©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Amino Acid Replacement of Asp in the Sheep 1 Isoform Eliminates ATP and Phosphate Stimulation of HOuabain Binding to the Na,K-ATPase without Altering the Cation Binding Properties of the Enzyme (*)

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

From the (1)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

Modification of aspartic acid 369 in the sheep 1 Na,K-ATPase to asparagine results in a membrane-associated form of Na,K-ATPase that can bind [H]ouabain with high affinity in the presence of Mg alone (K = 20.4 ± 2.6 nM). Ouabain binding to the D369N mutant is not stimulated by inorganic phosphate, confirming that Asp is both the catalytic phosphorylation site and the only P interaction site which stimulates ouabain binding. Cation inhibition of Mg-stimulated ouabain binding to the D369N mutant demonstrated that three Na and two K ions inhibit [H]ouabain binding and suggests that this inhibition must occur via a cation-sensitive conformational change which does not directly involve dephosphorylation of the enzyme. In the presence of 10 mM Mg, ATP stimulates ouabain binding to the wild type protein, (AC = 21.4 ± 2.7 µM) but inhibits the binding to the D369N mutant (IC = 2.52 ± 0.17 µM) indicating that the mutation does not destroy the high affinity site for MgATP but does change the nature of the protein conformation normally induced by a nucleotide-Na,K-ATPase interaction. Increasing the Mg from 1 to 10 mM did not alter the AC or IC values for ATP and reveals that the Mg interaction which stimulates ouabain binding in the absence of nucleotide involves a distinct divalent cation site not associated with the binding of the magnesium-nucleotide complex. Thus, altering the catalytic phosphorylation site of Na,K-ATPase does not affect the expression of the ouabain-sensitive protein in the membrane fraction of NIH 3T3 cells and does not disrupt the binding of Na, K, Mg, ouabain, or ATP to the enzyme. However, the D369N substitution does inhibit the formation of a nucleotide-protein complex with high affinity for ouabain.


INTRODUCTION

The Na,K-ATPase is a member of a class of active cation transporters which form a characteristic -aspartyl phosphate intermediate during the ATP hydrolysis/ion translocation cycle. Amino acid replacement of this conserved aspartic acid residue in the Na,K-ATPase and in related active cation transporters results in complete loss of cation transport (1-3). This loss of cation transport was attributed to either loss of ATPase activity (1, 2) or lack of mutant protein expression and/or stability in the system being utilized(3) . In addition to loss of ATPase activity, this amino acid replacement in the Na,K-ATPase expressed in oocytes was reported to abolish cardiac glycoside binding to the exterior surface of these cells(2) .

Cardiac glycosides (such as ouabain) specifically inhibit the Na,K-ATPase through a mechanism which is not completely understood. The affinity of the Na,K-ATPase for ouabain is closely linked to enzyme cycling as demonstrated by the effects of enzyme substrates on the protein-drug interaction. For example, Mg and inorganic phosphate (P) stimulate ouabain binding in the absence of monovalent cations (4, 5) and Mg and ATP stimulate ouabain binding in the presence of Na(6, 7) . Mg alone increases the affinity of the ATPase for ouabain 300-fold over Tris buffer alone(8) . Based on this substrate-dependent affinity of the enzyme for ouabain, it has been established that the interaction site for P and ATP which stimulates ouabain binding is the conserved catalytic phosphorylation site(9, 10, 11, 12, 13) . Moreover, although ATP and P increase the enzyme's affinity for ouabain an additional 10- and 20-fold, respectively, over magnesium alone, Mg appears to be the essential element for high affinity ouabain binding.

In this work the conserved aspartic acid residue (Asp) was replaced with an uncharged amino acid, asparagine, in the ouabain-sensitive sheep 1 isoform of Na,K-ATPase. The mutant protein was expressed in NIH 3T3 cells which contain a relatively ouabain-insensitive endogenous isoform of Na,K-ATPase. In this system, [H]ouabain binding can be utilized as a highly specific probe for the exogenous protein, even though the mutant D369N is inactive(14, 15, 16) . The purpose of this study was to determine if D369N is expressed in NIH 3T3 cells in a form which can bind ouabain and to decipher whether the amino acid substitution alters the mechanism by which ligands (Na, K, Mg, ATP, and ADP) regulate the binding of ouabain.


