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J. Biol. Chem., Vol. 277, Issue 23, 20270-20276, June 7, 2002

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Distinct Regulatory Effects of the Na,K-ATPase  Subunit^*

Helen X. Pu, Rosemarie Scanzano, and Rhoda Blostein

From the Departments of Medicine and Biochemistry, McGill University, Montreal, Quebec H3G 1Y6, Canada

Received for publication, January 30, 2002, and in revised form, March 28, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The two variants of the  subunit of the rat renal sodium pump, _a and _b, have similar effects on the Na,K-ATPase. Both increase the affinity for ATP due to a shift in the enzyme's E₁  E₂ conformational equilibrium toward E₁. In addition, both increase K⁺ antagonism of cytoplasmic Na⁺ activation. To gain insight into the structural basis for these distinct effects, extramembranous N-terminal and C-terminal mutants of  were expressed in rat 1-transfected HeLa cells. At the N terminus, the variant-distinct region was deleted (N7) or replaced by alanine residues (N7A). At the C terminus, four (_aC4) or ten (_aC10) residues were deleted. None of these mutations abrogates the K⁺/Na⁺ antagonism as evidenced in a similar increase in K'_Na seen at high (100 mM) K⁺ concentration. In contrast, the C-terminal as well as N-terminal deletions (N7, _aC4, and _aC10) abolished the decrease in K'_ATP seen with wild-type _a or _b. It is concluded that different regions of the  chain mediate the distinct functional effects of , and the effects can be long-range. In the transmembrane region, the impact of G41R replacement was analyzed since this mutation is associated with autosomal dominant renal Mg²⁺-wasting in man (Meij, I. C., Koenderink, J. B., van Bokhoven, H., Assink, K. F. H., Groenestege, W. T., de Pont, J. J. H. H. M., Bindels, R. J. M., Monnens, L. A. H., Van den Heuvel, L. P. W. J., and Knoers, N. V. A. M. (2000) Nat. Genet. 26, 265-266). The results show that Gly-41  Arg prevents trafficking of  but not pumps to the cell surface and abrogates functional effects of  on pumps. These findings underscore a potentially important role of  in affecting solute transport, in this instance Mg²⁺ reabsorption, consequent to its primary effect on the sodium pump.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The Na,K-ATPase, or sodium pump, maintains the high Na⁺ and K⁺ gradients across the plasma membrane of animal cells. Accordingly, this pump plays a major role in determining the cytoplasmic Na⁺ concentration and hence the cytoplasmic concentration of protons and Ca²⁺, as well as other solutes whose accumulation is driven by secondary countertransport systems. The kinetic properties of the sodium pump are, in turn, subject to complex mechanisms of short- and long-term regulation. While the nature of the catalytic  subunit isoform may be a primary determinant of tissue-specific behavior of the pump, there are also diverse mechanisms underlying pump regulation. (For review, see Refs. 1 and 2).

There is an increasing body of evidence that a family of small, single transmembrane proteins characterized by the motif FXYD are expressed in a tissue-specific manner. To date, at least two members have been identified in kidney, FXYD2 or  (4, 5) and FXYD4 or the corticosteroid hormone-induced factor, CHIF¹ (6-8). Both modulate the kinetic behavior of the Na,K-ATPase (see Refs. 5, 9, 10-13 for  and 13, 14 for CHIF). Another related protein, phospholemman-like protein of shark (PLMS) (15), related to FXYD1 (phospholemman) in mammalian heart (16), also modulates function in a phosphokinase C-dependent manner. To date, at least seven members of this family have been identified (3).

The  subunit of the Na,K-ATPase was discovered over 20 years ago (17, 18) and was shown recently to exist as two major variants in the kidney, _a and _b (19), consistent with predictions based on the Expressed Sequence Tag (EST) data base (20). These are splice variants and differ only in their N-terminal residues. In the rat, the seven N-terminal amino acids TELSANH of _a are replaced by Ac-MDRWYL in _b (19). Expression studies in fetal tissues suggest that a third form may be present (21).

