl(2), pH 7.4). After a 30-min incubation on ice, cells were centrifuged (2,000 times g) and resuspended at a density of 2 times 10^7 cells/ml in flux medium (50 mM NaCl, 75 mM choline chloride, 25 mM HEPES/Tris, 100 µM bumetanide, pH 7.4), in the presence or absence of 1 mM ouabain. Flame photometry demonstrated that the concentration of free K in the flux medium was approximately 30 µM. After an additional 5-min incubation at 37 °C, the reaction was started by adding an equal volume of flux medium containing Na (1 µCi/ml) in the presence or absence of 1 mM ouabain. At prescribed intervals, 150-µl aliquots (1 times 10^6 cells) were removed and layered on 0.5 ml of silicone oil (Versilube F-50; Harwich Chemical Corp.) in a 1.5-ml microcentrifuge tube and centrifuged for 30 s. Cell separation occurred in <5 s. The aqueous and oil phases were removed by aspiration and discarded. The cell pellets were excised and the radioactivity determined by liquid scintillation. The rates of ouabain-sensitive sodium-uptake shown were determined at the 2-min time point.

Data Analysis

Curve fitting of the experimental data was carried out using a Marquardt least-squares nonlinear regression computing program (Sigma Plot, Jandel Scientific, San Rafael, CA) as described previously (Blanco et al., 1995).


RESULTS

Expression and Assembly of Mutant Na,K-ATPase Polypeptides in Sf9 Insect Cells

To determine the role of the DEAC-modified glutamate in Na,K-ATPase function, site-directed mutagenesis was used to replace Glu in the rodent Na,K-ATPase with an alanine, aspartate, or lysine residue. Single recombinant baculoviruses containing the cDNAs coding for both the mutant alpha1 (Glu Ala, Glu Asp, or Glu Lys) and native beta1 subunits were used to infect Sf9 insect cells, a cell line derived from the ovary of the fall armyworm, S. frugiperda. As shown in Fig. 1A, insect cells infected with the respective alphabeta recombinant baculoviruses express stoichiometric levels of mutant alpha1 and native beta1 polypeptides that are recognized by the anti-alphabeta polyclonal antiserum, poly(alphaA). The Na,K-ATPase from rat kidney is shown for comparison.


Figure 1: Expression of rat Na,K-ATPase mutant polypeptides in infected Sf9 cells. A, immunoblot analysis of Sf9 proteins. Recombinant baculoviruses directing the coexpression of the mutant alpha (Glu Asp, Glu Lys, or Glu Ala) and native beta1 subunits were used to infect Sf9 insect cells. After 72 h Sf9 proteins (20 µg) were separated by SDS-PAGE (7.5% gel) and transferred to nitrocellulose. The alpha and beta polypeptides were detected with a polyclonal anti-alphabeta antiserum (poly(alphaA)) that immunoreacts with both the rat alpha1 and beta1 subunits. B, association of mutant alpha and native beta subunits coexpressed in Sf9 cells. Proteins from mutant alphabeta infected Sf9 cells, and combined proteins from cells individually expressing alpha1 and beta1 (alpha1 + beta1) were immunoprecipitated with a monoclonal antibody (C464) against the alpha-subunit (IP AB). The precipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with the anti-beta1 antiserum (IB AB). The native beta1 subunit from kidney membranes (15 µg) is shown as a standard.



To determine if the individual alpha polypeptides can assemble with the beta1 subunit, infected Sf9 cells were solubilized in 1% CHAPS and immunoprecipitated with an alpha-specific monoclonal antibody. If the mutant alpha1 and beta1 subunits are in a stable detergent-resistant association, then the beta1 subunit should coimmunoprecipitate with the mutant alpha1 polypeptide. Using an anti-beta antiserum, the beta subunit can be identified in the immunoprecipitate. As shown in Fig. 1B, the alpha mutants and the beta1 polypeptide can form detergent-stable complexes when coexpressed in the cells. In contrast, when cells singly infected with either an alpha1 or beta1 baculovirus are combined prior to solubilization, the beta1 polypeptide is not identified in the immunoprecipitate (Fig. 1B, lane 2). Thus, the site-directed alpha mutants are expressed at high levels in the Sf9 cells and are structurally competent as judged by their ability to stably assemble with the beta1 subunit in cells coexpressing both the alpha and beta polypeptides.