EXPERIMENTAL PROCEDURES

Materials

Companies from which reagents were purchased were previously reported(15, 16, 17) . The specific radioactivity of [H]ouabain was determined by the method of Wallick and Schwartz(1988)(18) .

General Methods

Site-directed mutagenesis(19) , establishment of stable 3T3 cell lines(15, 16, 17, 20) , isolation of crude plasma membranes(17, 21) , and immunological detection (22) were all performed using methods previously described.

[H]Ouabain Binding to Crude Membranes

All ouabain binding studies were conducted under the following conditions unless otherwise indicated in the figure legends or text: 5 mM MgCl and 50 mM Tris-HCl (pH 7.4) in a final volume of 0.5 ml. The amount of protein used was typically 50 µg of total protein/assay tube, depending on the specific activity of the individual membrane preparation. The affinities for ouabain of both the D369N and the wild type proteins under phosphate-free conditions were determined using unlabeled ouabain in a self-competition assay with [H]ouabain (See Equation 1, Ref. 16). The apparent K for wild type sheep 1 was calculated to be 50 nM while the D369N protein had a K for ouabain of 20 nM. For the competition curves with unlabeled ouabain, eight concentrations of unlabeled ouabain (including zero) were examined in triplicate (Fig. 3). Ligand stimulation or inhibition of [H]ouabain binding was characterized using at least 11 concentrations (including zero) of each ligand in duplicate. To optimize the determinations of the different ATPase-ligand interactions, the concentration of [H]ouabain utilized was approximately equal to the ouabain K values calculated for each protein. Aliquots of each reaction mixture (50 µl) were taken with each experiment to determine the exact concentration of [H]ouabain utilized. The samples were filtered, and radioactivity analyzed as described previously(16) . Data describing the competition of [H]ouabain with unlabeled ouabain was fit to the self-competition model as described previously(16) . P, Mg, Na, K, ATP, and ADP data were fit to a four parameter logistic function for either stimulation of binding: {[(B - B)/(1 + (AC/x))] + B} or inhibition of binding: {[(B - B)/(1 + (x/IC))] + B}. B and B represent the maximum and minimum amounts of bound [H]ouabain, respectively. n represents the approximate number of ligands responsible for the inhibition or activation of [H]ouabain binding, a factor which is similar to a Hill coefficient. The AC or IC value is the concentration of ligand which produces 50% of the activation or inhibition, respectively. x is the concentration of inhibiting or stimulating ligand (i.e. P, Mg, Na, K, or nucleotide). All data analysis was done using the KaleidaGraph program by Abelbeck Software.


Figure 3: Ouabain competition curves. [H]Ouabain binding was measured in the presence of various concentrations of unlabeled ouabain. The assay contained 5 mM MgCl and 50 mM Tris-Cl (pH 7.4). The symbols represent the mean of triplicate determinations and are coded as follows: wild type () and D369N (). The error was calculated for each point and is shown unless it is smaller than the symbol size. The data were fit to a simple self-competition model with three adjustable parameters: K (dissociation constant for ouabain), ET (total number of ouabain-binding sites), and NS (proportionality constant for nonspecific binding) (see ``Experimental Procedures''). The fitted parameters calculated for the wild type: (K = 49.6 ± 5.7 nM; ET = 0.244 ± 0.010 nM; NS = 0.000324 ± 0.000011) and for the D369N mutant: (K = 27.2 ± 2.0 nM; ET = 0.252 ± 0.016 nM; NS = 0.000244 ± 0.000012).




RESULTS

Expression of the D369N Mutant

Initially, these experiments were designed to directly address whether a mutant form of Na,K-ATPase in which the catalytic phosphorylation site was altered could be stably expressed and could bind ouabain with high affinity. To this end, the sequence encoding the amino acid replacement D369N was introduced into two cDNAs: one which encodes a ouabain-resistant form of Na,K-ATPase (sheep 1(RD))()and a second which encodes a ouabain-sensitive form of Na,K-ATPase (sheep 1). The mutant sheep 1(RD) cDNA was transfected into HeLa cells and grown in the presence of 1 µM ouabain. In this expression system, only the exogenous ouabain-resistant Na,K-ATPase can support cell viability. Thus, if a mutation impairs the functional activity of the exogenous Na,K-ATPase, no transfected HeLa cells will survive the ouabain selection. No ouabain resistant colonies were observed for the D369N mutant (data not shown) and thus confirmed that this amino acid replacement disrupts ATPase activity presumably by inhibiting the formation of the -aspartyl-phosphorylated intermediate as previously suggested by Ohtsubo et al.(2) .

