K+/Na+ antagonism at cytoplasmic sites of Na+-K+-ATPase: a tissue-specific mechanism of sodium pump regulation

Alex G. Therien and Rhoda Blostein

Department of Biochemistry, McGill University, Montreal, Canada H3G 1A4


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue-distinct interactions of the Na+-K+-ATPase with Na+ and K+, independent of isoform-specific properties, were reported previously (A. G. Therien, N. B. Nestor, W. J. Ball, and R. Blostein. J. Biol. Chem. 271: 7104-7112, 1996). In this paper, we describe a detailed analysis of tissue-specific kinetics particularly relevant to regulation of pump activity by intracellular K+, namely K+ inhibition at cytoplasmic Na+ sites. Our results show that the order of susceptibilities of alpha 1 pumps of various rat tissues to K+/Na+ antagonism, represented by the ratio of the apparent affinity for Na+ binding at cytoplasmic activation sites in the absence of K+ to the affinity constant for K+ as a competitive inhibitor of Na+ binding at cytoplasmic sites, is red blood cell < axolemma approx  rat alpha 1-transfected HeLa cells < small intestine < kidney < heart. In addition, we have carried out an extensive analysis of the kinetics of K+ binding and occlusion to the cytoplasmic cation binding site and find that, for most tissues, there is a relationship between the rate of K+ binding/occlusion and the apparent affinity for K+ as a competitive inhibitor of Na+ activation, the order for both parameters being heart >=  kidney > small intestine approx  rat alpha 1-transfected HeLa cells. The notion that modulations in cytoplasmic K+/Na+ antagonism are a potential mode of pump regulation is underscored by evidence of its reversibility. Thus the relatively high K+/Na+ antagonism characteristic of kidney pumps was reduced when rat kidney microsomal membranes were fused into the dog red blood cell.

alpha 1-isoform; heart; kidney


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE NA+-k+-atpase, or sodium pump, is a ubiquitous membrane protein complex that maintains the high electrochemical gradient of Na+ and K+ across the plasma membrane of animal cells (for reviews, see Refs. 13, 20, and 29). It comprises two essential subunits, alpha  and beta . The catalytic alpha -subunit encompasses the sites of nucleotide and cation binding and undergoes conformational transitions associated with the coupling of ATP hydrolysis to the translocation of Na+ and K+. The beta -subunit is required for proper insertion and stability of the enzyme in the plasma membrane and also has a role in modulating cation affinity (reviewed in Ref. 4). Multiple isoforms of both the alpha  (alpha 1, alpha 2, alpha 3, alpha 4)- and beta  (beta 1, beta 2, beta 3)-subunits are expressed in a tissue- and development-specific manner (3, 19).

Although the basic function of the Na+-K+-ATPase is the maintenance of cation homeostasis, modification of its behavior in certain tissues may be critical to specialized functions such as Na+ reabsorption across epithelia, plasma K+ clearance by skeletal muscle, adjustment of the set point for Na+/Ca2+ exchange in the heart, and restoration of the electrochemical cation gradient after propagation of the nerve impulse. Relevant to such diversity of function in various tissues is an increasing body of evidence suggesting that the Na+-K+-ATPase is subject to complex short- and long-term regulation. In intact cells, sodium pump activity may be modulated by alterations in 1) intrinsic kinetic behavior, 2) cell surface expression, and 3) de novo pump synthesis (for reviews, see Refs. 2 and 8). Furthermore, the distinct properties of the different isoforms of the catalytic subunit and their putative distinct susceptibilities to regulatory processes comprise a diverse and elaborate set of modulatory mechanisms.

