©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Glutamic Acid 327 in the Sheep 1 Isoform of Na,K-ATPase Stabilizes a K-induced Conformational Change (*)

(Received for publication, October 5, 1994; and in revised form, December 1, 1994)

Theresa A. Kuntzweiler (§) Earl T. Wallick (1) Carl L. Johnson (1) Jerry B Lingrel (¶)

From the Department of Molecular Genetics, Biochemistry, and Microbiology Department of Pharmacology and Cell Biophysics, University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267-0524

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

By combining the tools of site-directed mutagenesis and [^3H]ouabain binding, the functional role of glutamic acid 327 in the fourth transmembrane domain of the sheep alpha1 isoform of Na,K-ATPase was examined with respect to its interactions with ouabain, Na, K, Mg, and inorganic phosphate. Using site-directed mutagenesis, this glutamic acid was substituted with alanine, aspartic acid, glutamine, and leucine. The mutant proteins were constructed in a sheep alpha1 protein background such that [^3H]ouabain binding could be utilized as a highly specific probe of the exogenous protein expressed in NIH 3T3 cells. Na competition of [^3H]ouabain binding to the mutant forms of Na,K-ATPase revealed only slight alterations in their affinities for Na and in their abilities to undergo Na-induced conformational changes which inhibit ouabain binding. In contrast, K competition of [^3H]ouabain binding to all four mutant forms of Na,K-ATPase displayed severely altered interactions between these proteins and K. Interestingly, [^3H]ouabain binding to the mutant E327Q was not inhibited by the presence of K. This mutant was previously reported to be functionally able to support cation transport with a 5-fold reduced K(0.5) for K-dependent ATPase activity (Jewell-Motz, E. A., and Lingrel, J. B.(1993) Biochemistry 32, 13523-13530; Vilsen, B.(1993) Biochemistry 32, 13340-13349). Thus, it appears that this glutamic acid in the fourth transmembrane domain may be important for stabilizing a K-induced conformation within the catalytic cycle of Na,K-ATPase that is not rate-limiting in the overall ATPase cycle but that displays a greatly reduced affinity for ouabain.


INTRODUCTION

The Na,K-ATPase (^1)is an integral membrane protein found in nearly all eukaryotic cells that utilizes energy from the hydrolysis of intracellular ATP to transport three sodium ions out of the cell and two potassium ions into the cell (1, 2, 3) . The enzyme is composed of an alpha subunit containing 8-10 transmembrane domains and a beta subunit which has one transmembrane segment. The catalytic cycle of Na,K-ATPase involves an acid-stable phosphorylated form of the protein, a characteristic shared by other active cation transporters such as the sarcoplasmic reticulum Ca-ATPase, the plasma membrane Ca-ATPase, H,K-ATPase, and Mg-ATPase(4, 5, 6, 7) . Unlike other ATPases, Na,K-ATPase is inhibited by cardiac glycosides such as ouabain.

In recent years, structure-function studies of Na,K-ATPase have focused on identifying the amino acids involved in binding and translocating Na and K ions. Both chemical modification studies and site-directed mutagenesis studies have targeted anionic amino acid residues located in the transmembrane domains of the alpha subunit. These residues contain negatively charged side chains which are thought to neutralize the cations during transport through the hydrophobic lipid bilayer. Six oxygen-containing residues in the transmembrane domains of the sarcoplasmic reticulum Ca-ATPase have been implicated as essential amino acids for Ca transport using site-directed mutagenesis(8) . Three of these residues contain carboxylic acid side chains. These negatively charged residues are conserved in the Na,K-ATPase and include Glu, Glu, and Asp in the sheep alpha1 isoform.

N,N`-dicyclohexylcarbodiimide (DCCD) labels Na,K-ATPase in a K protectable manner and blocks cation occlusion upon modification(9) . Hence, DCCD is thought to bind to a residue essential for potassium occlusion. By combining DCCD labeling and limited proteolytic digestion, two sites of DCCD modification have been located within the transmembrane domains (10) . Glutamic acid 953 in the COOH-terminal half of the sheep alpha1 protein has been identified as one site of modification and glutamic acid 327 is hypothesized to be another site of modification. Site-directed mutagenesis studies, involving Glu and a neighboring glutamic acid residue, Glu, have demonstrated that altering the charged side chains at these positions does not dramatically change the cation-dependence properties of ATP hydrolysis by Na,K-ATPase(11) . Therefore, it is possible that the residue labeled by DCCD in the NH(2)-terminal half of the enzyme, Glu, is the carboxyl group important for cation occlusion.

