©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Tissue-specific Versus Isoform-specific Differences in Cation Activation Kinetics of the Na,K-ATPase (*)

(Received for publication, August 17, 1995; and in revised form, January 18, 1996)

Alex G. Therien (1) Nestor B. Nestor (1) William J. Ball (2) Rhoda Blostein (1)(§)

From the  (1)Department of Biochemistry, McGill University, Montreal, Canada and the (2)Department of Pharmacology, College of Medicine, University of Cincinnati, Cincinnati, Ohio 45267-0575

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The experiments described in this report reconcile some of the apparent differences in isoform-specific kinetics of the Na,K-ATPase reported in earlier studies. Thus, tissue-specific differences in Na and K activation kinetics of Na,K-ATPase activity of the same species (rat) were observed when the same isoform was assayed in different tissues or cells. In the case of alpha1, alpha1-transfected HeLa cell, rat kidney, and axolemma membranes were compared. For alpha3, the ouabain-insensitive alpha3*-transfected HeLa cell (cf. Jewell, E. A., and Lingrel, J. B.(1991) J. Biol. Chem. 266, 16925-16930), pineal gland, and axolemma (mainly alpha3) membranes were compared. The order of apparent affinities for Na of alpha1 pumps was axolemma approx rat alpha1-transfected HeLa > kidney, and for K, kidney approx alpha1-transfected HeLa > axolemma. For alpha3, the order of apparent affinities for Na was pineal gland approx axolemma > alpha3*-transfected HeLa, and for K, alpha3*-transfected HeLa > axolemma approx pineal gland. In addition, the differences in apparent affinities for Na of either kidney alpha1 or HeLa alpha3* as compared to the same isoform in other tissues were even greater when the K concentration was increased. A kinetic analysis of the apparent affinities for Na as a function of K concentration indicates that isoform-specific as well as tissue-specific differences are related to the apparent affinities for both Na and K, the latter acting as a competitive inhibitor at cytoplasmic Na activation sites. Although the nature of the tissue-specific modulation of K/Na antagonism remains unknown, an analysis of the nature of the beta isoform associated with alpha1 or alpha3 using isoform-specific immunoprecipitation indicates that the presence of distinct beta subunits does not account for differences of alpha1 of kidney, axolemma, and HeLa, and of alpha3 of axolemma and HeLa; in both instances beta1 is the predominant beta isoform present or associated with either alpha1 or alpha3. However, a kinetic difference in K/Na antagonism due to distinct betas may apply to alpha3 of axolemma (alpha3beta1) and pineal gland (alpha3beta2).


INTRODUCTION

The sodium potassium adenosine triphosphatase (Na,K-ATPase) or sodium pump is responsible for maintaining the electrochemical gradient of Na and K across the plasma membrane of animal cells. It normally couples the hydrolysis of one molecule of ATP to the transport of three Na ions out and two K ions into the cell (for reviews, see (1, 2, 3, 4) ). This cation pump is a heterodimer comprised of a catalytic alpha subunit (approx105 kDa) and a highly glycosylated beta subunit (45-55 kDa), and may (5, 6) or may not (7, 8) form larger oligomers. The alpha subunit contains the binding sites for Na, K, ATP, and the highly specific cardiac glycoside inhibitors such as ouabain, as well as the site of phosphorylation(1) . The function of the beta subunit is not completely understood; it appears to be essential for the normal delivery and correct insertion of alpha into the plasma membrane (9) and to have some influence on the catalytic activity of alpha(10, 11, 12, 13, 14) . A third peptide subunit known as the subunit (6.5 kDa) appears to exist in association with alpha and beta, at least in certain tissues, although its role is yet to be determined(15) .

In mammals, three isoforms of the alpha subunit (alpha1, alpha2, and alpha3) and two of the beta subunit (beta1 and beta2) are known to exist(4) . Isoforms of the alpha subunit are expressed in a tissue-specific manner: alpha1 is present ubiquitously; alpha2 is detected mainly in skeletal muscle, heart, and certain neuronal cells (neurons and astrocytes); and alpha3 is mainly in neurons(4, 16) .

Earlier studies of cation activation of the Na,K-ATPase by Sweadner (17) using rat kidney and axolemma and later studies by Shyjan et al.(18) using kidney, brain, and pineal gland indicated a higher affinity for Na in preparations now known to be predominantly alpha3. Thus, the order of apparent affinities for Na in the former study was axolemma (predominantly alpha3) > kidney (alpha1 only) and in the latter, pineal gland (predominantly alpha3) geq brain (a mixture of alpha1, alpha2, and alpha3) > kidney. In contrast, Jewell and Lingrel(19) , using membranes isolated from HeLa cells transfected with the individual alpha isoforms, reported that the order of apparent affinities for Na is alpha1 approx alpha2* > alpha3*, and for K, alpha3* > alpha2* approx alpha1, where alpha2* and alpha3* denote ouabain-resistant mutants of alpha2 and alpha3, respectively. Moreover, studies of pump-mediated K (Rb) influx into these individual isoform-transfected cells confirmed the general conclusions drawn from the aforementioned work, except that considerably larger kinetic differences among the isoforms were observed(20) . Interestingly, in experiments carried out with kidney and axolemma microsomal membranes delivered by membrane fusion into red cells, the order of apparent affinities for cytoplasmic Na and K resembled those of alpha1- and alpha3*-transfected HeLa cells, respectively(20) .