We have previously cloned and expressed the _a and _b variants in mammalian cells and characterized their two main regulatory roles (Refs. 10 and 12 and reviewed in Ref. 22). One function of  is to increase cytoplasmic K⁺ antagonism of Na⁺ activation, which is apparent as an increase in K'_Na, particularly at elevated K⁺ concentrations. The other function is a -mediated increase in the apparent affinity for ATP, concordant with our earlier finding that antibodies raised against the C terminus of  decreased the affinity for ATP (5). We ascribed the latter decrease in K'_ATP to a -mediated shift in the poise of the steady-state E1  E2 equilibrium toward E₁. Consistent with this finding is the behavior of both  subunits expressed in Xenopus oocytes (13). Thus, in the presence but not absence of Na⁺, both subunits alter the apparent affinity for extracellular K⁺ in a membrane potential-dependent manner, indicative of a -mediated shift in conformational equilibrium toward E₁. Although no notable difference between _a and _b function could be detected (12), the significance of the presence of two major variants of  may be related to their partially overlapping but distinct patterns of expression (12, 23), which, in turn, may be relevant to specific functions along the nephron.

One goal of this study was to gain insight into the structural basis for the two distinct kinetics effects of . To this end, we examined the consequences of altering both N- and C-terminal extramembranous regions of  by deletion and alanine replacement of the variant-specific N terminus and deletion of up to ten residues from the C terminus. The other aim was to analyze the functional basis for the transmembrane Gly-41  Arg mutation associated with familial magnesium-wasting in man (24). This analysis underscores an important role of  in affecting secondary transport as a result of primary effects on Na,K-ATPase function.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Mutagenesis-- The  mutants analyzed in the present study were generated as follows. Using the _a cDNA as template, a series of PCR reactions was carried out with appropriate primers to generate the cDNAs coding for the deletion of the C-terminal four and ten residues of the _a variants, _aC4 and _aC10, respectively, as well as the cDNAs coding for deletion or alteration of the N terminus that is distinct in the two variants, i.e. N7 (first seven residues deleted) and N7A (first seven residues replaced by alanines). These mutants are schematized in Fig. 1A. The point mutation in the transmembrane region (Gly-41 replaced by Arg) was introduced into the _b cDNA using the QuikChange^TM site-directed mutagenesis kit (Stratagene).

Expression in Rat 1-HeLa Cells-- The above mutated cDNAs were subcloned into the pIRES expression vector as described previously (19, 12). All mutations were confirmed by DNA sequencing. The pIRES/cDNAs were then transfected into HeLa cells stably expressing the rat 1 subunit of Na,K-ATPase (1-HeLa cells, kindly provided by Dr. J. B. Lingrel) using the LipofectAMINE reagent (Invitrogen) as described (12, 19). Single clones expressing mutated  were selected in 400 µg/ml hygromycin B. Western blots were carried out to analyze the expression of each mutant.

Polyacrylamide Gel Electrophoresis and Western Blotting-- Unless otherwise indicated, SDS-PAGE was carried out using 10% NuPage gels (Novex) with SDS/MES running buffer. PFO-PAGE was also carried out with 10% NuPage gels in which the detergent perfluorooctanoate (PFO) replaced SDS. For both systems, the running and sample buffers were made according to the recipes supplied by the manufacturer (Novex). Antibodies used were anti-C (antibody C33, described in Ref. 10), anti-_a recognizing the N terminus of _a (19), anti-1 subunit obtained from Sigma (A277), anti-calnexin to recognize the endoplasmic reticulum (StressGen), and anti-giantin to detect the Golgi (a gift from Dr. Edward Chan).