Glutamic acid 327 is located in the fourth transmembrane domain of the Na,K-ATPase and is highly conserved throughout the amino acid sequences of related ATPases(4, 5, 6, 7) . Previously, site-directed mutagenesis was employed to change Glu to Gln, Leu, Ala, and Asp(12, 13, 14) . These mutagenesis studies utilized similar expression schemes which take advantage of species-specific variations in ouabain sensitivity to examine the effects of the mutation on cell viability and on the cation-dependent ATPase activity. These schemes involve introducing the mutation into a cDNA which encodes a ouabain-resistant isoform of Na,K-ATPase (K = 1 times 10M; i.e. rat alpha1, sheep alpha1(RD) (^2)or rat alpha2*(^3)), expressing this mutant in a cell line containing a ouabain-sensitive endogenous form of the ATPase (K = 1 times 10M; COS-1 or HeLa) and selecting for cells expressing the ouabain-resistant protein by growing the cells in the presence of ouabain (0.2, 1, or 5 µM). An active Na,K-ATPase is essential for the survival of mammalian cells due to its role in the establishment and maintenance of the electrochemical gradient across the cell membrane. By growing these transfected cells in the presence of ouabain, the endogenous Na,K-ATPase is inhibited, and only the exogenous ATPase can produce the essential cation gradient. Only if the transfected cDNA encodes for an active enzyme will cells be viable in ouabain. The mutant forms of Na,K-ATPase, E327Q and E327L, were able to transport cations, produce an electrochemical gradient, and maintain cell viability. The mutants E327D and E327A were unable to support cell viability in the presence of ouabain. The cation-dependence of Na,K-ATPase activity was examined in the presence of 1 times 10M ouabain for mutant proteins E327L and E327Q. The mutant E327L demonstrated a 4-fold higher K(0.5) for Na and a 2-fold higher K(0.5) for K than the wild type protein. The mutant E327Q displayed a 2-fold higher K(0.5) for Na and a 3-6-fold higher K(0.5) for K than the wild type ATPase. From these small changes in the K(0.5) values, it was concluded that the charge of this glutamic acid side chain was not essential for ATPase activity. However, based on the cell viability results, the length of the side chain was suggested to be important sterically in the enzymatic cycle. Although these original site-directed mutagenesis studies revealed some important facts about the glutamic acid at position 327, the mechanistic step (i.e. cation binding or conformational changes) that the substitutions E327D and E327A disrupt was not identified, since these mutant enzymes were inactive and could not be examined.

In order to study both inactive and active mutant enzymes, we have examined the cation-enzyme interactions of Na,K-ATPase by observing the effects of Na and K on ouabain binding to the enzyme. The affinity of the Na,K-ATPase for cardiac glycosides is closely linked to enzyme cycling. Two assay environments which are known to induce high affinity ouabain binding to the enzyme are Mg, ATP, and Na or Mg and P(i)(15, 16) . Both of these conditions promote formation of a phosphorylated form of the enzyme (E2-P) which is cation-sensitive. Thus, the role specific amino acids play in binding cations during the catalytic cycle of Na,K-ATPase may be probed by studying the effects of cations on [^3H]ouabain binding to mutant forms of the Na,K-ATPase.


EXPERIMENTAL PROCEDURES

Materials

Molecular biology reagents were purchased from New England Biolabs, Amersham Corp., Pharmacia, Promega, and Qiagen. Cell culture supplies were obtained from Life Technologies, Inc. and Fisher. [^3H]ouabain was purchased from DuPont NEN. The specific radioactivity of the [^3H]ouabain was determined as described previously(17) . Scintillation fluid was purchased from Research Products International Corp. All other reagents (NaCl, KCl, Tris base, HCl, phosphoric acid, MgCl(2), and ouabain) were of the highest quality available.

Site-directed Mutagenesis

Eukaryotic expression vectors, pKC4, containing either the wild type sheep Na,K-ATPase cDNA or a mutant cDNA were constructed as described previously(18) . Briefly, a cassette from the coding region of the sheep Na,K-ATPase alpha1 subunit cDNA was subcloned into the bacteriophage M13. The cassette contained an 847-base pair (bp) XbaI-PstI fragment including the region encoding amino acid resides 230-512. The Kunkel (19) method of site-directed mutagenesis was used to introduce the desired mutations into the cassette. Cassettes containing the appropriate mutations were sequenced in their entirety to screen for unwanted mutations. These cassettes were subcloned back into the context of the wild type sheep Na,K-ATPase alpha1 subunit cDNA in the pKC4 expression vector. Final constructs were analyzed by restriction analysis, as well as by sequencing across the mutated site. All plasmids were purified on Qiagen columns prior to their use for transfection.

Transfection of NIH 3T3 Cells

NIH 3T3 cells were maintained in Dulbecco's modified Eagle's medium containing 10% calf serum as described elsewhere(18) . Cotransfection of NIH 3T3 cells with 20 µg of plasmid DNA containing a 20:1 molar ratio of pKC4 vector to pSKNeoA was performed using a modified calcium phosphate procedure(20) . Two days after transfection the cells were split 1:100 and selected in 400 µg/ml G418. After 10 days, 10 colonies for each mutant form of sheep alpha1 were isolated and expanded into stable cell lines.

Sequence Analysis of Genomic DNA Isolated from Clonal NIH 3T3 Cell Lines

Genomic DNA was isolated from the neomycin-resistant cell lines cotransfected with pKC4 containing the mutant sheep alpha1 cDNAs and pSKNeoA. A polymerase chain reaction (PCR) was used to amplify an 821-bp fragment containing the mutated codon at position 1258-1260 bp. The primers utilized in the PRC reactions were designed to span a region which contains exons 8-11 in the genomic sequence of Na,K-ATPase. Thus, a product of the appropriate size (821 bp) was generated from the exogenous sheep alpha1 Na,K-ATPase cDNA and not from the endogenous mouse DNA. The PCR fragments were sequenced on an Applied Biosystems model 373 DNA sequenator (Applied Biosystems, Inc., Foster City, CA) using the Taq DyeDeoxy Sequencing protocol with dye-labeled terminators.

Isolation of Crude Plasma Membranes from NIH 3T3 Cells

Crude plasma membranes were isolated from transfected NIH 3T3 cells as described by Schultheis et al.(21) . Thirty confluent 150-mm tissue culture dishes were used for each NaI preparation. Between five and eight preparations of two clonal lines of each mutant were required to complete the [^3H]ouabain binding studies presented here. A modified Lowry procedure was used to determine the protein concentration of each membrane preparation(22) .

The crude plasma membrane preparations from NIH 3T3 cell lines were analyzed by Western immunoblotting. The proteins contained in the membrane preparations were resolved on a 10% SDS-polyacrylamide gel, electrophoretically transferred onto nitrocellulose, and stained with the monoclonal antibody M7-PB-E9 and an antimouse horseradish peroxidase-conjugated secondary antibody. M7-PB-E9 was raised against lamb kidney Na,K-ATPase and is alpha subunit specific such that it distinguishes the transfected sheep alpha1 isoform from the endogenous mouse alpha1 subunit(23, 24) . M7-PB-E9 was a generous gift from the laboratory of Dr. W. J. Ball (University of Cincinnati).