Enzymatic Analysis of Glu Mutants of the Na,K-ATPase

In order to determine whether the expressed mutant alpha polypeptides are functionally active, Na,K-ATPase activity was measured using saturating concentrations of ligands (120 mM NaCl, 20 mM KCl, and 3 mM MgbulletATP). The corresponding activities are presented in Table 1. Interestingly, replacement of Glu with an alanine, aspartate, or a positively charged lysine does not diminish Na,K-ATPase activity, with the mutant alphabeta complexes displaying activities 7-10-fold higher than the uninfected cells or cells expressing the unrelated integrin associated protein (Lindberg et al., 1993). (It is assumed that the lysine residue side chain, pK(a) 10.8, displays a net positive charge in the reaction buffer, pH 7.4.) Thus, the mutant-derived activities are comparable to the level of Na,K-ATPase observed with the wild-type enzyme. These results are consistent with previous studies using transfected mammalian cells demonstrating that the Glu Ala (Vilsen, 1995; Feng and Lingrel, 1995) and Glu Gln (Feng and Lingrel, 1995) mutants are functional. However, in transfected HeLa cells the substitution of Glu with aspartate did not confer ouabain resistance, suggesting that the modification impaired enzymatic activity. The functional expression of the Glu Asp mutation in insect cells suggests that in mammalian cells the substitution may influence processing or assembly of the enzyme. Dose-response curves for the ouabain inhibition of Na,K-ATPase activity of the mutants were determined on membranes from infected Sf9 cells under non-limiting ligand concentrations (120 mM Na, 20 mM K, and 3 mM Mg). All mutants displayed inhibition curves similar to the wild-type Na,K-ATPase (data not shown) with K(i) values in the order of 10M. These values are in good agreement with the low ouabain sensitivity previously reported for the rodent alpha1beta1 isozyme (Blanco et al., 1993; O'Brien et al., 1994) and suggest an unaltered ouabain-binding capacity for the Na,K-ATPase mutants.



To characterize the cation requirements of the mutant enzymes, ATPase assays were performed in medium free of both Na and K, in K-free medium with Na, or in medium containing both Na and K. Interestingly, as shown in Table 1, the Glu Ala mutant displays a significant ouabain-sensitive Na-ATPase activity in the absence of potassium ions. This Na-ATPase activity represents approximately 38% of the maximal activity and is not present at a sodium concentration of 1.5 mM. No ouabain-sensitive ATPase activity was observed with K alone (data not shown).

Na Uptake

One possible cause of the elevated Na-ATPase activity of the Glu Ala mutant is an increased efficacy of extracellular Na to act as a surrogate for K in stimulating enzyme turnover and transport. To analyze the cation transport properties of the mutant Na-ATPase activity at the extracellular surface, ouabain-sensitive uptake of external Na was characterized in infected Sf9 cells expressing the wild-type or mutant Na,K-ATPase. As shown in Fig. 2, after 72 h, Sf9 cells coexpressing the mutant alpha(Glu Ala) and the beta1-subunit exhibit a ouabain-sensitive sodium uptake (a maximal rate of 4.5 nmol of Na/10^6 cells/min) that is not present in uninfected cells (data not shown), in cells infected for 48 h, or in cells expressing the wild-type enzyme (Fig. 2, inset). It appears that the Na-ATPase activity and the Na uptake displayed by the Glu Ala mutant represents the well characterized Na/Na exchange mechanism in which, in the absence of K, extracellular Na is transported as K to stimulate enzyme dephosphorylation (Blostein, 1983; Campos and Beaugé, 1994). As Na/Na exchange accompanied by ATP hydrolysis has been shown to represent approximately 6 to 10% of the maximal Na/K exchange in the wild-type enzyme (Cornelius, 1991; Mercer and Dunham, 1981) (undetectable in our assays), the elevated level observed with the Glu Ala mutant suggests a reduction in the ability of the enzyme to discriminate between Na and K at the extracellular loading sites.