Subcellular Fractionation-- Transfected HeLa cells were grown to near confluence on 15-cm dishes and fractionated at 4 °C essentially as described by Simpson et al. (25). Briefly, the cells were scraped off the plate, washed twice with ice-cold 20 mM Tris-HCl, 1 mM EDTA, and 255 mM sucrose, pH 7.4 (TES), and then homogenized (30 strokes using a motor-driven teflon pestle/glass homogenizer). Nuclei and unbroken cells were removed by centrifugation at 1,000 × g. The supernatant was then centrifuged for 20 min at 19,000 × g after which the pellet was suspended, layered on a sucrose cushion (1.12 M sucrose, 20 mM Tris-HCl, 1 mM EDTA, pH 7.4), and centrifuged for 60 min at 100,000 × g (Beckman Ti70 rotor for this and subsequent centrifugations). The membrane-rich fraction at the interface was collected, resuspended in TES, and centrifuged for 30 min at 41,000 × g to obtain a pellet of plasma-rich membranes (PRM). The initial supernatant from the 19,000 × g supernatant was centrifuged at 41,000 × g for 30 min, yielding a pellet of high density microsomal membranes (HDM). The resulting supernatant was then centrifuged at 180,000 × g for 75 min, yielding a pellet of low density membranes (LDM). Each pellet (PRM, HDM, and LDM) was resuspended in 200 µl of TES, and aliquots were taken for the determination of protein concentration (Lowry assay) and Western blot analysis.

Cell Surface Biotinylation-- The method used is a modification of the method of Stephan et al. (26) used for HeLa cells. Transfected HeLa cells were grown to ~80% confluence in 6-well plates and washed twice with ice-cold PBS/CM (phosphate-buffered saline containing 0.1 mM CaCl₂ and 1 mM MgCl₂). All further steps were carried out on ice. Each well of cells was treated with NHS-SS-biotin (Pierce; 1.5 mg/ml in 10 mM HEPES, 2 mM CaCl₂, 150 mM NaCl, pH 8.5) for two successive 20 min incubations with gentle shaking. The reagent was freshly prepared for each incubation. After biotinylation, each well was briefly rinsed with PBS/CM containing 100 mM glycine and then treated with the same solution for 30 min on ice to ensure complete quenching of the unreacted NHS-SS-biotin. The cells were then lysed for 30 min with 500 µl of 1% Triton X-100, 0.1% SDS in L1 buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris-HCl, pH 7.5, containing 10 µg/ml (each) leupeptin and pepstatin and 200 µM phenylmethylsulfonyl fluoride. Each sample was then centrifuged at 18,000 × g for 10 min to remove cell debris. Protein determination on the supernatant (total cell lysate) was performed by the Lowry method. To recover the biotinylated proteins, 100 µg of supernatant protein was incubated with 100 µl of streptavidin-agarose beads (Pierce) overnight at 4 °C with end-over-end rotation. The beads were removed by centrifugation, and the supernatant representing the unbound fraction was saved for Western blot analysis. The beads were washed three times with L2 buffer (L1 buffer omitting the SDS), then twice with high salt L2 (L2 containing 500 mM NaCl and 0.1% Triton X-100), and once with 50 mM Tris-HCl, pH 7.5. The biotinylated proteins were eluted from the beads by incubation in 100 µl of SDS-PAGE sample buffer containing 5% -mercaptoethanol at 37 °C for 30 min.