[^3H]Ouabain Binding to Crude Membrane Fragments

All ouabain binding studies were conducted under the following conditions unless otherwise indicated in the figure or table legends: 5 mM MgCl(2), 5 mM Tris-phosphate, 50 mM Tris-HCl (pH 7.4) in a final volume of 0.5 ml. The samples were incubated for 6 h at 37 °C. The amount of protein used was dependent on the specific activity of the membrane preparation. Typically, 20-100 µg of total protein was present in each assay tube. Experiments were carried out in disposable plastic tubes. For the competition curves with unlabeled ouabain, eight concentrations of unlabeled ouabain (including zero) were examined in triplicate. The concentrations of unlabeled ouabain are indicated on the x axis of Fig. 2. The concentration of [^3H]ouabain was set at approximately 3 nM; however, aliquots of the reaction mixtures were taken with each experiment to determine the exact concentration of [^3H]ouabain used. Ligand and enzyme concentrations were adjusted such that no more than 10% of the added ouabain was bound. Thus, the free ligand concentration was essentially equal to the added ouabain concentration in all of the experiments presented, and the total concentration of ouabain was used in place of the free concentration in the equations used to analyze the competition data. Following incubation, the samples were aspirated onto glass fiber filters using a Brandel M24R cell harvester. The filters were washed four times with 5 ml of cold water. Filters were placed in RPI Budget-solve and counted in a Packard 2000 CA Scintillation counter with an efficiency of 42%. Monovalent cation inhibition experiments were conducted under the same incubation conditions described above unless otherwise indicated in the figure legends. Eleven to 15 concentrations of each cation (including zero) were used in duplicate to define the inhibition curves. The [^3H]ouabain concentration in the cation competition experiments was set at approximately 10 nM; however, the exact concentration was measured for each experimental mix and adjusted in the curve fits. Data were plotted and fit to either a simple Hill equation (n(a), n(i), and AC values) or to a cooperative model (K(i) and K(a) values) (see figure legends) using KaleidaGraph by Abelbeck Software. The results presented in Fig. 2Fig. 3Fig. 4have been normalized such that the maximum binding in the absence of competitor (unlabeled ouabain, Na, or K, respectively) was set at 100% to account for varying expression levels of the proteins.


Figure 2: Ouabain competition curves. [^3H]Ouabain binding was measured in the absence and presence of various concentrations of unlabeled ouabain as shown on the x axis. The symbols represent the mean of triplicate determinations. The error was calculated for each point and is shown unless it is smaller than the symbol size. Assay conditions were as described under ``Experimental Procedures,'' except for E327D which was assayed in the presence of 15 mM Tris-P(i) and 10 mM MgCl(2). Symbol representation is as follows: box, wild type sheep alpha1; circle, E327Q; , E327D; , E327L; and , E327A. The data were fit to a simple self-competition model:

where [I] is the concentration of unlabeled ouabain, E is the amount of enzyme, [O] is the concentration of [^3H]ouabain, and NS is the proportionality constant for nonspecific binding. Three adjustable parameters were calculated for each curve and include K (dissociation constant), Eand NS. The fitted parameters were calculated: wild type sheep alpha1 (K = 1.51 ± 0.18 nM; E = 0.106 ± 0.003 nM; NS = 0.000343 ± 0.000012); E327Q (K = 4.06 ± 0.35 nM; E = 0.130 ± 0.004 nM; NS = 0.000322 ± 0.0000096); E327D (K = 12.5 ± 0.8 nM; E = 0.268 ± 0.009 nM; NS = 0.000379 ± 0.0000146); E327L (K = 2.87 ± 0.31 nM; E = 0.142 ± 0.004 nM; NS = 0.000470 ± 0.0000143); E327A (K = 1.67 ± 0.13 nM; E = 0.110 ± 0.004 nM; NS = 0.000365 ± 0.000012).




Figure 3: Na competition curves. [^3H]Ouabain binding was measured for isolated membranes in the absence and presence of various concentrations of NaCl as shown on the x axis. The symbols represent the mean of duplicate determinations. The error bars represent the range of the duplicate determinations and are not shown if smaller than the symbol size. The assay conditions were as described under ``Experimental Procedures,'' except for E327D which was assayed in the presence of 15 mM Tris-P(i) and 10 mM MgCl(2). The symbol representation is as follows: box, wild type sheep alpha1; bullet, E327Q; , E327D; , E327L; and , E327A. The curves displaying the data of E327Q and the wild type were fit to a competitive model involving three Na sites:

where K is the dissociation constant for ouabain obtained from the ouabain competition experiments (Fig. 2) and [O] is the concentration of [^3H]ouabain. E327D, E327L, and E327A were fit to a similar competitive equation involving two Na sites such that the fourth term in the denominator of this equation was eliminated. The two adjustable parameters calculated were: K, (Na inhibition constant) and E (amount of ouabain-binding sites). The fitted values obtained were: wild type sheep alpha1 (K = 15.7 ± 0.1 mM; E = 0.241 ± 0.003 nM); E327Q (K = 20.8 ± 0.3 mM; E = 0.167 ± 0.003 nM); E327D (K = 7.51 ± 0.16 mM; E = 0.359 ± 0.008 nM); E327L (K = 19.2 ± 0.4 mM; E = 0.106 ± 0.002 nM); and E327A (K = 24.2 ± 0.4 mM; E = 0.289 ± 0.003 nM).