The mutated sheep 1 cDNA which encodes the D369N amino acid substitution in a ouabain-sensitive form of Na,K-ATPase was cotransfected into 3T3 cells with a plasmid encoding a neomycin resistance protein. The transfected 3T3 cells were grown in the presence of 400 µg/ml G418 (a neomycin analog), and G418-resistant colonies were isolated. This expression system does not require that the exogenous Na,K-ATPase be functionally capable of supporting cell viability. Immunological detection was utilized to screen the isolated cell lines for expression of sheep 1 protein. Three clonal cell lines which expressed the sheep 1 proteins were expanded, and crude membranes were prepared using NaI treatment. Twenty µg of total protein from these membrane preparations (as determined by a Lowry assay) were separated on a 7.5% SDS-polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose. The M7-PB-E8 monoclonal antibody grown against sheep kidney Na,K-ATPase (23) was used to probe the exogenously expressed sheep 1 protein without cross-reacting with the endogenous mouse protein from 3T3 cells (Fig. 1). A single band of protein migrating at a molecular mass of approximately 110 kDa was detected by the monoclonal antibody in the stable transfectants but not in untransfected 3T3 cells. Several membrane preparations of two clonal cell lines transfected with either the D369N cDNA or the wild type cDNA showed relatively high expression levels of sheep 1 protein and were utilized in the ouabain binding experiments.


Figure 1: Western analysis of sheep 1 cDNA transfectants. Twenty µg of total protein isolated from the neomycin-resistant NIH 3T3 cell lines were probed with the monoclonal antibody, M7-PB-E8, which specifically reacts with the exogenous sheep 1 protein and does not cross-react with the endogenous mouse 1 protein of NIH 3T3 cells. This autoradiograph of an immunoblot shows the content of sheep 1 protein in membrane preparations from one wild type clonal line and two D369N clonal lines. The arrows indicate the positions of the prestained molecular weight markers (Bio-Rad), phosphorylase b (106,000 Da) and -galactosidase (116,500 Da) which were used as a reference to identify the Na,K-ATPase (110,000 Da).



Interaction of Inorganic Phosphate with the Expressed Proteins

To test for additional phosphate interaction sites which might stimulate ouabain binding, [H]ouabain binding to both the expressed wild type sheep 1 and the D369N mutant protein was measured at 23 concentrations of inorganic phosphate ranging from 0 to 30 mM. A portion of the curve defining this P effect is displayed in Fig. 2. No stimulation of [H]ouabain binding was observed for the mutant D369N for P concentrations up to 30 mM. P stimulation of the wild type sheep 1 displayed a half-maximum stimulation (AC) of 0.12 ± 0.02 mM P, which is identical to the value previously reported(15) . These data are consistent with this aspartic acid at position 369 in the sheep 1 isoform being the only interaction site for inorganic phosphate which stimulates ouabain binding as well as being the site of -aspartyl phosphorylation during the enzymatic cycle(24) .


Figure 2: Inorganic phosphate stimulation of [H]ouabain binding. [H]Ouabain binding was measured in the absence and presence of various concentrations of inorganic phosphate as displayed on the x axis. The symbols represent the mean of triplicate determinations, and the calculated standard error is shown unless it was less than the symbol size. All assay solutions contained 5 mM MgCl, 50 mM Tris-Cl (pH 7.4), and 20 nM [H]ouabain and were incubated for 6 h at 37 °C. The data characterizing the expressed wild type sheep 1 are shown by the open squares () while the data for the D369N mutant are symbolized by closed circles (). The wild type data were fit to the four parameter logistic function for activation (see ``Experimental Procedures''). The fitted parameters for the wild type curve were: B = 0.402 ± 0.012 nM; B = 0.147 ± 0.001 nM; AC = 0.127 ± 0.017 mM; and n = 0.91 ± 0.05.