Kinetic Assays and Data Analysis-- Kinetic assays of Na,K-ATPase were carried out in triplicate as described previously (12) with either mutant or WT -transfected rat 1-HeLa cells assayed concomitantly with mock-transfected rat 1-HeLa cells. As in those previous studies, K'_ATP and V_max were obtained by fitting the data to a simple Michaelis-Menten model; K'_Na and V_max were obtained by fitting the data to the 3-site non-cooperative model described by Garay and Garrahan (27) in their classic studies with red cells. The model assumes that Na⁺ ions bind randomly at three equivalent cytoplasmic sites. Unless indicated otherwise, values of K'_ATP and K'_Na were obtained from at least three separate paired experiments (WT or mutant  analyzed concurrently with mock-transfected control; compare representative experiments shown in Figs. 2 and 4) carried out with each of at least two different clones of the same wild-type or mutant-transfected 1-HeLa cells. These values were used to quantify the effects of WT and mutant  subunits as described in Figs. 3 and 5.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression of Mutants-- Our earlier studies with _a- and _b-transfected HeLa cells stably expressing the rat 1 isoform (1-HeLa cells) showed that the  subunit has at least two kinetic effects on Na,K-ATPase: a decrease in K'_ATP and an increase in K'_Na due to K⁺/Na⁺ antagonism. To assess the structural basis for these distinct kinetic effects, extramembranous mutants as well as the intramembranous G41R mutant were constructed and expressed in rat 1-transfected HeLa cells. Several clones with high expression were used for preparation of the membranes. Fig. 1, panel A depicts the constructs, and panels B-D provide verification of their expression following Western blotting of representative clones of each mutant together with rat kidney enzyme, control mock-transfected 1-HeLa, and _a-1-HeLa membranes. In the blot shown in panel B, the C-terminal deletion mutants _aC4 and _aC10 were detected with anti-_a raised against the N terminus of _a but not anti-C. In panel C, mutants with the _a and _b distinct residues of the N terminus either deleted (mutant N7) or replaced by seven alanine residues (N7A) were detected with anti-C raised against the C terminus. In all lanes of each blot, similar units of activity were analyzed indicating that the levels of  expression of the mutants were at least as high as seen in the WT _a control, which, in turn, is at least as high as that of kidney. It is noted, however, that with the present NuPage system, a species that migrates significantly slower than _a is seen in the lanes showing _a and _a mutants. In our previous studies (19), this species was barely detectable. Its size could reflect the presence of undissociated _a dimers using the NOVEX system. To date, we have not pursued this issue further.

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Mutants of the rat  subunit and their expression in rat 1-HeLa cells. A, mutations introduced into the extramembranous N and C termini and transmembrane region. B, comparison of expression of extramembranous mutants with -transfected 1-HeLa cells, control (mock-transfected) 1-HeLa cells, and kidney probed with anti-a-specific (N-terminal) antibodies. C, comparison of expression of extramembranous mutants with a-transfected 1-HeLa cells, control (mock-transfected) 1-HeLa cells, and kidney probed with anti- (C-terminal) antibodies that recognize both  variants. D, comparison of expression of b and bG41R using SDS-PAGE and PFO-PAGE and probed with anti- (C-terminal) antibodies. For each blot shown, similar amounts (activity) of enzyme were analyzed.

The blot in Fig. 1, panel D shows that the G41R substitution in the transmembrane domain of _b alters the mobility seen in immunoblots. The difference in mobility of the _bG41R mutant compared with WT _b is barely detected using SDS-PAGE but is clearly seen when electrophoresis is carried out in PFO-PAGE (see "Experimental Procedures"). The reduced mobility probably reflects a structural change due to the charge alteration introduced by the Gly  Arg substitution.

Effect of Mutations on K⁺/Na⁺ Antagonism-- _a and _b both increase K⁺ antagonism of cytoplasmic Na⁺ activation (12). As shown in that study, this effect is evidenced in kinetic assays that show: (i) greater K⁺ inhibition of Na,K-ATPase of either _a- or _b-1-HeLa compared with mock (vector alone)-transfected rat 1-HeLa cells, when K⁺ is varied at a constant, relatively low (5 mM) Na⁺ concentration and (ii) an increase in K'_Na as K⁺ concentration is increased to a greater extent with either _a or _b than with mock-transfected 1-HeLa cells. Thus, when K'_Na is determined as a function of K⁺ concentration and analyzed using the Garay-Garrahan non-cooperative 3-site model for Na⁺ cytoplasmic activation based on the Albers-Post reaction mechanism, the data adhere closely to the predicted linear relationship K'_Na = K_Na(1 + [K⁺]_in/K_K) in which the slope, K_Na, is the affinity for Na⁺ in the absence of K⁺ and K_K is the affinity for K⁺ as a competitor of cytoplasmic Na⁺.² (Compare the similar K⁺/Na⁺ competition reported for red cells by Sachs, Ref. 28). The analysis (12) showed that both  variants caused a similar ~2-fold decrease in K_K but had no detectable effect on K_Na. This effect translates into a ~60% increase in K'_Na determined at 100 mM K⁺.