Although the nature of the alpha -subunit isoform may be the primary determinant of the intrinsic kinetic properties of the enzyme, other cell-specific components may interact with and modulate kinetic behavior. In an earlier study, we described tissue-specific differences in the interactions of the enzyme with Na+ and K+ (28). A particular intriguing finding, albeit rudimentary, was a notable difference in the effects of K+ as a competitive inhibitor at cytoplasmic Na+ activation sites of pumps comprising either alpha 1beta 1 or alpha 3beta 1. In this report, we describe a more extensive analysis of this tissue-specific K+/Na+ antagonism and its mechanistic basis as it pertains to alpha 1beta 1 pumps. Using the technique of fusing alpha 1beta 1 pumps from one tissue (kidney medulla) into another (red blood cell), we show that the tissue-specific effects are, in at least one instance, subject to modification.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture and membrane preparations. Rat alpha 1-transfected HeLa cells were grown and maintained in culture as described elsewhere (27). Membranes from kidney, axolemma, and heart and alpha 1-transfected HeLa cells and red blood cells were prepared as described previously (27, 28). Epithelial cells from small intestine were isolated by an adapted method (18). Briefly, rat small intestines were sliced longitudinally and washed with ice-cold 340 mM NaCl, and epithelial cells were detached from the intestine by incubation in 240 mM NaCl containing 2.5 mM EDTA for 1 h at 4°C. After removal of the remaining intestine, the detached cells and cellular debris were pelleted by centrifugation for 15 min at 39,000 g. Membranes were prepared from the pellet by the method used for kidney medulla. All membrane preparations were stored at -70°C in a solution containing 1 mM EDTA.

Enzyme assays. Before all experiments, membranes were permeabilized by preincubation for 10 min at room temperature in 15 mM Tris-Cl (pH 7.4) containing 1% BSA and 0.65 mg/ml SDS, followed by dilution with 15 mM Tris-Cl (pH 7.4) containing 0.3% BSA, essentially as described by Forbush (10). Such treatment was previously shown to yield maximally permeabilized membranes, at least for kidney (data not shown). Assays of Na+-K+-ATPase activity were carried out essentially as described previously (28), except that 5 µM ouabain was included to inhibit the activity of isoforms other than the rat alpha 1. Accordingly, alpha 1-specific activities were determined as the difference in ATP hydrolysis measured in the presence of 5 µM ouabain and either 5 mM ouabain or 100 mM KCl in the absence of NaCl, with no detectable differences between these two baselines. Average activities of the membrane preparations of rat kidney medulla, axolemma, heart, alpha 1-transfected HeLa cell, red blood cell, and small intestine membranes (µmol Pi · mg protein-1 · min-1) were as follows: 2.8, 0.23, 0.14, 0.15, 0.05, and 0.5, respectively. For membrane preparations of mouse kidney (whole), axolemma, and heart, the activities were: 0.84, 0.15, and 0.10, respectively. Assays of the K+ dependence of K+ occlusion [E1 + K+ left-right-arrow  E2(K)], where E1 is the K+-free enzyme and E2(K) is the occluded enzyme, and the rate of deocclusion [E2(K) right-arrow right-arrow E1 + K+] were also carried out as described elsewhere (6), with the following modifications: 1) the final assay volume was 100 µl and contained 5 µM ouabain, 2) ionic strength was kept constant at 4 mM with choline chloride during the preincubations with K+, and 3) for the deocclusion assays, enzyme was preequilibrated with 4 mM K+, which was found to be sufficient to form maximal E2(K) for all tissues used.

Polyethylene glycol-mediated fusion of kidney microsomes to dog red blood cells. Fusion of rat kidney microsomes to dog red blood cells was carried out essentially as described by Munzer et al. (22) with minor modifications. Dog blood was collected into 1/10 volume of 0.1 M EDTA. The red blood cells were isolated by centrifugation (2 min at 500 g) and washed four times at 4°C with 10 volumes of wash buffer (140 mM NaCl, 10 mM KCl, 5 mM glucose, 68 mM sucrose, and 20 mM Tris-PO4, pH 7.4). The cells were then suspended and incubated in wash buffer for 1 h at 37°C, centrifuged, and washed one time at 4°C with wash buffer containing 1 mM adenosine and 0.5 mM adenine. One hundred fifty microliters of SHE solution (0.25 M sucrose, 0.03 M histidine, 1.0 mM EDTA-Tris, pH 7.4) or SHE containing 0.20-0.45 mg rat kidney microsomes was added to 1 ml of the packed red blood cells, and the cells were then incubated for 15 min at room temperature, followed by dropwise addition of 70% polyethylene glycol to a final concentration of 45%. The suspension was further incubated with gentle mixing for 45 s at room temperature, then 90 s at 37°C, followed by successive dilutions with 6, 14, and 26 ml of repletion buffer (wash buffer containing 1 mM adenosine, 0.5 mM adenine, and 2 mM MgCl2), with 30-s incubations at room temperature before each dilution. Cells were then incubated further for 1 h at 37°C, centrifuged for 5 min at 100 g, and washed four times at 4°C with 10 vol of wash buffer containing 2 mM MgCl2, by resuspension and centrifugation at 100 g. These last steps resulted in the isolation of cells free of unfused microsomes. Membranes from fused and mock-fused red blood cells were prepared by the same method as that used for rat red blood cells.