Mg Stimulated [H]Ouabain Binding

In the presence of Mg alone, both the wild type sheep 1 protein and the D369N mutant protein were able to bind [H]ouabain present in nanomolar concentrations. This is demonstrated clearly at 0 mM P in Fig. 2, keeping in mind that nonspecific binding (NS) represents a value which is less than 10% of the total amount bound (wild type, NS = 0.0126 ± 0.0006 nM and D369N, NS = 0.0049 ± 0.0002 nM as determined from unlabeled ouabain self-competition data). This observation suggested that the affinity of both proteins for ouabain is relatively high in the presence of Mg alone and that [H]ouabain binding could be utilized as a probe for these expressed proteins. In order to determine the K values characterizing these protein-drug interactions, competition curves were carried out in the presence of 5 mM MgCl and 50 mM Tris-Cl using [H]ouabain and unlabeled ouabain. Fig. 3shows a comparison of the competition curves for one membrane preparation of D369N and of the wild type protein. The mean K values obtained for three different preparations of each protein are presented in . In the presence of 5 mM Mg, the affinity of the wild type sheep 1 protein for ouabain is 34-fold lower than the affinity in the presence of P and Mg (K = 1.53 ± 0.08 nM, 5 mM P, and 5 mM Mg(15) ). This increase in apparent K for ouabain caused by the absence of P is similar to the 23-fold increase previously calculated from rate constants observed for purified sheep Na,K-ATPase(8) . In the presence of Mg alone, the mutant D369N displayed a 2.5-fold higher affinity for ouabain compared to the wild type protein. The high affinity of D369N for ouabain is evidence that the ouabain interaction site on the mutant (presumably involving the extracellular surface of the protein) has not been markedly altered compared to the wild type protein.

To maximize the accuracy of the calculated K values for all ligands, all competition curves characterizing the wild type protein were done in the presence of approximately 60 nM [H]ouabain, and those describing the D369N mutant were done in the presence of approximately 20 nM [H]ouabain. The observed nonspecific binding is primarily due to [H]ouabain binding to the glass fiber filters and is essentially independent of the enzyme concentration employed. The maximum level of [H]ouabain bound to the membranes is dependent on the expression levels of the exogenous proteins and differs slightly between membrane preparations.

To ensure that the Mg site(s) on these proteins were saturated at 5 mM Mg, activation of [H]ouabain binding to the Na,K-ATPase was examined with respect to Mg concentrations (data not shown). In previous studies, an apparent AC value for Mg stimulation of ouabain binding to the wild type protein was determined to be 0.20 ± 0.03 mM in the presence of 5 mM P and 50 mM Tris (pH 7.4)(15) . In the absence of P, the apparent AC value for Mg stimulation of ouabain binding to the wild type Na,K-ATPase is increased 3.6-fold to 0.71 mM (). The AC value for Mg stimulation of ouabain binding in the absence of P to the D369N mutant (0.12 mM) is 5.9-fold lower than that of the wild type. The Mg AC values and n values shown in are averages of three experiments done in duplicate on three separate membrane preparations of two clones for each protein. Although the cause for the alterations in the Mg AC values are not completely understood, it appears that 5 mM Mg provides an excess of divalent cation such that all detectable Mg activation sites are occupied in the absence of additional ligands. Therefore, the changes in the amount of [H]ouabain bound at various inhibitor concentrations (i.e. unlabeled ouabain, Na or K, in the upcoming experiments) reflect protein inhibitor (ligand) interactions and are not a direct result of unoccupied Mg sites on the protein. It is interesting to note that the number of Mg interactions (n) which stimulate ouabain binding to the two proteins were calculated to be greater than one and may be an indication that at least two Mg activation sites are located on the Na,K-ATPase.