In the present study, several clones of each mutant were analyzed, each paired with control mock-transfected 1-HeLa membranes. Thus, Na⁺ activation profiles determined at 100 mM K⁺ of mutants as well as the wild-type _a- or _b-1-HeLa cells are shown in Fig. 2. Each paired experiment shown is representative of one of several experiments carried out with membranes from several clones of the same mutant. Fig. 3 summarizes the results of all paired experiments (control together with mutant or WT ). In the presentation of the kinetic effects of each  mutant relative to WT _a and _b, we have normalized all the data as follows. Values of the ratios of K'_Na (wild-type _a- or _b- or mutant -transfected 1-HeLa cells) to K'_Na (control 1-HeLa cells) for all experiments from all clones of the same mutant were averaged, and for each mutant or WT  the mean ± S.E. are shown. These data show clearly that only the mutation in the transmembrane domain (G41R) abrogated the wild-type _a- or _b-mediated decrease in apparent Na⁺ affinity.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Effect of mutations on K'Na determined at 100 mM K+. Representative paired experiments (control mock-transfected with either wild-type b or mutant ) are shown. Dashed line, control; solid line, WT or mutant .

View larger version (10K): [in this window] [in a new window]   Fig. 3.   Summary of comparative effects of mutations on K'Na determined at 100 mM K+. The kinetic effects of each  mutant relative to WT a and b are normalized and presented as follows: the ratios of K'Na (wild-type a- or b- or mutant -transfected 1-HeLa cells) to K'Na (control 1-HeLa cells) for all experiments from all clones of the same mutant were averaged. Each point shown is the mean ± S.E. Differences between mutants and wild-type  subunits are not significant (p > 0.1) except for bG41R (p  0.02).

In other experiments (not shown), we observed that compared with control mock-transfected cells the aforementioned extramembranous mutants, as well as _a and _b, but not the intramembranous _bG41R mutant, increased K⁺ inhibition of activity measured at low Na⁺ concentration (see Fig. 4 in Ref. 12).

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Effect of mutations on K'ATP. Representative paired experiments (control mock-transfected with either wild-type b or mutant ) are shown. Dashed lines, control; solid lines, WT or mutant .

Effect of Mutations on Apparent Affinity for ATP-- The apparent affinity for ATP of each mutant was compared with control 1-HeLa cells in a series of experiments carried out with several clones of each mutant analyzed as described above for determination of K'_Na. Single representative experiments for each mutant are shown in Fig. 4. Fig. 5 summarizes the results of all of the experiments. As for the analysis of effects on K'_Na, for each of the WT or mutant  subunits the ratio of K'_ATP (wild-type _a- or _b- or mutant -transfected 1-HeLa cells) to K'_ATP (control 1-HeLa cells) was obtained, and the mean ± S.E. is presented. As shown previously (12), both  variants reduce K'_ATP. However, Gly-41  Arg replacement or deletion of ten or even four of the penultimate C-terminal residues abrogates the -mediated increase in ATP affinity. An unexpected finding is the abrogation of the effect on K'_ATP by removal of the variant-distinct N terminus (N7), particularly since the two  variants have similar effects despite the notable structural divergence of their N termini.

View larger version (9K): [in this window] [in a new window]   Fig. 5.   Summary of comparative effects of mutations on K'ATP. The kinetic effects of each  mutant relative to WT a and b are normalized and presented as follows. The ratios of K'ATP (wild-type a or b- or mutant -transfected 1-HeLa cells) to K'ATP (control 1-HeLa cells) for all experiments from all clones of the same mutant were averaged. Each point shown is the mean ± S.E. For mutants aC4, aC10, N7, and bG41R, the ratios are significantly different from that of the wild-type  subunits (p  0.01).

Evidence that the Gly-41  Arg Substitution in the Transmembrane Domain Alters Trafficking of  to the Cell Surface-- From previous immunolocalization studies, we (12) and others (23) have shown that  is highly expressed in kidney tubules. In regions where it is present, it is not seen alone but always together with . In other regions,  is present without .