Figure 4: K-Induced effects on ouabain binding. [^3H]Ouabain binding was measured in NaI-treated membranes in the presence of various concentrations of KCl as shown on the x axis. The symbols represent the mean of duplicate determinations. The error bars represent the range of the duplicate determinations and are not shown if smaller than the symbol size. The assay conditions were (5 mM P(i) and 5 mM Mg) as described under ``Experimental Procedures.'' The assay conditions for the inset data were the same with the exception that the Mg concentration was 10 mM and the Tris-P(i) concentration was 15 mM. The symbol representation is as follows: box, wild type sheep alpha1; bullet, E327Q; , E327D; , E327L; and , E327A. The amount of bound [^3H]ouabain (B) as a function of K concentration was calculated with the following equation:

where E is the total enzyme concentration, [O] is the concentration of [^3H]ouabain, alpha is the interaction factor of inhibition, and NS is the proportionality constant for nonspecific binding. Fitted parameters for the inhibition phase were: wild type sheep alpha1 (at 5 mM P(i) and 5 mM Mg) (K = 1.02 ± 0.03 mM; E = 0.115 ± 0.002 nM; alpha = 10.1 ± 0.2); E327A (at 5 mM P(i) and 5 mM Mg) (K = 0.879 ± 0.009 mM; E = 0.117 ± 0.003 nM; alpha = 2.76 ± 0.11); E327L (at 5 mM P(i) and 5 mM Mg) (K = 1.18 ± 0.07 mM; E = 0.139 ± 0.003 nM; alpha = 3.35 ± 0.11); wild type sheep alpha1 (at 15 mM P(i) and 10 mM Mg) (K = 1.17 ± 0.06 mM; E = 0.112 ± 0.002 nM; alpha = 12.9 ± 0.3). Fitted parameters for the activation phases: E327A (K = 84 ± 28 mM; beta = 1.40 ± 0.31); E327D (at 5 mM P(i) and 5 mM Mg) (AC = 2.71 ± 0.54 mM); E327D (at 15 mM P(i) and 10 mM Mg) (AC = 2.23 ± 0.51 mM). K values for E327A were calculated by fitting both the inhibition and the activation phase of these data to the following equation:

where beta is the interaction factor of activation. Data describing the KCl effects on E327Q were not fit to any activation or inhibition models, and a smooth curve was drawn through the data.



[^3H]Ouabain Binding to Whole NIH 3T3 Cells

Neomycin-resistant cell lines were distributed onto 24-well plates with approximately 2.5 times 10^5 cells/well. Six wells of each cell line were plated and allowed to grow for 2 days prior to [^3H]ouabain binding. [^3H]ouabain binding to transfected 3T3 cells was measured by incubating the cells in 0.5 ml of K/Ca-free buffer containing 60 nM [^3H]ouabain (for total binding) or 60 nM [^3H]ouabain plus 1 µM unlabeled ouabain (for nonspecific binding) at 37 °C for 30 min. The details of this procedure have been described elsewhere(25) .


RESULTS

Expression of Glu Mutants

Previous site-directed mutagenesis studies have produced conflicting conclusions concerning the importance of Glu in the mechanism of Na,K-ATPase(11, 12, 13, 14) . Based solely on the inability of E327A and E327D to support cell viability, it might be concluded that Glu is functionally essential. However, E327Q and E327L yield active enzymes suggesting that this residue may not be essential. In order to compare all 4 Glu substitutions in the same system, each mutant was remade in a ouabain-sensitive protein and characterized with respect to its ability to interact with Na and K using [^3H]ouabain binding as a probe. This system of characterizing the mutant proteins is not dependent on the Na,K-ATPase being catalytically active.

The mutations were constructed in a sheep alpha1 cDNA which encodes a ouabain-sensitive enzyme, and these cDNAs were transfected into NIH 3T3 cells containing an endogenous protein with a low affinity for ouabain. The exogenous proteins bind ouabain with a 1000-fold higher affinity than the endogenous enzyme. Thus, at the low concentrations of [^3H]ouabain utilized in these experiments, there is no interference from the binding of [^3H]ouabain to the endogenous Na,K-ATPase(21) . Since the exogenous protein is ouabain sensitive, no selectable function is conferred to the cells upon expression of these cDNAs; therefore, an alternative to ouabain selection was required. The mutated cDNAs were cotransfected with a gene which codes for a neomycin resistance protein and selected in 400 µg/ml of G418. cDNAs encoding all four substitutions (E327A, E327D, E327Q, and E327L) were transfected into NIH 3T3 cells, and clonal cell lines were established for each mutant. Western analysis of crude membrane preparations from these cell lines indicated that the sheep alpha1 proteins (wild type and mutants) were being expressed. To establish that the transfected cDNAs were integrated into the cell genome, genomic DNA was isolated from each clonal cell line. Using sheep alpha1-specific primers, PCR was employed to amplify a region of the DNA which encodes amino acids 230-512 of the exogenous DNA. The DNA fragment from each cell line was sequenced and revealed a single altered codon which encoded the desired amino acid at position 327. No additional mutations were detected in the DNA fragment surrounding the mutated codon.