The aim of the experiments described in this study was to reconcile the discordant results obtained in the foregoing studies, as well as numerous earlier reports, regarding the order of apparent affinities for Na and K of different tissues and/or isoforms (for review, see (4) ). In particular, the question of isoform-specific versus tissue-specific properties of the rat Na,K-ATPase has been addressed by studying the same isoform, either alpha1 or alpha3, in the membranes of various cells. Thus, the properties of the alpha1 isoform were examined in kidney, axolemma and rat alpha1-transfected HeLa cells, and those of the alpha3 isoform, in axolemma, pineal gland, and alpha3*-transfected HeLa cells. The results provide evidence for isoform-independent, tissue-specific modulation of the kinetic behavior of the Na,K-ATPase, the most striking being the differences in the effects of intracellular K as a competitive inhibitor of Na at cytoplasmic Na activation sites.


EXPERIMENTAL PROCEDURES

Antibodies

Antibodies used include M7-PB-E9, a rat alpha3-specific monoclonal antibody, and polyclonal antisera 757 and 50946 which recognize rat beta1 and beta2, respectively(21, 22) , and 754 which was raised against the NH(2)-terminal (amino acids 1-13) sequence of lamb alpha1. A polyclonal antiserum specific for rat alpha3 isoform and monoclonal antibodies specific for alpha1 (6H) were generous gifts from Dr. Michael Caplan, Yale University. Goat anti-mouse antibodies used for immunoprecipitations were purchased from Tago Immunologicals, and horseradish peroxidase-labeled secondary antibodies (donkey anti-rabbit) from Bio/Can Scientific.

Cell Culture and Membrane Preparations

Rat kidney microsomes were prepared as described by Jørgensen (23) and stored in a sucrose-histidine-EDTA buffer (SHE buffer: 0.25 M sucrose, 0.03 M histidine, 1.0 mM Tris-EDTA, pH 7.5) at -70 °C. Rat axolemma membranes were prepared as described by Sweadner (24) and stored at -70 °C in a solution comprising 0.315 M Sucrose, 10 mM Tris, and 1 mM EDTA, at pH 7.4. Rat pineal gland membranes were prepared as described by Ceña et al.(25) , with the following modifications. After sonication (Braun-Sonic 1510 sonicator) four times at low setting for 3 s in SHE buffer, the protein was collected by centrifugation at 100,000 times g for 30 min at 4 °C using a TLA100 rotor in a Beckmann TL-100 centrifuge, resuspended in SHE buffer (approx500 µl/10 mg of original tissue), and stored at -70 °C. Membranes were isolated from rat alpha1- and alpha3*-transfected HeLa cells as described elsewhere (19, 26) and stored at -70 °C. Protein concentrations of the tissue preparations were determined using the Lowry assay as modified by Markwell et al.(27) . Specific activities are indicated in the legends to Fig. 1and Fig. 2.


Figure 1: Activation by Na or K of rat alpha1 Na,K-ATPase from kidney, axolemma, and transfected HeLa cells. Membranes were prepared and assayed as described under ``Experimental Procedures.'' Ouabain-sensitive activities are the difference between hydrolysis measured in the presence of 10 µM and 5 mM ouabain, and are the means ± S.D. (triplicate determinations) expressed as percentages of V(max). The curves were fitted to . Representative experiments are shown and values of K(0.5) and n for replicate experiments are shown in Table 1. A, activation by Na at 10 mM KCl (V(max) values are 3.72 ± 0.07, 0.260 ± 0.004, and 0.107 ± 0.003 µmol/(mgbulletmin) for kidney, axolemma, and HeLa cells, respectively); B, activation by K at 100 mM NaCl (V(max) values are 4.32 ± 0.14, 0.330 ± 0.030, and 0.108 ± 0.003 µmol/(mgbulletmin) for kidney, axolemma, and HeLa cells, respectively). bullet, kidney; circle, axolemma; up triangle, transfected HeLa cells. In the insets the data were fitted to .




Figure 2: Activation by Na and K of rat alpha3 Na,K-ATPase from pineal glands, axolemma, and transfected HeLa cells. Assays were carried out and analyzed as described in Fig. 1, except that ouabain-sensitive activities attributed mainly to alpha3 are the differences between hydrolysis measured in the absence and presence of 5 mM ouabain (pineal gland) or the absence and presence of 10 µM ouabain (axolemma) or in the presence of 10 µM and 5 mM ouabain (alpha3*-transfected HeLa cells). A, activation by Na at 10 mM KCl (V(max) values are 0.353 ± 0.011, 3.34 ± 0.07, and 0.078 ± 0.002 µmol/(mgbulletmin) for pineal gland, axolemma, and HeLa cells, respectively); B, activation by K at 100 mM NaCl (V(max) values are 0.465 ± 0.008, 5.19 ± 0.07, and 0.075 ± 0.003 µmol/(mgbulletmin) for pineal gland, axolemma, and HeLa cells, respectively). bullet, pineal gland; circle, axolemma; up triangle, transfected HeLa cells. In the insets the data were fitted to .