Analysis of kinetic data. Data for Na+ activation of Na+-K+-ATPase activity and for K+ dependence of K+ occlusion were expressed as percentages of maximal activity or maximal occlusion, as determined by extrapolation of the curves, using the Kaleidagraph computer program (Synergy software) with the noninteractive model of cation binding described by Garay and Garrahan (12)
<IT>m</IT> = <IT>M</IT>/(1 + <IT>K</IT>′<SUB>cat</SUB>/[cat])<SUP><IT>n</IT></SUP> (1)
where m represents either the rate of the reaction or the level of K+-occluded enzyme [E2(K)]; M represents either maximal Na+-K+-ATPase activity or maximal K+ occlusion; K'cat represents either K'Na, the apparent affinity for Na+, or Kocc, the apparent affinity for K+ occlusion; [cat] represents the concentration of cation, either Na+ or K+; and n represents the number of binding sites, either three in the case of the Na+ activation experiments or two in the case of the K+ occlusion experiments. Variances are given as SDs, except for Table 1 in which case the SEs obtained from least square fits are shown. In the case of the estimations of the occlusion rate constant, ko (ko = kd/Kocc, where kd is the deocclusion rate constant; Table 2), the SDs were obtained from a Monte Carlo simulation of a joint probability distribution.

                              
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Table 1.   Affinities for Na+ and K+ binding to Na+ activation sites of alpha 1 pumps of various tissues


                              
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Table 2.   Comparison of kinetic parameters Kocc, kd and ko


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previous studies of the Na+ and K+ activation kinetics of the Na+-K+-ATPase have shown both isoform- and tissue-specific differences in apparent affinities for Na+ and K+ at activating cytoplasmic and extracellular sites (17, 24, 28). Our earlier analysis provided rudimentary evidence of tissue-specific interactions of Na+ and K+ at cytoplasmic sites that are particularly relevant to the behavior of the pump in vivo under the normal or resting steady-state condition of high K+ and low Na+ concentrations in the cytosolic milieu (28). Thus the kidney alpha 1beta 1 enzyme is notably more sensitive to K+ inhibition at cytoplasmic Na+ activation sites than pumps of the same enzyme (rat alpha 1beta 1) of either rat alpha 1-transfected HeLa cells or axolemma, the latter two assayed in the presence of low ouabain to inhibit activity due to ouabain-sensitive forms. The comparative behavior of pumps of these and of other tissues is shown in Fig. 1A. The plots depict the activities of rat pumps as a function of Na+ concentration under conditions of relatively high (50 mM) K+ concentration. The behavior suggests that pumps of heart, kidney, and intestine have lower apparent affinities for Na+ compared with pumps of alpha 1-transfected HeLa cells, axolemma, and red blood cells. Figure 1B shows a similar pattern, albeit with somewhat smaller differences, for pumps of another species (mouse), namely heart, kidney, and axolemma.


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Fig. 1.   Na+ activation of alpha 1 Na+-K+-ATPase from various tissues. Membranes were prepared and assayed at various NaCl concentrations and 50 mM KCl, as described in MATERIALS AND METHODS. Data were fitted to Eq. 1 (n = 3 experiments). Representative experiments are shown, and the data points are the means of triplicate determinations ± SD, expressed as %maximal velocity (Vmax). A: Na+ activation of rat alpha 1 pumps of kidney (open circle ), red blood cells (), small intestine (star ), heart (), alpha 1-transfected HeLa cells (), and axolemma (black-diamond ). B: Na+ activation of mouse alpha 1 pumps of kidney (open circle ), axolemma (black-diamond ), and heart (). Brackets denote concentration.