[^3H]Ouabain binding to intact 3T3 cells was performed to establish that the mutant sheep alpha1 enzymes were located in the plasma membrane. Fig. 1presents the amount of [^3H]ouabain (60 nM) bound/mg of total protein for untransfected NIH 3T3 cells and for two clonal cell lines of each mutant and wild type sheep alpha1 Na,K-ATPase. The nonspecific binding (NS) of [^3H]ouabain (60 nM) to each cell line was measured in the presence of 1 µM unlabeled ouabain. From these data, one can see that the cell lines which express sheep alpha1 proteins (mutant or wild type) bind 5-10-fold more [^3H]ouabain/mg of total protein than the untransfected 3T3 cells and at least 4-fold more than nonspecific binding. The presence of [^3H]ouabain binding in the mutant cell lines reveals that at least some of the translated mutant sheep alpha1 protein is located in the plasma membranes of these clonal cell lines. Moreover, the [^3H]ouabain binding to intact cells indicates that the mutant proteins are assembled with an extracellular protein conformation sufficiently similar to the native sheep alpha1 protein to permit ouabain binding. The variation in maximum binding levels for each mutant cell line may be due to different expression levels, secondary to variable insertional sites of the transfected cDNAs in the genomic DNA. It is interesting to note that E327L has the lowest expression level in 3T3 cells as measured by [^3H]ouabain binding to whole cells, yet it is translated at high enough levels in HeLa cells to support viability. Thus, it does not appear that the lower expression levels of the mutant sheep alpha1 proteins are the result of the amino acid substitution interrupting the protein processing steps of Na,K-ATPase.


Figure 1: [^3H]Ouabain binding to intact NIH 3T3 cells. The amount of [^3H]ouabain/mg of total protein is displayed for the clonal 3T3 cells expressing wild type sheep alpha1 protein (WT), untransfected NIH 3T3 cells (3T3), and clonal 3T3 lines expressing E327A, E327Q, E327D, and E327L. The maximum values represent the amount of 60 nM [^3H]ouabain bound to intact cells in the presence of K/Ca-free buffer with respect to the total protein within these cells. Nonspecific binding (NS) to the intact 3T3 cells was also measured in identical conditions with the addition of 1 µM unlabeled ouabain. WT-NS, A-NS, Q-NS, D-NS, and L-NS display the nonspecific binding observed for the clonal cell lines expressing wild type sheep alpha1 Na,K-ATPase, E327A, E327Q, E327D, and E327L, respectively. The values were obtained by averaging the amounts bound from three separate experiments performed in triplicate for two clonal cell lines of each mutant.



Ouabain Competition Curves in Glu Mutants

In the presence of Mg and P(i), the wild type Na,K-ATPase maintains a protein conformation which binds ouabain with high affinity. Monovalent cations (Na and K) inhibit ouabain binding under these conditions, presumably by inducing a conformational change in the enzyme that results in a markedly reduced affinity for ouabain. Prior to studying these cation-induced conformational changes, the affinity of each mutant for ouabain was determined in the presence of Mg and P(i). Competition curves were carried out using [^3H]ouabain and unlabeled ouabain and the data were fit to a simple self-competition model. Fig. 2shows a comparison of the ouabain competition curves for one membrane preparation from each Glu mutant and the wild type isoform. The mean K(D) values obtained for three different preparations of wild type and three different mutant membrane preparations for each substitution are presented in Table 1. Under these binding conditions, the E327A mutation did not significantly affect the affinity of the enzyme for ouabain. However, the amino acid substitutions E327L, E327Q, and E327D increased the K(D) of the enzyme for ouabain by 1.6-, 2.9-, and 2.1-fold, respectively. Nonspecific binding expressed as a percentage of total binding was less than 1.26 ± 0.34% for all membrane preparations. This nonspecific binding was due to [^3H]ouabain binding to the filters and was independent of the enzyme concentration in the binding study. These initial studies demonstrated that all the mutants are able to form a protein conformation which has a high affinity for ouabain in the presence of Mg and P(i).



Sodium Inhibition Curves for Wild Type and Glu Mutant Forms of Na,K-ATPase

In the presence of Mg and P(i), [^3H]ouabain binding to sheep alpha1 Na,K-ATPase (expressed in 3T3 cells or purified from sheep kidney) is decreased to the same extent by saturating concentrations of Na as by saturating concentrations of unlabeled ouabain(16) . Na-induced inhibition curves for the Glu mutants are presented in Fig. 3. These data represent a single curve for one membrane preparation from each mutant cell line and one from a wild type sheep alpha1 cell line. Na competition experiments were conducted with at least three different membrane preparations of each mutant. Na inhibition data were initially fit to a simple Hill equation from which a Hill coefficient for Na was determined for each mutant. These coefficients reflect the number of Na ions which interact with the proteins to inhibit the binding of [^3H]ouabain and, thus, were used to define the number of Na terms utilized in the competition models. Na inhibition data from E327A, E327L, and E327D were fit to a competition equation involving two Na ions whereas data from E327Q and the sheep alpha1 wild type were fit to a competition model involving three Na ions (see legend to Fig. 3). E327A, E327L, and E327Q all demonstrated a slight increase in K(i) values for Na inhibition of [^3H]ouabain binding as compared to the wild type enzyme (Table 1). In contrast, E327D displayed a slightly lower K(i) value for Na effects on [^3H]ouabain binding than that observed for the sheep alpha1 enzyme. All curves exhibited a complete inhibition of [^3H]ouabain binding at saturating concentrations of Na. Thus, it appears that all of the Glu mutant proteins have relatively unaltered affinities for Na (K(i) values increased only 2-fold) and are able to undergo Na-induced conformational changes which reduce ouabain binding (all display apparent simple competitive inhibition).

The effects of increasing ionic strength on [^3H]ouabain binding were examined in a series of equilibrium binding assays with increasing concentrations of choline chloride. Similar to previous studies(26) , choline chloride concentrations above 150 mM inhibited [^3H]ouabain binding to both mutant and wild type Na,K-ATPase membrane preparations (data not presented). IC values obtained from fitting these data to a simple Hill equation were approximately 195-210 mM for both the wild type and the mutant forms of the ATPase. Therefore, the effects of Na and K observed at cation concentrations leq100 mM can only be explained through specific interactions of these monovalent cations with the enzyme and do not reflect inhibition due to elevated ionic strength.