Figure 2: Na uptake in alpha(Glu Ala)beta1 infected Sf9 cells. Ouabain-sensitive sodium uptake was measured in Sf9 cells infected for 48 or 72 h, using Na as tracer. The transport assay was started with the addition of the tracer and stopped at the times indicated by centrifuging the cells through a layer of silicone oil. Bumetanide (100 µM) was present to inhibit the bumetanide-sensitive sodium uptake. The inset shows rates of ouabain-sensitive sodium uptake at the 2-min point in Sf9 cells infected with the alpha(Glu Ala)beta1 baculovirus as compared to the wild-type alphabeta baculovirus. Each value is the mean with error bars representing the standard error of triplicate determinations.



Trypsin Digestion of the Na,K-ATPase

The Na,K-ATPase exhibits a characteristic pattern of cleavage by trypsin, which differs depending on the presence of Na or K in the medium. Moreover, this property is taken as evidence that the cations stabilize different conformational states of the enzyme [E(1)(Na) versus E(2)(K)] (Jørgensen, 1975, 1977). In the presence of Na, tryptic fragmentation produces one major fragment of M(r) = 77,000, while digestion in K medium results in two specific peptide fragments of M(r) = 58,000 and M(r) = 41,000 (Castro and Farley, 1979). As extracellular Na exhibits an increased K-like effect on enzyme activation in the Glu Ala mutant, this effect might also be reflected in the proteolytic cleavage pattern of the enzyme resulting in the generation of the K-specific tryptic fragments in the presence of only Na. To analyze the tryptic digestion pattern of baculovirus-induced Na,K-ATPase, membrane proteins from alpha1beta1 or alpha1(Glu Ala)beta1 infected Sf9 cells were digested with trypsin, electrophoresed, and transferred to nitrocellulose. The immunoblot was probed with an anti-alpha antiserum (anti-NASE) that recognizes a 12-amino acid sequence that is common to both the sodium (M(r) = 77,000) and potassium (M(r) = 58,000) tryptic fragments. As shown in Fig. 3, when digested in Na medium, both the wild-type and mutant Na,K-ATPase generate the sodium tryptic fragment (M(r) = 77,000). However, in contrast to the wild-type enzyme, digestion of the Glu Ala mutant produces the normally K-induced tryptic fragment (58 kDa). The tryptic fragmentation pattern of the mutant and wild-type Na,K-ATPase in Na and K-free media is shown as a control (Fig. 3, lanes 1 and 3, respectively). As previously reported (Castro and Farley, 1979), trypsin digestion of the Na,K-ATPase in imidazole/EDTA buffer without salt generates a tryptic fragment of M(r) = 77,000 (lanes 1 and 3). However, the level of the 77-kDa fragment produced is significantly increased when the tryptic digestion is performed in a Na medium (lanes 2 and 4). Thus, this data suggests that in the mutant, Na can stabilize the E(2)(K) form of the enzyme and is consistent with an increased efficacy of Na at the extracellular binding sites.


Figure 3: Proteolysis of the Na,K-ATPase. Membrane proteins (200 µg) from alpha1beta1 or alpha1(Glu Ala)beta1 infected cells were digested with trypsin in a NaCl or salt-free medium, resolved on SDS-PAGE, and transferred to nitrocellulose. The immunoblot was probed with an anti-alpha antiserum (anti-NASE) that recognizes a sequence (KNPNASEPKHLL) common to both the 77- (Na) and 58-kDa (K) tryptic fragments. The position of the 77-kDa tryptic fragment and the 58-kDa tryptic fragment are shown by arrows. In the controls (lanes 1 and 3), the tryptic cleavage pattern produced in medium free of Na and K is shown.