K Inhibition of [H]Ouabain Binding

Ouabain binding stimulated by either Mg and P or by Mg, Na, and ATP is antagonized by the addition of K to the equilibrium medium (4, 6). Presumably, K induces a conformational change in the Na,K-ATPase such that the resulting form of the protein has a lower affinity for ouabain. As demonstrated in Fig. 4, K also inhibits ouabain binding in the presence of Mg alone. The average apparent IC and the pseudo-Hill coefficient (n) for K determined from four separate membrane preparations of each cell line are presented in . The D369N mutant demonstrates a 3-fold lower AC value for K compared to the wild type protein under P-free conditions. Thus, it appears that the two K inhibition sites previously observed in the presence of Mg and P(14, 15, 16) can be probed on the wild type sheep 1 protein in the presence of Mg alone, and these K sites remain intact in the D369N mutant form of the sheep 1 protein.


Figure 4: K inhibition of [H]ouabain binding. The amount of [H]ouabain bound was measured in the presence of various concentrations of KCl, 5 mM MgCl, and 50 mM Tris-Cl (pH 7.4). The symbols represent the mean of duplicate determinations. The error bars display the range of the duplicates and are not shown if smaller than the symbol size. The symbol representation is the same as in Fig. 3. These data were fit to a four parameter logistic function. The fitted parameters for the wild type were: B = 0.331 ± 0.006 nM; B = 0.034 ± 0.001 nM; IC = 1.41 ± 0.06 mM; and n = 2.00 ± 0.08. The calculated parameters for D369N were: B = 0.217 ± 0.007 nM; B = 0.0088 ± 0.0001 nM; IC = 0.445 ± 0.022 mM; and n = 1.75 ± 0.05.



Na Inhibition of [H]Ouabain Binding

Unlike K, Na stimulates ouabain binding in the presence of Mg and ATP and antagonizes ouabain binding in the presence of Mg and P (4-7). In the presence of Mg alone, Na induces a conformational change in the wild type Na,K-ATPase and the D369N mutant protein which reduces the affinities of the proteins for ouabain. In Fig. 5, examples of the inhibitory effects of Na on Mg-stimulated [H]ouabain binding are displayed. These inhibition data were fit to a four parameter logistic function (see ``Experimental Procedures''). The average IC and n values for Na of at least three trials on two clones are presented in .


Figure 5: Na inhibition of ouabain binding. The amount of [H]ouabain bound in the presence of various concentrations of NaCl was measured in the presence of 5 mM MgCl and 50 mM Tris-Cl (pH 7.4). The symbols represent the mean of duplicate determinations. The error bars represent the range of the duplicates and are not shown if smaller than the symbol size. The symbol representation is as follows: () wild type sheep 1 and () D369N. These data were fit to a four parameter logistic function. The calculated parameters for the wild type were: B = 0.161 ± 0.003 nM; B = 0.0204 ± 0.0004 nM; IC = 8.04 ± 0.36 mM; and n = 1.83 ± 0.07 and for D369N were: B = 0.195 ± 0.003 nM; B = 0.0080 ± 0.0002 nM; IC = 13.0 ± 0.3 mM; and n = 2.31 ± 0.06.



MgATP and MgADP Effects on [H]Ouabain Binding

In the absence of Na, MgATP was shown to stimulate ouabain binding to purified Na,K-ATPase by increasing the on-rate for ouabain and in turn decreasing the K for the drug (8). We have utilized this stimulatory effect to determine if the MgATP site has remained intact in the D369N mutant. To avoid the inhibitory effects of Na on Mg-stimulated [H]ouabain binding and to limit the amount of ATP hydrolysis due to ATPase turnover in the wild type protein, no Na was added to these equilibrium studies. As shown in Fig. 6A, addition of MgATP stimulated ouabain binding in the wild type protein both at 1 mM Mg and 10 mM Mg. Average AC values for three preparations at both concentrations of Mg are shown in . As shown in Fig. 6B, MgATP inhibits the Mg-stimulated ouabain binding to the D369N mutant in the presence of either 1 or 10 mM Mg. Average IC values are summarized in for three membrane preparations of the mutant D369N. This MgATP induced effect demonstrates that a high affinity ATP-binding site exists on the D369N mutant protein and suggests that the cytoplasmic loop in which the amino acid replacement is located resembles the wild type protein and specifically binds ATP with high affinity.