To gain insight into the mechanistic basis for these tissue-specific differences, we carried out a series of analyses of the kinetic behavior of alpha 1 pumps of each tissue, in which Na+ activation profiles were determined as a function of K+ concentration. As in our previous study and based on the Albers-Post model with the assumption that Na+ and K+ bind randomly at three equivalent cytoplasmic sites, the data were analyzed by the relationship described by Garay and Garrahan (12), i.e.
<IT>m</IT> = <FR><NU><IT>M</IT></NU><DE><FENCE>1 + <FR><NU><IT>K</IT><SUB>Na</SUB></NU><DE>[Na]<SUB>i</SUB></DE></FR> <FENCE> 1 + <FR><NU>[K]<SUB>i</SUB></NU><DE><IT>K</IT><SUB>K</SUB></DE></FR></FENCE></FENCE></DE></FR> (2)
where [Na]i and [K]i are the cytoplasmic concentrations of Na+ and K+, respectively; m and M represent velocity and maximal velocity, respectively; KNa is Na+ binding at cytoplasmic activation sites in the absence of K+; and KK is K+ acting as a competitive inhibitor of Na+ binding at cytosplasmic sites. This model predicts a linear relationship between the apparent affinity constant for cytoplasmic Na+, K'Na, and [K]i according to the following relationship
<IT>K</IT>′<SUB>Na</SUB> = <IT>K</IT><SUB>Na</SUB>(1 + [K]<SUB>i</SUB>/<IT>K</IT><SUB>K</SUB>) (3)
The good fits of the data to Eq. 3 (Fig. 2) indicate that this noninteractive model provides a valid basis for analyzing and quantifying K+/Na+ antagonism and deriving values for the apparent affinity constants 1) KNa and 2) KK. As shown by the data summarized in Table 1, the order of susceptibility to competitive inhibition by K+, expressed as the ratio KNa/KK, is heart > kidney > small intestine > axolemma alpha 1 approx  alpha 1-transfected HeLa cells > red blood cells (KNa/KK = 0.239 ± 0.025, 0.102 ± 0.003, 0.070 ± 0.005, 0.042 ± 0.002, 0.046 ± 0.001, and 0.027 ± 0.005, respectively). For heart and kidney, the high K+ inhibition can be attributed to higher affinities for K+ (lower values of KK) at the cytoplasmic Na+ binding sites (KK = 3.96 ± 3.05 and 10.0 ± 0.9 mM, respectively) compared with axolemma, alpha 1-transfected HeLa cells, and small intestine (KK = 18.7 ± 1.8, 19.9 ± 0.7, and 20.8 ± 3.6, respectively). With pumps of small intestine, however, the relatively high KNa/KK value is associated with a lower affinity for Na+ (KNa= 1.46 ± 0.16 mM) than those in kidney, axolemma alpha 1, and alpha 1-transfected HeLa cell pumps (KNa = 1.02 ± 0.09, 0.78 ± 0.05, and 0.91 ± 0.02 mM, respectively), whereas the relatively low K+/Na+ antagonism characteristic of red blood cells may be due primarily to a high intrinsic affinity for Na+ (KNa = 0.51 ± 0.16).


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Fig. 2.   Dependence of apparent affinity for Na+ (K'Na) on K+ concentration for rat alpha 1 Na+-K+-ATPase from various tissues. Na+ activation of Na+-K+-ATPase was determined as described in Fig. 1. K'Na obtained from plots of activity vs. Na+ concentration (Eq. 1 with n = 3) were plotted as a function of KCl concentration. Data points shown are means ± SD of at least 3 separate experiments, and the values obtained for Na+ binding at cytoplasmic activation sites in absence of K+ (KNa), K+ acting as a competitive inhibitor of Na+ binding at cytoplasmic sites (KK), and KNa/KK are shown in Table 1. Dashed lines are the curves obtained for kidney, axolemma, and alpha 1-transfected HeLa cells, taken from Fig. 4 of Therien et al. (28). Symbols are as described in the legend to Fig. 1.