There are three points of evidence that support the theory that the Gly-41  Arg mutation alters trafficking of  to the cell surface. The first is indirect and is inferred from the finding that post-translational modification of  is altered by this mutation. Thus, when a number of different clones of _b-1-HeLa and _bG41R-1-HeLa were analyzed by Western blotting with anti-C (Fig. 6), the  chain appeared as a doublet in _b-1-HeLa clones, i.e. a lower band shown previously to correspond to _b of kidney and an upper additional species referred to as _b'. In contrast, few, if any, of the _bG41R clones showed the additional upper _b' band, which is, presumably, a post-translationally modified form of _b.

View larger version (54K): [in this window] [in a new window]   Fig. 6.   Immunoblots of different clones expressing b and bG41R. Triton X-100-solubilized cells were analyzed by Western blotting using anti-C antibodies.

The second point of evidence for altered trafficking is the difference in distribution of _bG41R compared with _b in Golgi-rich membranes. This was apparent when the transfected 1-HeLa cell membranes were fractionated into putatively PRM, HDM, and LDM and then analyzed by Western blotting using anti- and anti-C antibodies, as well as anti-calnexin and anti-giantin, as markers of endoplasmic reticulum (Fig. 7, ER) and Golgi, respectively. A representative experiment using this fractionation procedure is shown in Fig. 7. Quantitative densitometry indicated that the relative proportion of  in the PRM fraction is reduced in _bG41R cells compared with that in WT _b cells, i.e. 25 and 45%, respectively, in the representative experiment shown. Although this rudimentary fractionation precluded good separation of PRM and ER as shown by the abundance of calnexin in both fractions, it is particularly noteworthy that the  subunit is present in much larger amounts in the Golgi-rich LDM of _bG41R compared with the LDM of WT _b. From quantitative densitometry, the percentage of total  present in the Golgi-rich LDM fraction was 22.5 ± 3.5% for _bG41R and 6.0 ± 0.7% for WT _b (average of two separate experiments).

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Effect of the Gly-41  Arg mutation on the distribution of  and  in membrane fractions. Cells were fractionated and analyzed for expression of  and  as described under "Experimental Procedures" except that 6% SDS-PAGE was used for gels blotted with anti-giantin. For all three fractions, volumes equivalent to the original cell homogenate were analyzed.

The third and most definitive point of evidence is the almost complete absence of mutant _bG41R protein in surface proteins isolated following biotinylation of surface-exposed lysine residues of intact cells. Using the impermeant biotinylation reagent NHS-SS-biotin, this procedure allowed the assessment of the relative amounts of WT _b and _bG41R at the surface of the transfected cells. Following removal of excess reagent and solubilization of the cells, the biotinylated proteins were captured on streptavidin beads and then analyzed by Western blotting with antibodies to  and . Fig. 8 depicts immunoblots of total detergent-solubilized cells (T), biotinylated surface proteins bound to streptavidin beads (B), and material that was unbound (U) to the beads. The total detergent-solubilized and unbound fractions were analyzed in equal amounts with respect to the original cells. For the bound fraction, the amount analyzed was 10 times that of detergent-solubilized and unbound in order to obtain comparable band densities. As shown in Fig. 8, the  subunit appears in surface membranes of both control _b-1-HeLa and _bG41R-1-HeLa cells. In contrast, _b appears primarily at the surface, whereas _bG41R remains inside the cells (unbound fraction) with little, if any, detectable at the surface.

View larger version (69K): [in this window] [in a new window]   Fig. 8.   Effect of the Gly-41  Arg mutation on cell surface expression of  and  determined by immunoblotting of streptavidin-isolated biotinylated surface membrane proteins. For total (T) and unbound (U) fractions, equivalent volumes with respect to the original cells were analyzed. For the fraction bound to the Streptavidin beads (B), the amount analyzed was 10 times the volume of T or U.