Figure 6: Nucleotide effects on [H]ouabain binding. [H]Ouabain binding was measured in the presence of various concentrations of ATP or ADP. The assays contained approximately 20 nM [H]ouabain, 50 mM Tris-Cl (pH 7.4), and either 1 mM MgCl (closed symbols) or 10 mM MgCl (open symbols). These assays were incubated for 3 h at 37 °C. Panel A shows the ATP activation of [H]ouabain binding to the wild type protein. These data were fit with a four parameter logistic function for activation, and the calculated constants were as follows: at 1 mM MgCl, B = 0.380 ± 0.014 nM; B = 0.050 ± 0.001 nM; AC = 19.2 ± 1.4 µM; and n = 0.896 ± 0.045 and at 10 mM MgCl, B = 0.349 ± 0.010 nM; B = 0.082 ± 0.002 nM; AC = 15.1 ± 1.4 µM; and n = 1.10 ± 0.08. Panel B displays the inhibitory effect of increasing amounts of ATP on [H]ouabain binding to the D369N mutant. These data were fit to a four parameter logistic function for inhibition (see ``Experimental Procedures''), and the calculated constants were: at 1 mM MgCl, B = 0.069 ± 0.002 nM; B = 0.011 ± 0.001 nM; IC = 2.03 ± 0.11 µM; and n = 1.38 ± 0.09 and at 10 mM MgCl, B = 0.065 ± 0.002 nM; B = 0.014 ± 0.001 nM; IC = 2.21 ± 0.11 µM; and n = 1.64 ± 0.13. Panel C displays the stimulatory effect of ADP on ouabain binding to the wild type protein () and the inhibitory effect of ADP on ouabain association with the D369N protein () at 10 mM MgCl and 50 mM Tris-Cl (pH 7.4). The wild type data were fit with the logistic function for activation, and the calculated constants are: B = 0.410 ± 0.011 nM; B = 0.177 ± 0.005 nM; AC = 7.54 ± 0.54 µM; and n = 1.47 ± 0.17. The D369N data were fit to the four parameter logistic function for inactivation with the calculated values being: B = 0.134 ± 0.004 nM; B = 0.036 ± 0.001 nM; IC = 3.52 ± 0.34 µM; and n = 1.30 ± 0.11.



To determine if the MgATP-induced stimulation of ouabain binding in the wild type protein is a result of nucleotide binding or a result of enzyme phosphorylation, MgADP was used to mimic simple nucleotide binding in the absence of Na. As shown in Fig. 6C, like MgATP, MgADP stimulates ouabain binding in the wild type protein and inhibits ouabain binding in the D369N mutant. Average AC and IC values for three experiments using two clonal cell lines for each protein are shown in . It is apparent that the nucleotide-induced conformational change normally observed as an increase in the affinity of the Na,K-ATPase for ouabain cannot occur if the aspartic acid residue at position 369 is altered to an asparagine.


DISCUSSION

Expression and Structure of the D369N Mutant

Previous mutagenesis studies involving this conserved aspartic acid residue in other P-type ATPases have demonstrated the loss of cation transport and phosphorylation upon amino acid replacement(2, 3, 25) . In the sarcoplasmic reticulum Ca-ATPase, this mutant protein (D351N) was transiently expressed in COS-1 cells and was characterized as impaired with respect to Ca transport and ATP-stimulated formation of a phosphorylated enzyme intermediate(25) . When a similar amino acid substitution was introduced in the yeast H-ATPase, the protein processing of the mutant was thought to be interrupted such that no protein was detected in the secretory vesicles of the yeast expression system(3) . Unlike the yeast H-ATPase, but similar to the SR Ca-ATPase, Na,K-ATPase in which the essential aspartyl residue has been replaced can be expressed in 3T3 cells as shown here and in oocytes as demonstrated by Ohtsubo et al.(2) . Both the immunological analysis and the presence of a ouabain-sensitive protein in the isolated membrane fraction of transfected 3T3 cells is direct evidence for the expression of the mutant protein. The interaction between the D369N mutant and ouabain suggests that the extracellular surface of the mutant resembles the wild type protein while the MgATP-D369N interaction shows the structural integrity of the cytoplasmic domain of the mutant. These data are consistent with the D369N mutant being expressed in a membrane of the 3T3 cells with an overall protein structure which is similar to the wild type sheep 1 Na,K-ATPase.