Analysis of K+ interactions: K+ occlusion and the rate of the E2(K) right-arrow right-arrow E1 + K+ reaction. To gain more insight into the interactions of K+ with cytoplasmic binding site(s), a detailed analysis of the binding and occlusion of K+ was carried out according to the following simplified equilibrium relationship, which does not distinguish the individual steps of sequential binding/occlusion of the two K+
(4)
The experimental approach involved indirect assays of K+ occlusion and deocclusion as described previously (6). Na+-K+-ATPase-containing membranes were equilibrated with various amounts of K+, under which condition K+ binds to E1 (presumably at the cytoplasmic binding site) and becomes occluded such that an equilibrium between E1 and E2(K) is established, as shown in Eq. 4 (11, 14). Taking advantage of the high rate of Na+-dependent phosphorylation of the K+-free enzyme, E1, and low rate of K+ deocclusion from E2(K) at 0°C, the enzyme is then phosphorylated at this temperature in the presence of [gamma -32P]ATP. The K+-dependent reduction in the amount of E1 susceptible to phosphorylation provides an estimate of K+-occluded enzyme, as described previously (6). Accordingly, Fig. 3A shows both the percentage phosphoenzyme (right axis) as measured directly, percentage K+-occluded enzyme (left axis) as estimated from the difference between maximal phosphoenzyme measured in the absence of K+, and phosphoenzyme formed after equilibration with K+ (see relationship given in Fig. 3A, top). For this series of experiments, we compared alpha 1 pumps of heart, kidney, small intestine, and alpha 1-transfected HeLa cells. The results indicate notable tissue-specific differences in the apparent affinity constant of the enzyme for K+ occlusion (Kocc). Thus the order of apparent affinities for K+ occlusion is heart approx  kidney > HeLa > small intestine (Kocc = 0.014 ± 0.001, 0.017 ± 0.003, 0.058 ± 0.014, and 0.158 ± 0.041 mM, respectively). A further series of experiments was carried out to determine whether and to what extent these differences are secondary to distinct rates of deocclusion. Accordingly, the rate of E1 formation from E2(K) was estimated at 10°C, as described previously (6). The results shown (Fig. 3B) indicate dramatic tissue-specific differences in the rates of deocclusion, represented in Eq. 4 by the deocclusion rate constant, kd. The occlusion rate constant, ko, was then obtained according to the relationship Kocc = kd/ko (see Eq. 4). Values of Kocc, kd, and ko for each tissue are given in Table 2. It is interesting, albeit possibly fortuitous, that a comparison of ko, obtained from experiments done at equilibrium, with the KK values obtained from the steady-state Na+-K+-ATPase reaction using the Garay-Garrahan model for K+/Na+ antagonism (see Table 1) shows a notable inverse correlation between the two. Thus the order of ko values is heart > kidney > HeLa approx  small intestine, and the order of KK calculated from Fig. 1 is heart <=  kidney < HeLa approx  small intestine.


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Fig. 3.   Occlusion/deocclusion of K+ for Na+-K+-ATPase of various tissues. Representative experiments are shown. A: formation of phosphoenzyme (EP) at 0°C after preincubation in the presence of various concentrations of KCl was carried out as described in MATERIALS AND METHODS. Values shown are the means ± SD of triplicate determinations of occluded enzyme [E2(K)], which is defined as the difference between 1) maximal EP formed in the absence of KCl [EPmax([K]=0)] and 2) EP formed in the presence of various KCl concentrations (EP[K]>0). Values are expressed as %maximal occluded enzyme on left, as determined by fitting the curves to Eq. 1 (n = 2). %EP is indicated on right. Values for Kocc, the apparent affinity constant for K+ occlusion, are shown in Table 2. B: formation of EP at 10°C was measured at the indicated times as described in MATERIALS AND METHODS. Points are the means ± SD of triplicate determinations of occluded enzyme, as defined in A, and are expressed as %maximal occluded enzyme, defined as the difference between EP formed in the absence of KCl and EP formed in the presence of 4 mM KCl at 0°C. Measured values of kd, the rate constant for K+ deocclusion, are shown in Table 2. star , Small intestine; , heart; , alpha 1-transfected HeLa cells; open circle , kidney.