Enzyme Assays

Membranes were permeabilized as described by Forbush(28) . Briefly, they were diluted to 0.06-0.5 mg/ml and treated for 10 min at 22 °C with 1% BSA, 0.65 mg/ml SDS and 25 mM imidazole, after which they were diluted 6-fold with 0.3% bovine serum albumin, 25 mM imidazole. ATP hydrolysis was measured as described previously(29) , in a final volume of 100 µl containing 30 mM Tris-HCl (pH 7.4), 1 mM EDTA, 3 mM MgCl(2), and, unless indicated otherwise, concentrations of NaCl varying from 0.5 to 100 mM with KCl kept constant at 10 mM, or KCl concentrations varying from 0.2 to 50 mM with NaCl kept constant at 100 mM, with choline chloride added so that ([NaCl] + [KCl] + [ChCl]) was constant at 150 mM. Prior to the assay, membranes were preincubated in the reaction medium without or with 10 µM or 5 mM ouabain for 10 min at 37 °C. The reaction was initiated by adding [-P]ATP (final concentration of 1 mM) and NaCl, KCl, and choline chloride to the concentrations listed above.

Immunoprecipitation and Immunoblotting

Axolemma membranes (0.4 mg/ml) were solubilized for 20 min at room temperature in solubilizing buffer comprising 1% Triton X-100 or 1% CHAPS, (^1)0.32% bovine serum albumin, 5 mM EDTA, dissolved in phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10.1 mM Na(2)HPO(4), 1.8 mM KH(2)PO(4)). The insoluble material was removed by centrifugation for 1 min at 500 times g, and the supernatant was then incubated for 1 h at 4 °C with monoclonal mouse antibodies specific for alpha1 or alpha3 (6 µg/150 µl solubilized axolemma). Protein G covalently linked to agarose beads (Pharmacia Biotech Inc.), pretreated for 3 h at 4 °C with goat anti-mouse antibody (100 µl of antibody added to 125 µl of dry beads), were added to the antibody-treated solubilized axolemma (25 µl of the original dry beads added to 150 µl of solubilized axolemma) and incubated overnight at 4 °C. After several washes of the beads (suspension in 200 µl of solubilizing buffer, centrifugation for 1 min at 500 times g), the protein was eluted with 60 µl of sample buffer (0.06 M Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 5% beta-mercaptoethanol, 0.00125% bromphenol blue), incubated at 37 °C for 5 min, and then separated on a 12% SDS-polyacrylamide gel using a Bio-Rad mini gel apparatus as described by Laemmli(30) . Proteins were transferred to a polyvinylidine difluoride membrane (Millipore) which was then blocked for 1 h at 37 °C in blocking buffer (PBS containing 5% milk powder and 0.1% Tween 20) and probed overnight at 4 °C with polyclonal rabbit anti-alpha1, -alpha3, -beta1, or -beta2 antisera diluted in blocking buffer. After several washes with 0.1% Tween 20 in PBS, the membranes were probed (1 h, 37 °C) with horseradish peroxidase-labeled donkey anti-rabbit antibody (diluted 1:5000 in blocking buffer) and exposed for 1 min to the Enhanced ChemiLuminescence (ECL) reagents obtained from Amersham Corp. Densitometry was carried out on several exposures of the Kodak imaging film using a SciScan 5000 scanner and software (U. S. Biochemical Corp.).

Analysis of Kinetic Data

Results are expressed as percentages of V(max) and were analyzed using the Kaleidagraph computer program with either (i) the equation for the noncooperative, three-site model described by Garay and Garrahan (31) :

where K is the apparent affinity for Na, and [Na] is the Na concentration or (ii) the cooperative, n-site model described by the following form of the Hill equation (cf. (32) ):

where ``cat'' represents the cation (K or Na). was fitted to the experimental points and the values of V(max), K, and n obtained. K representing K or K was obtained from through the relationship K = (K)^1^n.


RESULTS

Naand KActivation Profiles of Distinct alpha Isoforms

To gain insight into the basis for the discrepancies in apparent cation affinities, a series of experiments were carried out in which the cation activation profiles of pumps of the same alpha isoform but from different tissues were compared. This comparison was confined to alpha1 and alpha3 of the rat. It was technically not feasible to include alpha2 in the analysis since there are virtually no suitable tissues with predominantly this isoform. (Although alpha2 may predominate in adult skeletal muscle, a high background Mg-ATPase activity precludes meaningful kinetic analysis of Na,K-ATPase.) For alpha1, the tissues compared were kidney, alpha1-transfected HeLa cells, and axolemma. The activity of alpha1 in axolemma was determined by taking advantage of the low sensitivity of the rodent alpha1 isoform to cardiac glycosides. Thus, axolemma alpha1 was assayed in the presence of 10 µM ouabain which effectively inhibits alpha2 and alpha3(33) . The alpha3-rich tissues compared were pineal gland, alpha3*-transfected HeLa cells, and axolemma. The difference in activity observed in the absence and presence of 10 µM ouabain was ascribed mainly to alpha3 since the proportion of alpha2 in axolemma is relatively low (20) . In the case of the pineal gland, we have confirmed the report by Shyjan et al.(18) showing that the predominant alpha isoform detected in immunoblots of the adult rat pineal gland is alpha3 (results not shown). In addition, alpha1 is also detected, but the immunoblots do not provide information regarding the relative activities of the two isoforms. Therefore, assays to quantify ATPase activity sensitive to low (10 µM) versus high (5 mM) ouabain concentrations were carried out, and the results indicated that the activity of alpha1 is less than 5% that of alpha3 in pineal gland (experiment not shown).