K, Na, and ATP Dependence of Na,K-ATPase Activities

To determine the affinity of the Glu Asp, Glu Lys, and Glu Ala mutants toward physiological ligands, activation curves of Na,K-ATPase activity by K, Na, and ATP were performed. For the K dependence of Na,K-ATPase, enzyme activity was measured at varying concentrations of K (0-30 mM) with Na fixed at 120 mM. The obtained K activation curves of the three mutants are presented in Panel A of Fig. 4, Fig. 5, and Fig. 6, where the corresponding activation curve for the wild-type enzyme has been included for comparison. Values describing the kinetic parameters of the different Na,KATPases are presented in Table 2. As shown, all three mutants exhibited a modest decrease in the apparent affinity for K. While replacement of Glu with an aspartate residue reduces the K affinity of the enzyme around 1.5-fold (K(0.5) value of 2.8 mM compared to 1.9 mM for the wild-type enzyme), both the Glu Ala and Glu Lys mutants displayed lower apparent affinities for K which correspond to a 2-3-fold reduction relative to the wild-type enzyme (K(0.5) values of 6.3 and 4.7 mM for the Glu Ala and Glu Lys mutants, respectively). For the Glu Ala mutant, the apparent K affinity was calculated by subtracting the Na-ATPase component from the total ouabain-sensitive activity and expressing the remaining K-dependent activity as a percentage of the maximal Na,K-ATPase. As Na is postulated to compete more efficiently with K for binding at the extracellular activation sites in the mutant, the calculated apparent affinity for K in this case may be overestimated.


Figure 4: K, Na, and ATP activation of the alpha1(Glu Asp)beta1 mutant enzyme. A, K activation of Glu Asp mutant. Na,K-ATPase activity of Sf9 membranes expressing the mutant alpha(Glu Asp)beta1 (bullet, , and ) or wild-type alpha1beta1 (circle, box, and &cjs2105;), was determined in a reaction medium containing: 120 mM NaCl, 3 mM ATP, 3 mM MgCl(2), 0.2 mM EGTA, 30 mM Tris-HCl, pH 7.4, and KCl as indicated, in the absence or presence of 1 mM ouabain. B, Na activation of Glu Asp mutant enzyme. Ouabain-sensitive ATPase activity assays were performed as described in A but with a reaction medium containing 30 mM KCl and varying concentrations of Na from 2.5 to 122.5 mM. In the Na and K activation experiments, ionic strength was kept constant with choline chloride. C, ATP stimulation of the Glu Asp mutant enzyme. Ouabain-sensitive ATPase activity assays were performed in the reaction medium described in A but containing constant Na and K (30 mM KCl, 120 mM NaCl) and the indicated ATP concentrations. All data are expressed as percent of the maximal Na,K-ATPase activity obtained. Each value is the mean and error bars represent the standard errors of the mean of at least three experiments performed in triplicate on samples obtained from different infections.




Figure 5: K, Na, and ATP activation of the alpha1(Glu Lys)beta1 mutant enzyme. A, K activation of Glu Lys mutant. Na,K-ATPase activity of Sf9 membranes expressing the mutant alpha(Glu Lys)beta1 (bullet, , and ) or wild-type alpha1beta1 (circle, box, and &cjs2105;), was determined as described in the legend to Fig. 4. B, Na activation of Glu Lys mutant enzyme. C, ATP stimulation of the Glu Lys mutant enzyme. All data are expressed as percent of the maximal Na,K-ATPase activity obtained. Each value is the mean and error bars represent the standard errors of the mean of at least three experiments performed in triplicate on samples obtained from different infections.




Figure 6: K, Na, and ATP activation of the alpha1(Glu Ala)beta1 mutant enzyme. A, K activation of Glu Ala mutant enzyme. Na,K-ATPase activity of Sf9 membranes expressing the mutant alpha(Glu Ala)beta1 (bullet, , and ) or wild-type alpha1beta1 (circle, box, and &cjs2105;), was determined as described in the legend to Fig. 4. B, Na activation of Glu Ala mutant enzyme. C, ATP stimulation of the Glu Ala mutant enzyme. All data are expressed as percent of the maximal Na,K-ATPase activity obtained. Each value is the mean and error bars represent the standard errors of the mean of at least three experiments performed in triplicate on samples obtained from different infections.