In order to unequivocally determine if the D369N protein is assembled into the plasma membrane of the 3T3 cells, one must probe the extracellular surface of the intact transfected cells either with a monoclonal antibody or with a specific extracellular probe (i.e. ouabain). Due to the lack of an available monoclonal antibody that binds to the extracellular surface of the sheep 1 protein, whole cell ouabain binding was utilized to examine if the mutant was located in the plasma membrane. No [H]ouabain binding was detected when intact 3T3 cells expressing the D369N mutant were incubated with the radioligand (data not presented). Ouabain binding to intact 3T3 cells is regulated by the presence of intracellular MgATP and subsequent intracellular Na binding. Both of these substrates, MgATP and Na, when added independently ( Fig. 5and Fig. 6B) or together (data not presented) were shown to inhibit Mg-stimulated ouabain binding in the studies employing membrane fragments from 3T3 cells expressing the D369N mutant. Thus, due to the presence of these intracellular components in the intact 3T3 cells, it was not surprising that ouabain did not bind to the extracellular surface of whole cells expressing the D369N mutant. Whether or not the D369N mutant is present in the plasma membrane of 3T3 cells or oocytes(2) , our ouabain binding data on isolated membrane preparations does explain why ouabain interactions with whole cells expressing the D369N mutant may not have been observed.

Ligand Sites of the Na,K-ATPase

This work suggests that phosphate interaction at the catalytic phosphorylation site, aspartic acid 369 in the sheep 1 isoform, induces a conformational change in the Na,K-ATPase which increases the affinity of the enzyme for ouabain. Thus, catalytic ATPase activity and ouabain binding are linked through this phosphorylation site which supports the use of [H]ouabain binding as a method for probing the functional sites of Na,K-ATPase. In addition, this double role for aspartic acid 369 contradicts the theory that phosphate interactions at a second site on Na,K-ATPase increase the affinity of the enzyme for ouabain(26, 27) .

Models for the mechanism of Na,K-ATPase have been proposed in which the chemical moieties associated with position 369 (i.e. aspartyl group or covalently bound phosphate) act as binding sites for the transported Na and K ions, directly linking the formation of the phosphorylated intermediate with cation transport(28, 29) . From the data presented here, three Na and two K ions interact with the Na,K-ATPase when both of these moieties are removed either by site-directed mutagenesis or simply by eliminating P from the equilibrium medium. Thus, assuming that the cation sites which inhibit ouabain binding are also the cation transport sites, it does not appear that either of these chemical groups are directly associated with the binding of the monovalent cations being transported.

Many investigators have questioned whether a phosphorylated intermediate form of the Na,K-ATPase is necessary for ouabain binding(30, 31, 32, 33) . Conclusions from these investigations have been met with some skepticism due to the possibility that contaminating inorganic phosphate promotes phosphoenzyme formation. No phosphoenzyme was present in the D369N studies presented here due not only to the absence of added inorganic phosphate but also due to the alteration of the catalytic phosphorylation site. Thus, our data unequivocally demonstrate that a phosphorylated Asp site is not required for ouabain binding to the Na,K-ATPase.

Proposed models for the inhibitory effects of Na and K on the ouabain binding properties of Na,K-ATPase often postulate cation stabilization and destabilization of the phosphorylated intermediate (10, 13). Although our results do not discount these effects of monovalent cations on phosphoenzyme formation, our studies suggest that this is not the only mechanism by which Na and K affect the ouabain binding properties of the ATPase. The cation inhibition experiments involving the D369N mutant ( Fig. 4and 5) had no phosphoenzyme present due to the absence of added P and substitution of the catalytic phosphorylation site. Thus, the Na inhibition and K inhibition of ouabain binding to the D369N protein are due to cation-induced conformational changes in the protein and cannot directly reflect the cation effects on the phosphoenzyme intermediate.