The results of kinetic experiments carried out with alpha1-containing membranes isolated from rat kidney, axolemma, and rat alpha1-transfected HeLa cells are shown in Fig. 1, and the results for membranes rich in alpha3, in Fig. 2. The data are expressed as percentages of V(max) and the curves are best fits to . The insets in Fig. 1A and Fig. 2A represent the same data fitted to .

As shown in Fig. 1A, the apparent Na affinity of alpha1 from kidney, with the K concentration held constant at 10 mM, appears somewhat lower than that of alpha1 from either axolemma or HeLa; K values were 6.6 ± 0.6, 4.7 ± 0.9, and 5.0 ± 0.3 mM for the three tissues, respectively. At a higher K concentration (20 mM; cf. (18) ), the difference in the K value for alpha1 of kidney became greater as indicated below (see Fig. 4). In the case of K activation (Fig. 1B), the order of apparent affinities (assayed at 100 mM Na) are as follows: axolemma < kidney approx HeLa, with K values of 2.4 ± 0.5, 0.9 ± 0.1, and 1.1 ± 0.3 mM, respectively. For the alpha3 isoform of the enzyme, Fig. 2A shows that the apparent affinity for Na of alpha3* from HeLa cells is markedly lower than that of alpha3 from either pineal gland or axolemma; K values were 11.1 ± 1.5, 4.9 ± 0.8, and 5.7 ± 0.9 mM, respectively. The K-activation profiles of alpha3 pumps (Fig. 2B) show that the apparent affinity for K of HeLa alpha3* pumps is higher than that of other tissues (K values were 0.7 ± 0.1, 1.6 ± 0.1, and 1.4 ± 0.4 mM, respectively). The kinetic constants and Hill coefficients (n) are shown in Table 1.


Figure 4: Dependance of K ` on K concentration for rat pumps from kidney, axolemma, pineal gland, and transfected HeLa cells. Assays were carried out as described in Fig. 1, but at varying concentrations of KCl (5, 10, 20, 35, and 50 mM). K were first determined by fitting the data obtained for each Na-activation curve to and were then plotted as a function of KCl concentration. Each point represents an average ± S.D. of at least three separate experiments, and the values of K and K(K) obtained are shown in Table 2. A, alpha1 pumps: bullet, kidney; circle, axolemma; , transfected HeLa cells. B, alpha3 pumps: bullet, pineal gland; circle, axolemma; , transfected HeLa cells.





In other experiments (not shown), the possibility that the results from axolemma membranes were flawed by an incomplete distinction of alpha1 from alpha3 was tested. Thus, since axolemma alpha1 activity is measured as the difference in ATPase activity in the presence of low and high ouabain concentrations, and alpha3, as the difference in activity in the absence and presence of low ouabain, a spuriously lower-than-true apparent affinity of axolemma alpha1 and higher-than-true apparent affinity of alpha3 for K may have resulted from the well documented K-mediated decrease in ouabain binding(34) . In other words, incomplete inhibition of alpha3 at high K concentrations would effect an apparent affinity decrease in the former and affinity increase in the latter curves relating activity to K concentration. In order to rule out this possibility, even though this K/ouabain antagonism is minimal in rat brain preparations(34) , an experiment was carried out in which the K concentration was varied in the presence of different concentrations of ouabain (5, 10, and 20 µM). It was found that K for the alpha1 enzyme remained constant at all three ouabain concentrations. Moreover, it should be noted that the enzyme was preincubated with ouabain in the absence of K (see ``Experimental Procedures''), and that the enzyme activity measured thereafter remained constant as a function of time.

KInteractions at Cytoplasmic NaBinding Sites

One of the inherent problems in kinetic studies of Na,K-ATPase in membrane fragments is the lack of control of the composition of cations at the cytoplasmic versus extracellular milieu. Specifically, it has been shown that K binding and inhibition at the cytoplasmic Na activation sites alters the enzyme's apparent affinity for Na(31) . To determine whether Na/K interactions are, indeed, distinct for the sodium pumps of different tissues, a series of activity measurements were carried out at varying K concentration and Na maintained constant at a low 5 mM rather than 100 mM concentration. The results shown in Fig. 3, A and B, indicate that the extent of K-inhibition at the presumably cytoplasmic Na binding site is at least partly affected by the nature of the tissue. As shown in Fig. 3A, the kidney alpha1 enzyme is significantly more sensitive to K inhibition than alpha1 from either HeLa cells or axolemma; at 20 mM KCl, a concentration at which the [K]/[Na] ratio of 4 is still lower than the normal physiological value (>10), the activity of kidney alpha1 is reduced by 40%, whereas that of the other tissues is minimally affected. The results for alpha3 (Fig. 3B) show that the transfected HeLa enzyme is much more sensitive to inhibition by K than either the axolemma or pineal gland enzymes.


Figure 3: Potassium inhibition of Na,K-ATPase of kidney, axolemma, pineal gland, and transfected HeLa cells at low Na concentration. Membranes were prepared and ATPase activity assays performed as described in Fig. 1and Fig. 2, but in the presence of 5 mM NaCl. The representative experiments show the mean ± S.D. of triplicate determinations expressed as percentages of the activity measured at 2 mM KCl. A, alpha1 pumps: bullet, kidney; circle, axolemma; up triangle, transfected HeLa cells. B, alpha3 pumps: bullet, pineal gland; circle, axolemma; up triangle, transfected HeLa cells.