To determine the requirement of the Glu Asp, Glu Lys, and Glu Ala mutants for sodium, Na,K-ATPase activity was measured at varying concentrations of Na (2.5-122.5 mM) with K fixed at 30 mM. In all cases the mutant Na,K-pumps display a shift in the apparent affinity for Na relative to the wild-type enzyme (Panel B, Fig. 4, Fig. 5, and Fig. 6). As listed in Table 2, the conservative substitution of glutamate with an aspartate residue resulted in approximately a 1.5-fold reduction in Na affinity (compare the K(0.5) value of 23.2 mM with 16.4 mM for the wild-type Na,K-ATPase). Whereas, replacement with an alanine resulted in a lower apparent affinity for Na, corresponding to a 2-fold reduction relative to the alpha1beta1 enzyme (K(0.5) value of 33.5 mM for the Glu Ala mutant). Surprisingly, in contrast to the Glu Ala and Glu Asp mutants, the replacement of Glu with the positively charged lysine residue demonstrates a modest, 1.5-fold increase in the K(0.5) value for Na activation (K(0.5) value of 9.5 mM). As the lysine substitution exhibits opposing effects on cation affinity and an undiminished level of Na,K-ATPase activity (see Table 1), a direct role of Glu in charge neutralization and cation binding is unlikely.

To characterize the kinetics at the low affinity ATP site, dose-response curves of Na,K-ATPase activity at millimolar concentrations of ATP were performed. The ATP dependence of the baculovirus-induced alpha mutants is presented in Panel C of Fig. 4, Fig. 5, and Fig. 6and the K(m) values are described in Table 2. As shown, all three mutant pumps display a 3-9-fold increase in apparent affinity for ATP over the wild-type enzyme (K(m) values of 0.11, 0.11, and 0.05 for the Glu Asp, Glu Ala, and Glu Lys mutants, respectively, compared to 0.46 for the wild-type enzyme).

CsDependence of Na,K-ATPase Activity

At the extracellular K site, other alkali metal ions have been shown to replace K and activate ouabain-sensitive Na efflux with a sequence of affinities of Rb > K > Cs > Li (McConaghey and Maizels, 1962). If replacement of Glu with an alanine residue reduces the selectivity of the enzyme at the extracellular surface, a change in the Cs half-maximal activation of Na,K-ATPase activity over the wild-type enzyme should be observed. To determine the affinity of the Glu Ala mutant for Cs, ouabain-sensitive ATPase activity was measured with varying concentrations of Cs (0-30 mM) in the absence of K and with Na fixed at 120 mM. Fig. 7shows that the Glu Ala mutant exhibits a 2-fold increase in the apparent affinity for Cs compared to the wild-type enzyme (K(0.5) values of 5.7 ± 0.7 mM for the Glu Ala mutant compared to 13 ± 2 mM for the wild-type Na,K-ATPase). Interestingly, the affinity of the mutant enzyme for Cs is similar to that for K in supporting ATPase activity. The apparent Cs affinity of the mutant was calculated by subtracting the Na-ATPase activity and expressing the remaining Cs-dependent activity as a percentage of the maximal Na,Cs-ATPase. Thus, this data is consistent with a reduction in the ability of the mutant Na,K-ATPase to discriminate between the monovalent cations at the extracellular site.


Figure 7: Cs activation of alpha1(Glu Ala)beta1 and native alpha1beta1 enzymes. Na,Cs-ATPase activity of Sf9 membranes expressing the mutant alpha(Glu Ala)beta1 (bullet) or wild-type alpha1beta1 (circle) was determined in a reaction medium containing: 120 mM NaCl, 3 mM ATP, 3 mM MgCl(2), 0.2 mM EGTA, 30 mM Tris-HCl, pH 7.4, and CsCl as indicated, in the absence or presence of 1 mM ouabain. Apparent cesium affinity was calculated as a percent of the maximal Na,Cs-ATPase activity obtained after subtraction of the Na-ATPase activity. Each value is the mean and error bars represent the standard errors of the mean of at least three experiments performed in triplicate on samples obtained from different infections. Total ionic strength was kept constant with choline chloride.