Mg and MgATP Interactions with the Na,K-ATPase

The importance of Mg in active cation transport by Na,K-ATPase was recognized in the original characterization of this protein by Skou(34) . Subsequent to these studies, both kinetic analysis and physical chemical studies have suggested that Mg interacts with the Na,K-ATPase at two distinct sites. The first site is observed in the presence of nucleotide and is thought to be a site related to the binding of MgATP. This site demonstrates a K for Mg of 5 µM as measured by ATP-ADP exchange in the presence of 125 mM Na, 5 mM ATP, and 1.25 mM [C]ADP(35) . The second Mg site is a separate divalent cation site. This site displays a K for Mg of 1-3 mM and is observed in the absence of nucleotide and Na in para-nitrophenylphosphate hydrolysis, Mn competition, and Rb release experiments (28, 35, 36). Mg binding to the distinct divalent cation site is thought to induce a conformation in Na,K-ATPase characterized by a high affinity for P and K and a lower affinity for Na(36) . In addition, the affinity of this distinct divalent cation site for Mg is increased 10-fold in the presence of P(36) .

We hypothesize that Mg interaction with the low affinity site on the wild type Na,K-ATPase induces a protein conformation with a high affinity for ouabain. This hypothesis is based on three characteristics of the Mg-wild type interaction. First, the AC value calculated for Mg stimulation of ouabain binding was 0.71 mM, similar to the K value which characterizes this low affinity site(28, 35, 36) . Second, this AC value was decreased approximately 3.5-fold in the presence of P, consistent with the effect of P on this low affinity site observed with a Rb-release technique(36) . Third, ouabain binding to the wild type protein was stimulated further upon formation of a E-MgATP complex (Fig. 6A). The absence of a Mg-induced shift in this MgATP stimulation of ouabain binding demonstrates that the two Mg interactions are not directly competitive on the wild type Na,K-ATPase. These data support the theory that the stimulation of ouabain binding observed in the presence of Mg alone is promoted by Mg interacting at a divalent cation site distinct from the MgATP site.

MgATP inhibited Mg-induced ouabain binding in the D369N mutant suggesting that the D369NMgATP complex has a reduced affinity for ouabain. The IC value for MgATP was approximately 2 µM indicative of a high affinity ATP site in this mutant form of the Na,K-ATPase. Thus, although Mg can interact with the D369N mutant to form an enzyme complex with a high affinity for ouabain, neither MgATP nor P can induce a conformation in the D369N mutant which displays a higher affinity for ouabain. Similar to the wild type protein, the absence of a Mg-induced shift in the MgATP inhibition curve implies that the Mg site which stimulates ouabain binding and the MgATP site are kinetically distinct. In addition, the dramatically different effects of Mg and MgATP association with the D369N protein suggest that the binding sites for Mg and MgATP are mechanistically distinct. Similar to the effects of MgATP, MgADP stimulated ouabain binding in the wild type protein and inhibited binding to D369N (Fig. 6C). The similarity between the effects of MgATP and MgADP suggests that the difference in nucleotide-induced conformational changes between the two enzymes is not a direct result of the inability of D369N to form a phosphorylated intermediate but may be a direct result of magnesium-nucleotide binding to the protein.

The different characteristics induced by MgATP interactions with the wild typeMg complex and the D369NMg complex suggest that the charged character of this aspartic acid is required to induce an enzyme complex with high affinity for ouabain in the presence of nucleotide. Therefore, we suggest that D369N is inhibited both by its inability to form a -aspartyl-phosphorylated enzyme intermediate and by its inability to undergo the normal conformational change induced by the binding of a magnesium-nucleotide complex. However, the tight association of MgATP to D369N demonstrates that the nucleotide-binding site of Na,K-ATPase is not destroyed by the amino acid substitution. In future structural investigations, the mutant D369N may serve as a stable model of the Na,K-ATPase in which the ligand-binding sites can be probed without the effects of varying turnover numbers altering calculated affinity constants.

  
Table: Apparent binding constants for protein-ligand interactions



FOOTNOTES

*
This work was supported in part by National Institutes 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 HL 08612-01.

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

The abbreviations used are: sheep 1(RD), sheep 1 isoform modified to a ouabain-resistant protein with the following amino acid substitutions: Q111R and N122D (37); Na,K-ATPase, sodium and potassium-activated adenosine triphosphatase; G418, geneticin, a neomycin analog.


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