It has been observed that the antagonistic effect of vanadate, a potent inhibitor of the sodium pump, is facilitated by the presence of K ions(35, 36) . To ensure that the K-mediated inhibition observed in this study is not the result of vanadate present in the kidney preparation, two control experiments were performed: (i) in one, assays were carried out in the presence of 2.5 mM norepinephrine, which reverses the effect of vanadate(36) , and (ii) in the other, the assay time was reduced 10-fold and the amount of kidney microsome sample increased 10-fold; if vanadate were present in the microsome suspension, such an increase in endogenous vanadate concentration should result in greater inhibition of activity. Neither of these conditions altered the K-inhibition profiles, which argues against an apparent K inhibition secondary to the presence of vanadate in the kidney preparation.

Based on the Albers-Post model of the Na,K-ATPase reaction mechanism and, more specifically, on the model which assumes random binding of Na and K to (the same) three equivalent sites on the cytoplasmic side of the enzyme, Garay and Garrahan(31) , in their studies on Na efflux in red cells, and Sachs(37) , in studies of ouabain-sensitive ATPase activity in broken red cell ghosts, showed that activity adhered closely to the following relationship:

where [Na] and [K] are the cytoplasmic concentrations of Na and K, respectively. In accordance with this model, the plot of K, the apparent affinity for sodium, as a function of K concentration yields the linear relationship K = K(1 + [K]/K(K)) (Equation 4; see (37) ). From this plot, K, the apparent affinity for Na when the K concentration is zero, as well as K(K), the apparent affinity for K at the cytoplasmic Na binding site, are readily obtained (cf. (37) ). In order to apply this analysis to our system, a series of experiments were carried out in which K was determined at various K concentrations for each of the tissues studied in Fig. 1and Fig. 2. It should be noted that the plots of K shown in Fig. 4, A and B, are best fits to the model described by (see insets of Fig. 1A and 2A), which adheres to the noncooperative assumptions of Garay and Garrahan, while the data of Fig. 1and Fig. 2were best fits to . The values of K and K(K), representing the cytoplasmic binding constants for Na and K, respectively, and calculated from the Fig. 4plots, are shown in Table 2.

In comparing these values, it is evident that the main difference between the alpha1 pumps of kidney and those of HeLa and axolemma is the higher apparent affinity for K as a competitive inhibitor of Na. In the case of the alpha3 isoforms, however, the lower apparent affinity for Na characteristic of transfected HeLa cells (Fig. 2A) is a function of both a lower affinity for Na as an activator as well as a higher affinity for K as a competitive inhibitor, as compared to either the pineal gland or axolemma alpha3 enzymes. Thus, the ratio K/K(K) reflects the ability of K to compete with Na for the cytosolic cation binding site and the larger the ratio, the more susceptible the enzyme is to competitive inhibition by K. A large K/K(K) ratio explains the greater K inhibition, at low Na concentration, observed in the case of kidney pumps and HeLa alpha3* pumps, as depicted in Fig. 3, A and B. As well, since K/Na antagonism should decrease as the Na concentration is increased, the curves of enzyme activity versus Na concentration shift to the right, resulting in the lower apparent affinities for Na, as noted in Fig. 1A and Fig. 2A.

Are the Tissue-specific Kinetic Differences the Result of alpha Associations with Different beta Isoforms?

The question as to whether differences in cation activation are due, at least to some extent, to differences in the beta isoform which associates with alpha was approached by carrying out a series of experiments involving immunoprecipitation of alpha1 and alpha3 from axolemma, followed by immunoblotting with alpha and beta isoform-specific antibodies to determine the nature of the associated beta subunits. The results of these experiments are shown in Fig. 5and summarized below. This question is relevant only to axolemma membranes since only beta1 is present in kidney and only beta2, in the adult pineal gland. In addition, HeLa cells contain beta1 message (38, 39) and beta1 protein has been detected by Western blotting; (^2)neither beta2 message nor protein were detected by polymerase chain reaction, Northern analysis, or Western blotting. (^3)


Figure 5: Coimmunoprecipitation of the beta subunit with alpha1 and alpha3 from rat axolemma. Membranes were prepared, solubilized in 1% Triton X-100 and immunoprecipitated with either an alpha1- or alpha3-specific monoclonal antibody as described under ``Experimental Procedures.'' Following SDS-polyacrylamide gel electrophoresis, the proteins were analyzed by Western blotting using polyclonal antisera specific for: A, alpha1; B, alpha3; C, beta1; and D, beta2. Lanes are: 1, axolemma; 2, kidney; 3, Immunoprecipitate from axolemma using alpha1-specific monoclonal antibody 6H; 4, Immunoprecipitate from axolemma using alpha3-specific monoclonal antibody M7-PB-E9; and 5, control: immunoprecipitation performed in the absence of the primary antibody. Molecular masses are given in kilodaltons.