DISCUSSION

Previous chemical modification studies using DEAC have implicated a glutamate residue of the Na,K-ATPase alpha subunit in cation binding (Argüello and Kaplan, 1994). In the present study, this glutamate residue, which in the rat enzyme corresponds to Glu, was replaced with an aspartate, alanine, or lysine residue and the Na,K-ATPase mutants were coexpressed with the beta1 subunit in Sf9 cells and characterized. All mutants are able to support ouabain-sensitive ATPase activity at levels comparable to that of the wild-type alpha1beta1 enzyme. In addition, studies using transfected mammalian cells demonstrate that the Glu Ala and Glu Gln mutants have maximal turnover numbers similar to the wild-type enzyme (Vilsen, 1995; Feng and Lingrel, 1995). Analysis of the kinetic properties of the mutant Na,K-ATPases, however, revealed altered dependences for Na, K, and ATP as compared to the wild-type Na,K-ATPase. In the case of K, all mutants display a decrease in the apparent affinity for the cation, while for Na, the affinity is reduced in the Glu Ala and Glu Asp substitutions and, unexpectedly, is increased slightly in the Glu Lys mutant. A similar decrease in the relative Na affinity was observed for the alpha(Glu Ala)beta1 enzyme in transfected COS and HeLa cells (Vilsen, 1995; Feng and Lingrel, 1995). All three mutants exhibit a significantly higher affinity for ATP compared to the wild-type enzyme, while sensitivity to the cardiotonic inhibitor, ouabain, remains relatively unaltered.

The observation that replacement of intramembrane Glu with the positively charged lysine residue does not inactivate Na,K-ATPase function nor exhibit a greater effect on cation affinities, argues against a primary role for Glu as a ligating residue within the cation binding domain. Rather, it seems likely that the glutamate residue participates indirectly in transport function by contributing to the overall structural integrity of the cation binding domain by possibly allowing the proper positioning of the ligating residues within the binding pocket. Hence, amino acid substitutions at position 781 that modify cation dependence are predicted to alter the proper active site architecture required for high-affinity binding. Furthermore, the observation that mutations of Glu affect both Na and K affinities is consistent with the notion that the residues involved in Na binding and transport are also involved in binding and transport of K. This is also supported by the finding that K, and to a lesser extent Na, can protect against modification by DEAC (Argüello and Kaplan, 1991). In addition, the ability of all three mutants to stably associate with the beta subunit to form active enzymes, along with the unmodified ouabain sensitivities, demonstrates that mutation of Glu does not introduce gross conformational changes in the enzyme. Rather, the effect of the Glu mutations appears to be localized to the cation binding domain and, secondarily, to the catalytic site.

Site-specific mutagenesis was recently used by Vilsen(1995) to replace Glu of the rat Na,K-ATPase alpha subunit with an alanine residue. When expressed in COS cells, the Glu Ala mutant was shown to exhibit a significant K-independent Na-ATPase activity, although the exact mechanism underlying the Na-ATPase was not determined. A similar mutation in the sheep alpha1 subunit, however, did not exhibit a detectable K-independent Na-ATPase activity (Feng and Lingrel, 1995). In the present study, using the rat alpha1 subunit, we report a significant ouabain-sensitive Na-ATPase activity in Sf9 cells expressing the alpha(Glu Ala)beta1 isozyme and provide evidence for a Na/Na exchange mechanism, with Na acting as a surrogate for K, to account for this aberrant activity. This conclusion is based on the finding that (i) Na in the absence of K stimulates the ATPase activity of the Na,K-ATPase mutant, (ii) in flux assays, the alpha(Glu Ala)beta1 mutant can mediate the inward transport of extracellular Na, and (iii) trypsin digestion of the alpha(Glu Ala)beta1 mutant in Na medium produces a peptide fragment (M(r) = 58,000) normally associated with the E(2)(K) conformation of the enzyme. These results preclude both the ADP-sensitive form of Na/Na exchange, which is not accompanied by appreciable hydrolysis of ATP, and uncoupled Na efflux in which only intracellular sodium is bound and transported (reviewed in Glynn, 1985).

The elevated Na influx associated with the Glu Ala substitution can be readily explained by a decreased capacity of the enzyme to discriminate between Na and K at the extracellular loading sites. As a result, extracellular Na substitutes for K, promoting its uptake and enzyme turnover. A rearrangement of the ligating residues within the cation binding pocket that alter the constants for K and Na binding could also account for the decreased capacity of the Glu Ala mutant enzyme to discriminate among alkali metal ions at the outer surface. Consistent with a reduction in cation selectivity is the finding that the affinity for Cs, a weak congener of K in enzyme activation, is increased in the Glu Ala mutant as compared to the wild-type enzyme. This increase in Cs affinity gives the mutant enzyme a nearly identical affinity for Cs and K. Interestingly, this effect appears to be limited to the extracellular loading sites, as K alone cannot substitute for Na in mutant enzyme activation. This indicates a greater cation selective capacity at the cytoplasmic surface, and is consistent with the inability of cytoplasmic K to substitute for Na in the reaction cycle of the wild-type enzyme (Dunham and Senyk, 1977).