The much lower proportion of  compared with  at the cell surface, as seen by the greater intensity of  in the unbound fraction (Fig. 8, U), is probably not because of fewer  subunits at the surface. More likely, the limited accessibility of  lysines to NHS-SS-biotin results in inefficient biotinylation and consequent underestimation of  subunits at the surface. The results do, however, permit comparison of the proportion of biotin-accessible  subunits in _bG41R- versus _b-transfected cells. With this proviso, it is clear that the Gly-41  Arg mutation prevents the  subunit from reaching the cell surface without a major effect on pump trafficking.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The  subunit of the sodium pump is a member of the FXYD family of small single-span transmembrane proteins. There are at least seven members of this gene family in mammals (3). In the kidney, two members, CHIF and , are regulators of the pump with opposite effects on apparent affinity for Na⁺ (13, 30). Immunolocalization studies indicate that their expression along the nephron is mutually exclusive. For example, CHIF is present exclusively in cortical and medullary collecting ducts and  primarily in the medullary thick ascending limb (14). The two major  variants have similar, if not identical, functional effects on the sodium pump complex. On the other hand, there are differences in their localization along the nephron. Although both co-localize to certain segments of the nephron and are abundant in the medullary thick ascending limb where rates of Na⁺ reabsorption are particularly high, they also exhibit distinct segment localizations. Thus, Pu et al. (12) found that _b but not _a was visualized in cortical thick ascending limb, whereas _a is present in the region of the macular densa in which _b is absent. More recent studies by Wetzel and Sweadner (23) have shown that _b is present in the distal convoluted tubule and connecting tubule; _a, if present, is less abundant.

The functional effects of  described earlier include the following: (i) an increase in apparent affinity for ATP reflecting a shift in the steady-state E₁/E₂ distribution toward the E₁ conformation and (ii) an increase in K⁺/Na⁺ competition at cytoplasmic Na⁺ activation sites. Considering these effects in terms of the Albers-Post reaction mechanism, in particular E₂(K⁺)  E₁ + K⁺ and E₁ + ATP + Na⁺  Na·E₁P + ADP, it is intriguing that the two effects of  are paradoxically opposing. A higher affinity for K⁺ at cytoplasmic Na⁺ activation sites should shift the E₁/E₂ poise away from E₁ and, conversely, a higher affinity for ATP should shift the poise away from E₂, toward E₁. The implications of these dichotomies are considered below.

From our earlier observation that anti-C treatment of the renal enzyme abrogated the effect of  on K'_ATP but had no effect on K⁺/Na⁺ antagonism, we suggested that the two effects of  are relevant to different regions of the  chain. The present mutagenesis study provides definitive evidence in support of this theory. Thus, deletion of ten and as few as four residues from the C terminus, as well as deletion of the variant-specific N terminus, completely abrogates the -mediated decrease in K'_ATP seen with both WT variants but not the increase in K⁺/Na⁺ antagonism. An intriguing possibility is that there is interplay between the two opposing modifying effects of  whereby the -mediated increase in K'_ATP affinity may counteract and hence minimize the true K⁺/Na⁺ antagonism and vice versa. This may come about if  effects on Na,K-ATPase behavior are, in turn, modulated by cell-specific interactions of // complexes with other cell elements such as those of the cytoskeleton.

The observation that none of the extramembranous mutants abrogated both effects of  indicates that all of these mutant  subunits associate with Na,K-ATPase / dimers. This finding is consistent with the recent report of Beguin et al. (13), which showed that the FXYD motif that is present in these mutants is critical for stable association.

The finding that deletion of the N terminus, like removal of the C terminus (or addition of anti-C-terminal antibodies), abrogates the effect of  on the E₁  E₂ conformational equilibrium points to long-range effects of / interactions on K'_ATP. Because the N-terminal deletion but not N7A replacement abrogates the K'_ATP effect, the  effect to stabilize E₁ does not involve TELSANH or MDRWYL interactions with but rather the remainder of the  chain.