Potassium Competition Curves for Wild Type and Glu Mutant Forms of Na,K-ATPase

The wild type sheep alpha1 protein (expressed in 3T3 cells or purified from sheep kidney) was previously demonstrated to have a reduced affinity for [^3H]ouabain when K was present in the Mg-P(i) medium(16, 26) . An example of the effects of K on [^3H]ouabain binding to the Glu mutants is presented in Fig. 4. The K-induced effects range from inhibition to activation of ouabain binding. The data presented are from one membrane preparation of each mutant; however, these K curves were repeated eight times with at least four different membrane preparations and two separate clonal cell lines. The K-effects presented in Fig. 4are highly reproducible and reflect the effects of the amino acid substitutions on the interaction between Na,K-ATPase and ouabain. The nonspecific binding was similar to that described under the ouabain competition results.

Previously, a partially competitive model for K effects on [^3H]ouabain binding to wild type sheep alpha1 protein was used to describe the incomplete inhibition of ouabain binding at saturating K concentrations(26) . The data obtained for the wild type sheep alpha1 enzyme and for the K inhibition phase of E327L and E327A fit this model (see Fig. 4legend). The Hill coefficients and the K(i) values characterizing the K inhibition of ouabain binding to E327L and E327A are presented in Table 1. The potassium K(i) values are approximately 1 mM for E327L and E327A, similar to the K(i) value for the wild type Na,K-ATPase. Unlike the wild type and E327L proteins, higher concentrations of K (geq10 mM) stimulate [^3H]ouabain binding to the mutant E327A protein. This increase in [^3H]ouabain binding is characterized by a K(a) value of 84 ± 28 mM and a Hill coefficient of 0.97 ± 0.35. Generally, it appears that both E327A and E327L bind low concentrations of K in a manner similar to the wild type (similar K(i) values) and undergo a K-induced conformational change which reduces their affinity for [^3H]ouabain.

In contrast to the results with E327A and E327L, K did not inhibit [^3H]ouabain binding to the E327Q and E327D mutants. The effect of K on E327Q was not fit to any model. The data describing K interactions with E327D demonstrated an increase in [^3H]ouabain binding with increasing concentrations of K and were fit to a simple Hill equation yielding a Hill coefficient of 1.9 ± 0.3 and an AC of 3.14 ± 0.19 mM. Overall, it appears that either the ability of E327D and E327Q to bind K has been altered or that these mutants do not undergo the K-induced conformational change which normally reduces the affinity of the enzymes for [^3H]ouabain.

Stimulation of [^3H]Ouabain Binding by Mg and P(i)

Activation of [^3H]ouabain binding to Na,K-ATPase was examined with respect to Mg and P(i) concentrations to ensure that the amino acid substitutions did not dramatically alter the affinity of the enzyme for these components. If the enzyme affinity for either Mg or P(i) had been drastically reduced, the amount of [^3H]ouabain bound in the absence of inhibitor (unlabeled ouabain, Na, or K) would not be dependent solely on the level of expressed enzyme but also on the number of occupied Mg or P(i) sites. As can be seen in Table 1, the apparent AC values for Mg stimulation of [^3H]ouabain binding in the presence of 5 mM P(i) were between 0.20 and 0.85 mM. The cation competition curves presented in Fig. 3and Fig. 4were performed in the presence of 5 mM Mg. Thus, all Mg activation site(s) were saturated in the absence of monovalent cations.

Apparent AC values for P(i) stimulation of [^3H]ouabain binding in the presence of 5 mM Mg are also presented in Table 1. These values range from 0.08 to 1.25 mM. Cation competition data presented in Fig. 3and Fig. 4were obtained in 5 mM P(i). Thus, all the inorganic phosphate site(s) which stimulate ouabain binding were occupied for the mutant proteins E327A, E327L, and E327Q in the absence of monovalent cations. The E327D mutant demonstrated the largest change in its AC value for P(i) which was approximately 10-fold higher than the wild type Na,K-ATPase. The ouabain, Na, and K competition curves were therefore repeated in the presence of 15 mM P(i) and 10 mM Mg for the E327D mutant enzyme, and these calculated kinetic constants are reported in Table 1. The K competition constants were not significantly changed by the higher P(i) and Mg concentrations for either the E327D or wild type proteins (see inset of Fig. 4). This is consistent with the concept that the inhibition of K is a direct effect and not an indirect effect due to a change in the degree of saturation by Mg and/or P(i). Additional K and Na inhibition curves at various Mg and P(i) concentrations must be done to fully understand the interaction of all these ions (monovalent or divalent cations or P(i)) with the Na,K-ATPase. Since all Mg and P(i) activation sites were saturated in the absence of monovalent cations under these equilibrium condition, we conclude that the inhibition and activation of [^3H]ouabain binding observed in Fig. 2Fig. 3Fig. 4were due to ouabain, Na, and K, respectively.


DISCUSSION

The exact role that glutamic acid 327 plays in the sheep alpha1 Na,K-ATPase is unknown. ATPase activity studies characterizing proteins containing amino acid substitutions at this site have been limited by the nonfunctional character of some of the mutants. In this work, the cation-protein interactions of four mutant proteins, E327Q, E327D, E327L, and E327A, were examined using Na and K inhibition of [^3H]ouabain binding. This radiolabeling technique can in principle detect any mutation in the Na,K-ATPase which alters either the number of cations associated with the enzyme or which alters the stability of an intermediate within the Na,K-ATPase pathway which is normally sensitive to cation binding.