When a mouse monoclonal antibody specific for alpha1 was used to immunoprecipitate the enzyme of Triton X-100-solubilized axolemma membranes, the only subunit isoforms detected on Western blots using rabbit polyclonal antisera specific for alpha1, alpha3, beta1, and beta2 as primary antibodies, were alpha1 and beta1 (Fig. 5, A-D, lanes 3). Further, when a mouse monoclonal antibody specific for alpha3 was used, alpha3 and beta1 were detected along with a barely visible band corresponding to beta2 (Fig. 5, A-D, lanes 4). Control experiments carried out omitting the precipitating antibody showed minimal amounts of nonspecific binding of the axolemma Na,K-ATPase subunits to the protein G-linked agarose beads (Fig. 5, A-D, lanes 5). The fact that the appearance of two bands, one of slightly higher mobility than alpha1 and alpha3 (Fig. 5, A and B, lane 3), the other at approx50 kDa (Fig. 5C, lanes 3-5), reflect nonspecific reactions was evidenced in the following controls (not shown). (i) The first band was present even when the primary detecting antibody was omitted, thus indicating that it is the result of nonspecific binding of the secondary blotting antibody to the primary immunoprecipitating antibodies. (ii) The second nonspecific band appeared even when the primary antibody (mouse anti-alpha1 or -alpha3) was omitted (Fig. 5C) or when the procedure was carried out in the absence of solubilized axolemma (not shown), indicating that it probably represents a nonspecific reaction involving the primary blotting antibody to beta1 and goat anti-mouse antibodies.

alpha-beta Stoichiometries in Axolemma

The stoichiometry of the alpha-beta associations was then evaluated in order to assess whether these associations may have been disrupted as a result of the membrane solubilization procedure. These analyses were done utilizing different exposures of the Western blots from several replicate experiments which were quantified as described under ``Experimental Procedures.'' In this work, two assumptions were made; first, that the alpha1:beta1 subunit stoichiometry of the kidney enzyme, as based on studies of the purified enzyme, is 1:1 (for example, see (40) ) and second, that heterodimers comprising beta1 are not preferentially immunoprecipitated compared to those comprising beta2.

The estimate of alpha1:beta1 stoichiometry in axolemma was based on a comparison of the densities of the alpha and beta bands of axolemma immunoprecipitated with anti-alpha1 monoclonal antibody with those of unprecipitated kidney microsomes, following exposures to anti-alpha1 and anti-beta1 antisera as shown in Fig. 5, A and C (lanes 2 and 3). The ratio of alpha1 to beta1 in precipitated axolemma was found to be 1.08 ± 0.17 (S.E. for five independent experiments).

Because there is no tissue in which alpha3 and beta1 have been shown to be expressed in a 1:1 ratio, the question of possible alpha3-beta1 versus alpha3-beta2 associations was evaluated as follows. The alpha3:beta1 ratio as detected in anti-alpha3-immunoprecipitated axolemma samples was compared to that found in unprecipitated axolemma membranes, after correcting the ratio for the proportion of beta1 presumed to associate with alpha1. The ratio in the immunoprecipitate of axolemma sample was found to be reasonably close to the ``corrected'' ratio observed in the unprecipitated membranes. Thus, if the corrected alpha3:beta1 ratio of unprecipitated axolemma is normalized at 1.00, the ratio of the precipitate is 1.08 ± 0.09 (S.E. for four independent experiments).

It should also be mentioned that in other experiments (not shown) aimed to determine whether the detergent Triton X-100 interfered with subunit interactions, immunoprecipitations were also carried out with a 4-fold lower concentration of Triton X-100 (0.25%) and with 1% CHAPS. Under both conditions, the alpha:beta ratios obtained were not significantly different from those observed with 1% Triton X-100 (data not shown).


DISCUSSION

In this study we show that the divergent results regarding the relative affinities of the Na,K-ATPase of the different rat isoforms as reported in different laboratories are not simply accounted for by differences in the experimental conditions used. Thus, we have reproduced the relative cation affinities for alpha1 versus alpha3 as reported by Jewell and Lingrel (19) and Munzer et al.(20) on the one hand, and those of Sweadner (17) and Shyjan et al.(18) , on the other. To gain insight into the basis for this dichotomy, we have assessed the apparent cation affinities of pumps of the same catalytic isoform, either alpha1 or alpha3, but from different tissues and, therefore, membrane environments. Marked differences in the apparent affinities for both Na and K were observed in pumps of the same alpha isoforms isolated from different cellular sources. These data and previous work in lamb (22) and dog (41) tissues are consistent with the conclusion that factors other than the type of catalytic isoform influence interactions of the pump with Na and K.

The most obvious tissue-specific protein component which interacts with the pump is the beta subunit. In fact, effects of different beta subunits on both K(10, 11, 12) and Na(13, 14) affinities have been described. Accordingly, one question is whether the kidney enzyme's lower apparent affinity for Na and higher apparent affinity for K as compared to other alpha1 pumps are the result of interactions of alpha1 with different beta subunits. To address this question, particularly in axolemma, in which both beta1 and beta2 have been identified, the nature of the beta subunit which coimmunoprecipitates with the distinct alpha subunits was assessed. The results of these experiments indicate that beta1 associates with alpha1 in axolemma and that the stoichiometry of the association is close to 1.0. The determination of alpha/beta1 stoichiometries imply that little, if any, beta2 associates with either alpha1 or alpha3. Whether beta2 associates preferentially with alpha2 in axolemma remains to be determined. Although HeLa cells contain human beta1, while kidney cells contain rat beta1, these subunits are 95% identical(39) . A difference in both type and amount of glycosylation has been observed between kidney and brain beta1 (42) and is presumably the basis for the differences in mobilities in immunoblots of kidney and axolemma as shown in Fig. 5C. The possibility remains that these differences are at least partly responsible for the distinct kinetics, even though there is evidence that the oligosaccharides are not essential for primary function (reviewed in (43) ).