A physiological basis for the dual effects of  is that it provides a fine-tuned, self-regulatory mechanism for balancing energy utilization and maintaining appropriate salt gradients across renal epithelial cells. Both  variants are particularly abundant in the medullary thick ascending limb. As reasoned elsewhere (22), it is in the anoxic regions of the medulla that the increased affinity for ATP effected by  would serve to maintain pump activity, and the moderate decrease in Na⁺ affinity would serve to balance ATP depletion and maintain an appropriately low intracellular Na⁺ concentration. Accordingly, its dual effect enables  to imbue the pump with the ability to handle ATP under energy-compromised conditions and yet be self-regulated by having an appropriately modest increase in the Na⁺ concentration set point. Recent studies by Garty et al. (14) have shown that in certain regions with little if any  in which the apparent affinity for Na⁺ is higher (29), in particular cortical and medullary collecting ducts, the renal pump is associated with CHIF. CHIF has the opposite effect of  on K'_Na (13); it increases the apparent affinity for Na⁺ at least 2-fold, which these authors suggest may be critical for aldosterone-responsive tissues, which have an important role in maintaining Na⁺ and K⁺ homeostasis. It is not known yet whether, in mirror image to the  effect, the increase in apparent Na⁺ affinity effected by CHIF reflects a decrease in the apparent affinity for K⁺ acting as a competitive inhibitor of cytoplasmic Na⁺ activation.

An important role of  in renal cation homeostasis secondary to its association with, and modulation of, Na,K-ATPase is demonstrated by our results showing the functional consequences of mutating Gly-41 to Arg. This study provides evidence that the G41R substitution alters  interaction with the pump, resulting in the failure of  both to traffic to the cell surface and to modulate pump kinetics. The former finding confirms the  routing defect reported by Meij et al. (24). In addition, our experiments indicate that pump trafficking per se is not notably affected.

The significance of the association of renal Mg²⁺-wasting with abrogation of  modulation of Na,K-ATPase is uncertain. It is evident that the consequences of changes in Na⁺, K⁺, and Cl transport along the different regions of the nephron are varied and complex. Reduced apparent ATP affinity of pumps by abrogation of their modulation by  may decrease pump activity and lead to secondary changes (reduction) in Mg²⁺ reabsorption. Accordingly, renal Mg²⁺-wasting seen in the dominant hypomagnesemia described by Meij et al. (24) appears to be secondary to the loss of  modulation of Na,K-ATPase function. The primary cellular mechanism remains to be determined. Also unexplained is the increase in renal calcium absorption and hypocalciuria that is consistently observed in these patients (24).

The experiments described in this study were carried out with cultured apolar cells. The extent to which  trafficking and abrogation of the  effects by G41R replacement are different in polar cells remains to be addressed. Current efforts are underway to address this aspect of  function in polarized renal epithelial cells.

	ACKNOWLEDGEMENTS

We thank Drs. Alex Therien, Steven Karlish, and Gary Quamme for helpful comments on the article and Dr. Edward Chan for the gift of anti-giantin.

	FOOTNOTES

^* This work was supported by operating grants from the Canadian Institutes of Health Research (MT-3876) and the Kidney Foundation of Canada.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. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Montreal General Hospital, 1650 Cedar Ave., Montreal, Quebec H3G 1A4, Canada. Tel.: 514-934-1934 (ext. 44501); Fax: 514-934-8332; E-mail: Rhoda. Blostein{at}mcgill.ca.

Published, JBC Papers in Press, April 19, 2002, DOI 10.1074/jbc.M201009200

²  K_Na and K_K are the affinity constants for Na⁺ (extrapolated to [K⁺] = 0) and for K⁺ at cytoplasmic site(s), respectively. K'_Na is the apparent affinity constant for Na⁺ at cytoplasmic sites.

	ABBREVIATIONS

The abbreviations used are: CHIF, corticosteroid hormone-induced factor; PFO, perfluorooctanoate; PRM, plasma-rich membranes; HDM, high density microsomal membranes; LDM, low density membranes; MES, 4-morpholineethanesulfonic acid; WT, wild-type; NHS, N-hydroxysuccinimide.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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