Effects of Substitutions on Na,K-ATPase Affinity for Ouabain

The affinity of Na,K-ATPase for ouabain is slightly altered by the mutations at Glu as shown by the ouabain competition curves. The higher K(D) values for ouabain associated with the mutants may reflect small structural rotations of the H(4) transmembrane domain. Previously, tyrosine 308 in the extracellular loop between H(3) and H(4) was shown to alter the affinity of Na,K-ATPase for ouabain(27) . The substitutions at Glu may change this putative contact point of the ouabain receptor site and weaken ouabain binding. Substitutions of other transmembrane amino acid residues (i.e. Cys, Tyr, and Thr) also reduce the affinity of Na,K-ATPase for ouabain(12, 28, 29) . Any mutation which disrupts the three-dimensional orientation of the transmembrane domains may allosterically influence other residues that compose the binding site for ouabain and thus affect the affinity of Na,K-ATPase for the drug.

Na Inhibition of [^3H]Ouabain Binding

Na inhibits ouabain binding to all four mutants in the presence of Mg and P(i). Three mutants, E327A, E327D, and E327L, display an apparent reduction in the number of Na ions which interact with the enzyme to inhibit ouabain binding (i.e. Hill coefficient approx2 rather than 3 as in the case of wild type and E327Q). This does not correlate with the ability of the mutants to support cell life. For example, E327L demonstrates a Hill coefficient of two as probed by inhibition of [^3H]ouabain binding, yet supports viability of HeLa cells. Thus, it appears that disrupting one inhibitory site for Na does not seriously disturb the binding of the remaining two sodium ions or their inhibitory effect on [^3H]ouabain binding. These observed alterations in the Hill coefficients may be reflective of altered cation stoichiometry for the mutant proteins as previously demonstrated under acidic conditions for purified Na,K-ATPase(30, 31) . However, it is possible that the reduced number of Na ions which inhibit ouabain binding does not directly relate to the number of Na ions being transported by the mutant enzymes but simply describes the number of Na ions which influence ouabain binding.

In addition to altered Hill coefficients, the K(i) values for Na inhibition of ouabain binding were increased nearly 2-fold for E327L, E327A, and E327Q. Previously, Na interactions with E327Q and E327L were investigated using cation-dependent ATPase assays in the presence of ATP, Mg, and saturating concentrations of K(13, 14) . The K(0.5) values for the Na dependence of ATPase activity for E327Q and E327L were reported to be 2-fold higher than that of the wild type ATPase. This similar increase in K(0.5) values and K(i) values for Na is consistent with the concept that the Na sites which activate the ATPase activity may be identical to the Na sites which inhibit ouabain binding. It appears that substitution of the carboxyl side chain of Glu disrupts Na binding to the enzyme by either reducing the number of Na inhibition sites (E327D, E327L, and E327A) or by increasing the inhibition constant of Na (E327Q, E327L, and E327A). No correlation exists between the inability of E327A and E327D to support cell viability and their ability to interact with Na as measured by inhibition of ouabain binding.

K Effects on [^3H]Ouabain Binding

Two aspects of the K inhibition of [^3H]ouabain binding to native Na,K-ATPase were previously observed(32, 33, 34) and must be emphasized when interpreting the data on E327L, E327D, E327Q, and E327A. First, in contrast to the situation with Na, even at high concentrations of K, complete inhibition of ouabain binding does not occur (i.e. K never decreases [^3H]ouabain binding to nonspecific levels). This characteristic suggests that ouabain can bind, albeit with low affinity, to a K-complexed ATPase (but not to a Na-complexed ATPase). Second, the calculated K(i) value for K inhibition of ouabain binding to the wild type sheep alpha1 protein is consistent with the apparent K(0.5) values for the expressed rat alpha isoforms as determined by measuring K dependence of Na,K-ATPase activity(35) . This similarity in the values of K(i) and K(0.5) supports the concept that K inhibition of Mg-P(i) supported ouabain binding is mediated by K binding to the same high affinity binding sites utilized by the native enzyme under enzyme turnover conditions (i.e. in the presence of Na, K, ATP, and Mg).

The K inhibition profile for E327L is similar to that observed for wild type Na,K-ATPase. The inhibition constant for K is approximately 2-fold higher than the K(i) value for K inhibition of the wild type and is similar to the K(0.5) value determined for this mutant by K-dependent ATPase measurements (K(i) = 1.24 ± 0.05 mM, K(0.5) = 1.25 ± 0.13 mM)(13) . When the K inhibition data for E327L was fit to the partially competitive equation (see legend of Fig. 4), the interaction factor (alpha) was determined to be 3-fold lower than the alpha for wild type. This change in the interaction factor is evident in the K inhibition profile of E327L as a higher level of [^3H]ouabain is bound at saturating K concentrations. This is consistent with the concept that the K-complexed mutant enzyme has a higher affinity for ouabain than does the K-complexed wild type Na,K-ATPase. Thus, upon the association of two K ions with E327L, the mutant undergoes only a portion of the conformational changes normally induced by K and retains an extracellular surface to which ouabain can bind more readily than to the wild type. E327L with two K ions bound also retains sufficient structural integrity in the membrane such that this mutant can support cell viability, possibly because other ligand-enzyme interactions compensate for the defect in the K-induced conformational change. For example, E327L exhibits a 5-fold decrease in the K(0.5) constant for ATP. (^4)

[^3H]Ouabain binding to E327Q is not inhibited by K. Previously, it was reported that E327Q demonstrated a 3-6-fold increase in its K(0.5) value for K-stimulated ATPase activity compared to the wild type protein(13, 14) , suggesting that the K-binding sites of this protein are intact but have altered affinities for K. Moreover, the mutant E327Q supports viability of HeLa cells and transports Rb from the extracellular to the cytoplasmic surface of the membrane as measured with Rb uptake experiments(36) . Thus, at the highest concentrations of K used in these [^3H]ouabain binding studies, one would predict that K is bound to the E327Q mutant but that the protein conformation induced by K binding contains an extracellular surface that can associate with ouabain. There appears to be a greater defect in the K-induced conformational change in the E327Q mutant enzyme than in the E327L mutant. Again, it is possible that the 10-fold decrease in the K(0.5) value for ATP observed for E327Q (14) compensates for the inability of the mutant to undergo K-induced conformational changes observed in the presence of Mg and P(i). This increase in ATP affinity along with the normal Hill coefficient for Na may enable E327Q to pump cations with enough efficiency to support cell life.