Can tissue-specific differences in K+/Na+ antagonism be attributed to distinct membrane environments? To test whether susceptibility to high K+/Na+ antagonism is reversible and related to the membrane environment of the Na+-K+-ATPase, pumps were transferred from the kidney to the red blood cell membrane by the fusion procedure described earlier (21, 22). In those studies, we showed that kidney microsomes can be functionally inserted in mature red blood cells; those of the dog have the advantage of not containing significant levels of endogenous Na+-K+-ATPase. In this study, we compared the Na+ activation profiles of fused and nonfused rat kidney pumps. We first carried out fusions in the presence or absence of kidney microsomes. Membranes were prepared, and the following three fusion conditions were used for subsequent functional assays: 1) kidney microsome-fused red blood cells, 2) kidney microsome/mock-fused red blood cells (kidney microsomes added after a mock fusion), and 3) mock-fused red blood cells. Membranes from mock-fused red blood cells (condition 3) contained no ouabain-sensitive ATPase activity (data not shown). Figure 4 shows that kidney microsome/mock-fused red blood cells have a typically high K'Na in the presence of 100 mM KCl [K'Na = 12.2 ± 2.4 mM, similar to values obtained with kidney pumps alone; K'Na = 13.1 ± 1.7 (average of 12 experiments), data not shown]. However, membranes of kidney microsome-fused red blood cells showed a 1.9-fold decrease in K'Na (6.3 ± 1.1 mM) toward that of alpha 1 pumps of rat red blood cells, axolemma, and alpha 1-transfected HeLa cells.


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Fig. 4.   Na+ activation of membranes of kidney pumps fused or mock fused into dog red blood cells. Pumps of kidney microsome-fused red blood cells and kidney microsome/mock-fused red blood cells (see text for definitions) were assayed as in Fig. 1, but in the presence of 100 mM KCl. Points are the means of triplicate determinations ± SD and are expressed as %Vmax, and curves were fitted to Eq. 1 (n = 3). Representative experiments are shown. , Kidney microsome-fused red blood cells; , kidney microsome/mock-fused red blood cells.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Several studies (17, 21) have clarified apparent anomalies noted in earlier analyses of the Na+-K+-ATPase kinetics of tissues comprising different pump isoforms. In essence, it is now clear that the distinct membrane environments of diverse tissues are an important determinant of kinetic behavior. Notable cases in point are the comparisons of alpha 1 pumps of kidney with alpha 3 pumps of either the pineal gland (24) or axolemma (26). Whereas alpha 3 pumps appeared to have a higher apparent affinity for Na+ at cytoplasmic activation sites compared with alpha 1 pumps of kidney, the order of apparent affinities was reversed for alpha 3 and alpha 1 pumps transfected into HeLa cells (17) or delivered from either rat axolemma or kidney into the dog red blood cell (21).

A more detailed evaluation of the tissue- versus isoform-specific behavior of pumps further underscored the importance of the membrane environment as a determinant of pump behavior (28). In that study, it became apparent also that there are notable tissue-specific differences in the extent to which K+ behaves as a competitive inhibitor at cytoplasmic Na+ activation sites of pumps comprising predominantly alpha 1 or alpha 3 catalytic isoforms. In fact, the relative concentrations of Na+ and K+ under which K+/Na+ antagonism is marked are those that prevail under steady-state physiological concentrations, and, as discussed below, this behavior may have important ramifications in relation to the regulation of intracellular Na+.