It is unlikely that the presence of distinct betas account for differences of alpha1 of kidney, axolemma, and HeLa, and of alpha3 of axolemma and HeLa; in both instances, beta1 is the predominant beta isoform present or associated with alpha1 or alpha3. However, tissue-specific differences in cation affinities, despite similar alphabeta pairing, does not imply that the beta subunit has no effect on function. In fact, a kinetic difference due to the distinct beta subunits is observed in the case of alpha3. Thus, as shown in Table 2, the ratio K/K(K) is 1.7-fold lower in pineal gland compared to axolemma, reflecting the 1.9-fold difference in K(K). That this difference is a result of beta2 association with alpha3 in the pineal gland and of beta1 with alpha3 in axolemma is supported by a recent report showing a 1.6-fold higher apparent affinity for Na of alpha3beta2 compared to alpha3beta1 in Sf-9 cells transfected with these isoform pairs (14) . In that study, the Na activation kinetics from which the kinetic constants were obtained were carried out in the presence of 30 mM K so that the difference in apparent affinity for Na may also reflect a difference in K(K). Other kinetic differences were not detected. Taken together, these results are consistent with a role for the distinct betas in modulating K interactions at cytoplasmic Na sites.

An important observation regarding the coimmunoprecipitation studies presented here is the isoform specificity of the reactions as evidenced in the coimmunoprecipitation of alpha with beta, but not of alpha1 with alpha3. This lack of coimmunoprecipitation between the different alpha isoforms is in contradiction with reports that pumps coimmunoprecipitate as alpha heterodimers in rat brain and in bacculovirus infected Sf-9 cells(5) . Although it is possible that the detergent (1% CHAPS) used in that study (5) did not fully solubilize the membranes, or that the Triton X-100 used in this study disrupted alpha-alpha interactions, these are unlikely explanations, since we confirmed our results using 1% CHAPS.

It can be argued that certain methodological procedures may be responsible for some of the differences observed for the alpha3 enzyme of axolemma as compared to that of other tissues. As described above, the activity ascribed to alpha3 in axolemma is that which is sensitive to 10 µM ouabain. Unfortunately, the ouabain-affinities of alpha2 and alpha3, both present in axolemma, are quite similar(4) , and it was technically difficult to distinguish the two on that basis. However, the amount of alpha2 in axolemma is relatively low (approx25%)(20) , so that its effect on the kinetic behavior cannot account for the magnitude of the differences in the observed kinetic constants as discussed below. As well, the similar fold difference in apparent affinity for external K ascribed to alpha1 versus alpha3 in two separate systems (transfected HeLa cells compared to axolemma- and kidney-fused red blood cells; see (20) ) argues against a substantial contribution of alpha2 to the behavior of the ouabain-sensitive pumps of axolemma. For the same reasons, it is unlikely that the mutation of alpha3 to render it ouabain-resistant in HeLa cells alters its behavior.

There has been some evidence that detergents, such as SDS used here to increase the permeability of membrane vesicles to substrates, can have an effect on cation activation kinetics of the Na,K-pump(44) . Although such an effect was not observed in the case of the Na-activation profile of axolemma enzyme, or in the case of the Na and K activation profiles of transfected HeLa cells (data not shown), it is entirely possible that other pumps might react differently to SDS treatment. However, the SDS concentration and the SDS:protein concentration ratio were identical in all of our experiments. Therefore, if SDS affects the different enzymes to varying extents, it should be the result of differential interactions with the surrounding environment, which would be consistent with the notion that the catalytic behavior of the pump does not depend solely on the isoform of the alpha subunit.

The most intriguing results of this study concern tissue-distinct K/Na antagonism. Differences in K(K), the apparent affinity for K at (cytoplasmic) Na activation sites, underlie tissue-specific differences in the sodium-activation profiles noted in both the present and earlier studies(17, 18, 19, 20) . There is evidence also of differences in apparent Na affinity, independent of K concentration, between pumps of the same catalytic isoform, although this difference was slight in the case of the kidney enzyme compared to other alpha1 pumps (see Table 2). In general, the ability of K to act as a competitive inhibitor of Na binding is reflected in the ratio of K to K(K), the cytosolic binding constants for Na and K, respectively. It is apparent from Table 2that, of the membrane systems examined, alpha1 in the kidney and alpha3 transfected into the HeLa cell have the largest K/K(K) ratios when compared to other pumps with the same alpha isoforms. It is these two enzyme preparations which exhibit the greatest sensitivity to inhibition by K as depicted in Fig. 3. Specifically, it seems that in the case of kidney alpha1, this inhibition is due to its relatively lower K(K) compared to the other alpha1 pumps, whereas with HeLa alpha3 pumps, it is due mainly to a higher K (Table 2). Whether these characteristics are intrinsic to the enzyme, for example due to tissue-specific co- or postranslational modification(s), or rather, the result of modulation of the enzyme by another associated protein remains unresolved.