Similar to E327Q in the Na,K-ATPase, the analogous amino acid substitution (E309Q) was previously constructed and characterized in the sarcoplamic reticulum Ca-ATPase (8, 37, 38, 39) . This mutant protein, E309Q, reacts with P(i) and Mg to form a phosphorylated protein in the presence of Ca (a ligand which normally inhibits this phosphorylation). In addition, this phosphorylated intermediate was shown to be extremely stable with a low rate of dephosphorylation. The [^3H]ouabain binding studies done on E327Q of Na,K-ATPase may indicate a possible inability of K to induce a conformational change necessary for dephosphorylation. Thus, it appears that the phosphorylated forms of these mutant proteins (E327Q or E309Q) in the Na,K-ATPase and the Ca-ATPase formed in the presence of Mg and P(i) are resistant to conformational changes normally induced by the binding of cation ligands.

Unlike E327Q and E327L, it is not known if E327D and E327A have intact K sites since these mutants could not support HeLa cell viability and do not have measurable ATPase activity. Hence, direct correlation of the K effects on [^3H]ouabain binding to the transport cycle of these mutants is not possible. As discussed with respect to E327Q, mutations of E327 inhibit K-induced conformational changes even in the cases where K sites are known to be intact. K interactions with E327A as probed by [^3H]ouabain binding demonstrate a normal inhibition constant at low K concentrations (leq10 mM). The stimulation of ouabain binding to E327A at higher concentrations of K suggests a possible third K interaction and major alterations in the Na,K-ATPase conformation resulting from this amino acid replacement. E327D does not demonstrate any ligand affinities similar to the wild type sheep alpha1 enzyme (Na, K, P(i), Mg, and ouabain affinities are all altered). Hence, the structural integrity of this E327D mutant of Na,K-ATPase must be questioned. Data which indicate that portions of the enzyme conformations of E327D and E327A may resemble wild type Na,K-ATPase include [^3H]ouabain binding to whole 3T3 cells and profiles of ouabain competition and Na competition. Previously, it had been observed that the hydrolytic activity of Na,K-ATPase is more sensitive to alteration of protein structure (as induced by chemical modification) than is ouabain binding to the enzyme(40) . Hence, we hypothesize that the amino acid substitutions E327A and E327D have changed the structure of the protein such that the hydrolytic activity is severely impaired; however, the mutants retain enough native structure that [^3H]ouabain can still bind to the proteins. Generally, we can conclude that the K interactions with E327D and E327A as probed by Mg-P(i) supported [^3H]ouabain binding are different from the K interactions with the wild type Na,K-ATPase; however, the cause of the different K-induced effects (absence of K-binding sites, unstable K-induced intermediates, or additional K-binding sites) is not fully understood.

Conclusions

Two features of the glutamic acid residue at position 327 in the sheep alpha1 isoform of Na,K-ATPase appear to be essential for unaltered cation interactions. First, the charge of this residue appears to be essential for stabilizing a K-induced intermediate with a low affinity for ouabain as observed by the altered minimum level of [^3H]ouabain binding at high K concentrations (as shown by E327L and E327Q). Second, the length of the Glu side chain may be important for maintaining the structural integrity of at least one intermediate in the Na,K-ATPase catalytic cycle (as suggested by E327D). Although the charge of Glu is important, removing the charge simply alters the rate-limiting step of the enzyme pathway and may be compensated for by the pump through an increased affinity for other ligands (e.g. ATP). In contrast, loss of structural integrity of a protein intermediate is irreversible and can lead to inability to sustain cell viability. Hence, E327D (and possibly E327A) are unable to support cell viability due to the presence of an unstable intermediate in the enzyme pathway of the mutant, as indicated by the appearance of an additional monovalent cation site.

The lowest ouabain affinity observed for a K-complexed enzyme was associated with the wild type protein in which the glutamic acid at position 327 is most likely responsible for stabilizing a K-induced conformation. This charged residue may stabilize this conformation by forming an internal salt bridge in which the charged side chain of Glu would be neutralized in the K-induced intermediate by a neighboring amino acid. This may explain the K protectable characteristic of DCCD modification of this residue(10) . In contrast to these chemical modification studies, we do not believe that the charged side chain of Glu is involved in ligating the K ions being transported since E327L and E327Q are able to transport K. However, it is significant that both chemical modification studies and these site-directed mutagenesis studies identified Glu in the fourth transmembrane domain of Na,K-ATPase as being essential for K interactions with the protein.


FOOTNOTES

*
This work was supported by National Institute of Health Grants HL 28573 (to J. B. L.) and HL 50613 (to E. T. W.). 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.

§
Recipient of National Institutes of Health Postdoctoral Fellowship HL08612-01.

To whom correspondence should be addressed. Tel.: 513-558-5324; Fax: 513-558-8474.

(^1)
The abbreviations used are: Na,K-ATPase, sodium and potassium-activated adenosine triphosphatase; P(i), inorganic phosphate; bp, base pairs; G418, geneticin; DCCD, N,N`-dicyclohexylcarbodiimide; PCR, polymerase chain reaction.

(^2)
Sheep alpha1 (RD) is the sheep isoform modified to a ouabain-resistant protein with the following amino acid substitutions: Q111R and N122D(18) .

(^3)
Rat alpha2 is the rat isoform modified to a ouabain-resistant protein with the following amino acid substitutions: L111R and N122D(35) .

(^4)
E. A. Jewell-Motz and J. B. Lingrel, personal communication.


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