In this study, the interaction of the enzyme with cytoplasmic K+ was analyzed in terms of the well-documented formation of K+-occluded enzyme by the direct binding of K+ to the E1 conformation of the enzyme (reviewed in Refs. 11 and 14). Based on the premise that the avidity of K+ for cytoplasmic cation binding sites should be evidenced in the rate of formation of the K+-occluded enzyme [E1 + K+ right-arrow E1 · K+ right-arrow E2(K); see Eq. 4], the present analysis of the K+ occlusion pathway of several tissues that vary in K+/Na+ antagonism (heart, kidney, small intestine, rat alpha 1-transfected HeLa) shows an intriguing inverse relationship between ko, calculated from determinations of Kocc and kd as defined by the simple equilibrium relationship in Eq. 4, and KK measured under steady-state conditions. Thus our data show a high rate of K+ occlusion in the tissue (heart) having a low KK (high affinity for K+ as a competitive inhibitor of Na+ binding), an intermediate rate in the tissue (kidney) with an intermediate KK, and lower rates in tissues (HeLa, small intestine) with the highest KK values. This analysis was not extended to either axolemma or red blood cells, since 1) in axolemma, conditions for measuring binding/occlusion and deocclusion of K+ could be confounded by the high proportion of the other (mainly alpha 3) isoforms and 2) in the red blood cell, the relatively high background activity and low specific activity of the enzyme precludes accurate measurements of these parameters.

The correlation between the affinity constants (KK) for K+ as a competitive inhibitor of Na+ binding (as determined by plotting K'Na of various tissues versus [K+]; Table 1) and the apparent rate constants for binding and occlusion of K+ (ko; Table 2) in pumps of kidney, heart, alpha 1-transfected HeLa cells, and small intestine membranes supports a model whereby K+/Na+ antagonism is related to the competing reactions represented by 1) binding/occlusion of Na+ to form E1P(Na) in the forward direction and 2) binding/occlusion of K+ to form E2(K) in the backward reaction. This result also supports a model of ion exchange whereby the K+ release site and the Na+ binding site are the same, consistent with several studies showing that mutational or biochemical alterations of residues deemed important for cation binding and occlusion can affect both Na+ and K+ interactions (23, 30).

Although Western blots of the various tissues showed a correlation between alpha 1 antibody reactivity and amount of alpha 1 activity analyzed in each lane (data not shown), there remains the possibility that our analysis is confounded by the presence of other ouabain-sensitive ATPases, such as the putative "nongastric" H+-K+-ATPases, namely the "colonic" H+-K+-ATPase found in the colon, kidney, and uterus of mammals (5), the H+-K+-ATPase found in amphibian bladder (16), and the gene encoding a human K+-dependent ATPase (ATP1AL1) first cloned from human skin (15). We consider this possibility unlikely, since, in the rat, mRNA for the colonic ATPase was not detected in brain or small intestine, and only trace amounts were detected in kidney and heart by Northern blot analysis, whereas the ATP1AL1 message is absent in all of these tissues (5).

To better understand the structural basis for the observed differences in K+/Na+ antagonism, we compared the Na+ activation profiles (at 100 mM K+) of kidney pumps fused into dog red blood cells with that of unfused kidney pumps in the presence of mock-fused dog red blood cell membranes. Kidney membranes and dog red blood cells were used since kidney microsomal membrane preparations are predominantly right-side-out and thus are conducive to efficient fusion, and dog red blood cells are devoid of endogenous Na+ pumps (see Ref. 22). Our results show a notable decrease in K'Na of kidney pumps fused into red blood cells, consistent with the conclusion that either components of the red blood cell membrane are interacting with the exogenous pumps or components of the kidney membrane are dissociating from these pumps, resulting in a decrease in their susceptibility to inhibition by K+. This effect requires intimate interactions between the pumps and the membrane environment since the mere presence of mock-fused red blood cell membranes in the assay medium had no effect on activity. Although our results suggest an effect of the membrane environment, we cannot rule out another possibility: since fusion requires a 1-h incubation of the fused cells at 37°C to ensure integration of the exogenous pumps, it is possible that the pumps are somehow altered during this period by a cytosolic factor present in the red blood cell, for example, through phosphorylation/dephosphorylation. In either case, these results show clearly that the high affinity for K+ acting as a competitive inhibitor of Na+ binding, which is characteristic of kidney pumps, is reversible and can be regulated by some cellular component(s). This type of reversible modulation of exogenous pumps by components of the red blood cell membrane is reminiscent of the observed regulation of pumps by the so-called Lp-antigen of low-K+ sheep red blood cells. Using the same fusion strategy, we showed that interaction of Lp antigen with exogenous kidney pumps conferred the distinctive K+ inhibition of pumps of genetically low-K+ sheep red blood cells (31).