The kinetic analyses of the sigmoid activation kinetics described in this and previous studies (17, 18, 19, 20) are based on conventional cooperative or noncooperative models. Using the noncooperative model, the apparent affinities of alpha1 and alpha3 for intracellular Na and K of the same tissue (HeLa) derived in the present study can account for the low affinities of alpha3 compared to alpha1 observed in studies of Munzer et al.(20) using intact cells, with the following provisos. Those authors pointed out that their data points for Na activation of alpha3 pumps could be obtained only in the region of the curve well below saturation due to the technical difficulty of raising intracellular Na above approx45 mM. This precluded a reliable estimate of the kinetic constants for alpha3 when using the noncooperative model. However, the data fitted well to a cooperative model (Equation 2 in (20) ) giving K(0.5) values for intracellular Na of 17.6 mM for alpha1 and and 63.5 for alpha3. In the present study, the K and K(K) values for rat alpha1 and alpha3 in HeLa membranes (Table 2) were used to obtain the observed apparent affinity, K, at 135 mM intracellular K, a concentration approximating that of the intact cells used by Munzer et al.(20) . Using these values of K (7.1 mM for alpha1 and 30.3 mM for alpha3) we derived curves of pump activation as a function of varying intracellular Na using the cooperative model (Fig. 6). The curves (solid lines) and the values of K(0.5) thus obtained (19.2 mM for alpha1 and 75.2 mM for alpha3) are similar to those derived from the data of previous studies (20) with intact cells (Fig. 6, dashed lines). Therefore, these results indicate that the physiologically significant extremely low apparent affinity of alpha3 reflects its much greater sensitivity to inhibition by intracellular K.


Figure 6: Comparison of Na-activation curves derived from kinetic constants in Table 2and those obtained in transport studies with intact cells. Using Equation 4, K and K(K) values from Table 2were used to obtain K for alpha1 and alpha3 at 135 mM K (cf. (20) ). The K values thus obtained (7.1 mM for alpha1 and 30.3 mM for alpha3) were used to derive curves (solid lines) of percent of V(max) as a function of Na concentration using the cooperative model () with n = 3.0 as in Munzer et al.(20) . Data points for alpha1 (bullet) and alpha3 () were taken from Fig. 6of Munzer et al.(20) and are also expressed as percent of V(max). The dashed curves were derived from the K(0.5) values for intracellular Na shown in Table 2of Munzer et al.(20) .



Modulation of pump behavior by the membrane environment is most likely the explanation for the discrepancies among previous reports concerned with isoform-specific behavior. A slightly higher Na affinity in axolemma compared to kidney was reported first by Sweadner (17) , and later by others including Shyjan et al.(18) who compared brain and kidney. This observation was replicated in the present study of kidney and axolemma Na,K-ATPase, with values very close to those reported by Sweadner, i.e. K values of 0.72 mM and 1.02 mM, respectively (Table 2) corresponding to K(0.5) values of 4.0 mM and 4.3 mM for axolemma and kidney, respectively (not shown).

Tissue-specific as well as isoform-specific behavior is evident not only in apparent affinities for cytoplasmic Na and K but also for K at extracellular sites (Table 1). Thus, it was only in the same (red cell or HeLa cell) environment that a higher affinity for extracellular K of alpha3 or axolemma compared to kidney (alpha1) was observed(19, 20) . It may be relevant that exogenous kidney pumps fused into red cells and endogenous red cell pumps behave identically with respect to the apparent affinity for extracellular K, K(45) . (The greater difference between alpha1 and alpha3 observed in studies with intact cells may reflect the limitation of kinetic studies with unsided preparations).

The foregoing considerations argue in favor of the conclusion that the primary structure of the alpha isoform is not the sole determinant of the magnitude of cation affinity/selectivity. Our observations are consistent with the existence of some pump modulator, for example one which interacts and effects a greater sensitivity of alpha1 to K inhibition in the kidney compared, for example, to alpha1 of axolemma; in the microsome-fused red cell system, association of the alpha subunit with the putative regulator may be interrupted following its association with other components of the new (red cell) environment. This kind of regulation is reminiscent of the effects of an intrinsic red cell membrane (blood group) antigen, Lp, found in genetically low potassium sheep red blood cells. This protein or glycoprotein interacts with the pump and effects K-inhibition (for review, see (46) ). Interestingly, when kidney pumps are delivered from microsomes into low potassium sheep red cells, the Lp antigen effects susceptibility to K inhibition(47) , which supports the idea that fusion into red blood cells confers a new membrane environment for the pump.

The study described in this report provides evidence in support of the conclusion that factors in addition to the primary structure of the alpha isoforms dictate the kinetic behavior of the Na,K-ATPase. Likely candidates include other membrane-bound components or modulation by co- or post-translational modifications of either subunit.


FOOTNOTES

*
This work was supported in part by the Medical Research Council of Canada Grant MT-3867 (to R. B.) and by the National American Heart Association Grant-in-aid (to W. J. B.). 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: Montreal General Hospital, 1650 Cedar Ave., Montreal, Quebec, Canada H3G 1A4. Tel.: 514-937-6011 (ext. 4501); Fax: 514-934-8332.

(^1)
The abbreviation used is: CHAPS, 3-[(3-cholamidopropyl)dimethylamonio]-1-propanesulfonic acid.

(^2)
W. J. Ball, unpublished results.

(^3)
R. Levenson, personal communication.


ACKNOWLEDGEMENTS

We thank Dr. Michael Caplan, Yale University School of Medicine, for his generous gifts of the polyclonal anti-alpha3 and monoclonal anti-alpha1 antibodies and Drs. E. A. Jewell-Motz and J. B. Lingrel, University of Cincinnati, for the gifts of the isoform-transfected HeLa cells. We are grateful to Dr. K. J. Sweadner, Massachusetts General Hospital, for helpful suggestions, Dr. R. Levenson, Pennsylvania State University, for communicating his unpublished results, and Dr. Stewart Daly for technical assistance.


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