During the reaction cycle, binding information is transmitted between the ATP domain in the major cytoplasmic loop and the cation sites. Communication between the two domains is critical for coordination of cation transport. For example, both ATP and P(i) have been shown to stimulate deocclusion of K, while binding of Na at the cytoplasmic activation site is required for the phosphorylation by ATP bound at the catalytic center. On the basis of the potential role of intramembrane Glu in cation binding and its proximity to the cytoplasmic ATP binding domain, it has been suggested that this residue may reside within a segment that actively links the two ligand binding domains (Argüello and Kaplan, 1994; Lutsenko et al., 1995). Consistent with this theory, we show that replacement of intramembrane Glu by an aspartate, lysine, or alanine residue results in an increase in the ATP affinity. Interestingly, substitution of Glu in the forth transmembrane segment has also been shown to modify the affinities for Na, K, and ATP (Vilsen, 1993). By analogy, this may also indicate a role for the fourth transmembrane segment in the transmission of information between the cation and ATP binding domains. Thus, ligand-induced conformational changes in one domain appear to be transmitted to the corresponding domain through the adjacent transmembrane segment. It should be noted that the ATP affinities reported here are for the low-affinity ATP site, as accurate calculation of the ATP dependence at the high-affinity site is not possible in our enzyme preparations.

Interestingly, our observations using infected Sf9 insect cells are similar to studies using site-directed mutagenesis and expression in mammalian cells (Vilsen, 1995; Feng and Lingrel, 1995). Substitutions of this glutamate in all three studies demonstrate a modified affinity for Na and an unaltered sensitivity to ouabain. Moreover, with the exception of the Glu Gln mutant expressed in HeLa cells (Feng and Lingrel, 1995), all glutamate mutants display a decreased apparent affinity for K. However, there are significant differences between the studies. For example, (i) while Glu mutants expressed in COS or Sf9 insect cells exhibit an increase in the apparent affinity for ATP at the low affinity site, no change in ATP activation is observed for the equivalent mutants expressed in HeLa cells, and (ii) the Glu Ala mutant when expressed in HeLa cells does not exhibit a K independent, Na-ATPase activity (Feng and Lingrel, 1995). It is possible that the modified sheep alpha1 subunit has different properties than the rodent subunit. This possibility will require further investigation. Finally, an advantage of the baculovirus expression system is that the mutant Na,K-ATPases do not have to confer ouabain resistance to be analyzed. Thus, although the Glu Asp substitution did not alter ouabain sensitivity in HeLa cells (Feng and Lingrel, 1995), it is catalytically competent.

In conclusion, Glu of the Na,K-ATPase alpha subunit is a critical coordinate of cation selectivity and activation, however, this residue does not appear to have a direct role in the binding and occlusion of cations. Similar conclusions have been obtained studying mutations in Glu using transfected mammalian cells (Vilsen, 1995; Feng and Lingrel, 1995). Additionally, the effect of Glu substitutions on the apparent ATP affinity suggests a role for the fifth membrane segment in the transmission of binding information between the ATP binding domain and the cation transport region of the alpha subunit.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM 39746 and the George M. O'Brien Kidney and Urological Diseases Center at Washington University School of Medicine. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Cell Biology and Physiology, Washington University School of Medicine, 660 S. Euclid Ave., Box 8228, St. Louis, MO 63110. Tel.: 314-362-6924; Fax: 314-362-7463.

(^1)
The abbreviations used are: DEAC, 4-(diazomethyl)-7-(diethylamino)coumarin; PAGE, polyacrylamide gel electrophoresis; CHAPS, 3-[(3-cholamidopropyl)dimethyl ammonio]-1-propanesulfonate.


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