It could be suggested that the membrane component that modulates susceptibility of the renal enzyme to K+/Na+ antagonism is the so-called gamma -subunit, since this peptide has been detected in this but not in other tissues (27). We consider this possibility unlikely for the following reasons: 1) gamma -subunit protein was not detected in heart (27) even though pumps of this tissue are even more susceptible to K+/Na+ antagonism than kidney pumps, and 2) the fusion experiments would indicate that the gamma -subunit dissociates from kidney pumps upon fusion into red blood cells, yet fused and unfused kidney pumps were inhibited by anti-gamma antiserum (cf., Ref. 27) to a similar extent (experiments not shown).

Association of alpha 1 pumps with distinct beta  isoforms is also unlikely to be the basis for tissue-specific differences in K+/Na+ antagonism. We have already shown in a previous report (28) that the alpha 1 isoform associates only with the beta 1 isoform in kidney, HeLa, and axolemma, and we have since determined that the beta 1, but not the beta 2, isoform is detected in Western blots of heart and small intestine (data not shown). In addition, we consider it unlikely that the beta -subunit could dissociate from alpha  upon fusion of pumps into the red blood cell. We cannot rule out, however, that distinct beta  isoforms are the basis for the high Na+ affinity (low KNa; see Table 1) of pumps of red blood cells, since message for the beta 2 and beta 3, but not the beta 1, isoforms was detected recently in human reticulocytes (25).

In previous studies of Na+-dependent Rb+ transport (21), we showed that kidney pumps have a higher affinity for cytoplasmic Na+ than axolemma pumps when fused into dog red blood cells. However, in a more recent paper (28), we showed that native kidney pumps have a lower affinity for Na+ than axolemma pumps in Na+-activated ATPase assays. We postulated that this discrepancy may be related to a lower K+/Na+ antagonism after fusion of the pumps. Here we show that this hypothesis is at least partly correct. Thus the high K+ inhibition characteristic of kidney pumps is reversible and related to the membrane environment of the pump.

Because the concentration of K+ in cells is generally high compared with that of Na+ (roughly 10-fold higher), modulation of K+/Na+ antagonism could be a physiologically important mechanism of pump regulation, especially in tissues in which the role of the pump is specialized, such as the heart, the kidney, and the small intestine. Although the sodium pump has a fundamental role as a "housekeeping" transporter responsible for maintaining Na+ and K+ homeostasis in the kidney and small intestine, it is also responsible for the absorption or reabsorption of Na+ and other solutes that are transported across the apical membrane by Na+-dependent transporters (for reviews, see Refs. 7 and 9). In the heart, the sodium pump is an indirect regulator of cardiac muscle contraction, as originally formulated by Baker et al. (1). Thus variations in K+/Na+ antagonism of heart enzyme could alter intracellular Ca2+ via Na+/Ca2+ exchange secondary to alterations in cytoplasmic Na+ concentration. It is likely that such a regulatory mechanism extends to species other than the rat, since similar results were obtained with the murine enzyme. The reversibility of K+/Na+ antagonism as evidenced in the experiments with kidney pumps fused into red blood cells underscores the potential importance of intracellular K+ as a regulator of pump activity.


    ACKNOWLEDGEMENTS

We thank Drs. E. A. Jewell-Motz and J. B. Lingrel, University of Cincinnati, for the generous gift of the rat alpha 1-transfected HeLa cells and Dr. Lawrence Joseph, McGill University, for help in the statistical analysis of the data. The assistance of Ania Wilczynska and Richard Daneman is gratefully acknowledged.


    FOOTNOTES

This work was supported by grants from the Medical Research Council of Canada (MT-3876) and the Quebec Heart and Stroke Foundation and by a predoctoral scholarship (to A. G. Therien) from the Fonds pour la Formation de Chercheurs et l'Aide à la Recherche.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: R. Blostein, Montreal General Hospital Research Institute, 1650 Cedar Ave., Rm. L11.124, Montreal, Quebec, Canada H3G 1A4 (E-mail: MIRB{at}musica.mcgill.ca).

Received 3 May 1999; accepted in final form 14 July 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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