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J. Biol. Chem., Vol. 277, Issue 12, 10162-10172, March 22, 2002

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Differential Regulation of Renal Na,K-ATPase by Splice Variants of the  Subunit^*

Elena Arystarkhova, Claudia Donnet, Natalya K. Asinovski, and Kathleen J. Sweadner

From the Laboratory of Membrane Biology, Neuroscience Center, Massachusetts General Hospital, Charlestown, Massachusetts 02129

Received for publication, December 4, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Sodium and potassium-exchanging adenosine triphosphatase (Na,K-ATPase) in the kidney is associated with the  subunit (, FXYD2), a single-span membrane protein that modulates ATPase properties. Rat and human  occur in two splice variants, a and b, with different N termini. Here we investigated their structural heterogeneity and functional effects on Na,K-ATPase properties. Both forms were post-translationally modified during in vitro translation with microsomes, indicating that there are four possible forms of . Site-directed mutagenesis revealed Thr² and Ser⁵ as potential sites for post-translational modification. Similar modification can occur in cells, with consequences for Na,K-ATPase properties. We showed previously that stable transfection of a into NRK-52E cells resulted in reduction of apparent affinities for Na⁺ and K⁺. Individual clones differed in  post-translational modification, however, and the effect on Na⁺ affinity was absent in clones with full modification. Here, transfection of b also resulted in clones with or without post-translational modification. Both groups showed a reduction in Na⁺ affinity, but modification was required for the effect on K⁺ affinity. There were minor increases in ATP affinity. The physiological importance of the reduction in Na⁺ affinity was shown by the slower growth of a, b, and b' transfectants in culture. The differential influence of the four structural variants of  on affinities of the Na,K-ATPase for Na⁺ and K⁺, together with our previous finding of different distributions of a and b along the rat nephron, suggests a highly specific mode of regulation of sodium pump properties in kidney.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The Na,K-ATPase,¹ or sodium pump, is the principal enzyme in animal cells that maintains ionic gradients of Na⁺ and K⁺ at the expense of ATP hydrolysis. In kidney, the Na⁺ gradient provides the driving force not only for the reabsorption of sodium, but also for secondary transepithelial transport of various essential solutes and water. Controlling sodium pump activity is thus an essential element of renal regulation.

The Na,K-ATPase has two obligatory subunits, the catalytic  subunit and a glycoprotein, , which are encoded by multigene families (1). Expression of  and  isoforms is developmentally regulated, and appears to be species- and tissue-specific (2, 3). In several experimental expression systems, the exchange of either  or  subunit isoforms affected enzymatic properties of the complex, most notably affinities for K⁺ and Na⁺ (3). Functional differences in intrinsic properties of Na,K-ATPase have been reported in the kidney; the affinity for Na⁺ varies significantly along the rat and rabbit nephron (4-7). This led to an effort in many laboratories to determine whether  and  isoforms could account for the differences, but no segment-specific localization of isoforms other than 1 and 1 was found for either mRNA or protein (8-12).

Two regulatory proteins that modulate intrinsic properties of the Na,K-ATPase have now been identified in kidney: the Na,K-ATPase  subunit (13, 14), and CHIF ("channel-inducing factor") (15, 16). Based on sequence homology, both proteins belong to the FXYD gene family, which unites small (5-15 kDa) single-span membrane proteins including phospholemman (17), MAT-8 ("mammary tumor antigen-8") (18), RIC ("related to ion channel") (19), as well as two new gene products, called FXYD6 and FXYD7 (20). Although Na,K-ATPase is widely distributed in kidney, expression of  and CHIF is segment-specific.  co-localizes with Na,K-ATPase in proximal tubules, distal convoluted tubules, and medullary thick ascending limb (21-23), whereas CHIF co-localizes with the pump in collecting duct (16).

The functional significance of  (FXYD2) for the Na,K-ATPase complex was a mystery for a long time. The key properties of the Na,K-ATPase (ouabain binding, enzymatic activity, and cation transport) can be obtained without it (24-26). It is also not required for assembly and transport of functional units to the plasma membrane (27). However, incorporation of a photoaffinity-labeled derivative of ouabain into  as well as  suggested that it may comprise part of the ouabain-binding site (13).

The first clear effect of  on Na,K-ATPase transport properties was detected by electrophysiological measurements in Xenopus oocytes. It influenced the apparent affinity of the Na,K-ATPase pump current for extracellular K⁺ (28). An antibody against  inhibited Na,K-ATPase in vitro and decreased affinity for ATP (29). The functional role of  was further assessed with -transfected mammalian cells. In HEK cells,  increased affinity for ATP (30). NRK-52E cells express the same 11 combination as kidney but no , and apparent affinities for Na⁺ and K⁺ were higher than for 11 from renal medulla (31). Stable transfection of a (the only known splice variant at the time) resulted in a reduction of affinities for Na⁺ and K⁺. This was observed with partially purified enzyme, indicating a stable functional alteration of enzyme properties. Interestingly, the modulation of Na⁺ affinity was abolished by a post-translational modification of  that occurred in cell culture and that shifted its gel mobility (31). A comparable decrease in apparent Na⁺ affinity has recently been ascribed to an increase in K⁺ antagonism of cytoplasmic Na⁺ activation in -transfected HeLa cells (21). Similarly,  was shown to influence Na,K-ATPase pump current by lowering the apparent affinity for intracellular Na⁺ (32). The injection of CHIF cRNA into Xenopus oocytes, in contrast, resulted in an increase in apparent affinity for Na⁺ (32). Taken together with the almost complementary distributions of  and CHIF along the rat nephron (16, 22), the functional data correlate very well with the differences in Na⁺ affinities in successive segments of the nephron (4, 6, 7, 33).

In rat kidney,  occurs as at least two splice variants, a and b, with different N termini (20, 34, 35). The splice forms colocalized in the inner stripe of the outer medulla in the thick ascending limb of the loop of Henle (21, 23), and co-immunoprecipitation with each other and  showed that they are associated in macromolecular complexes (23). Our observations indicate that their distribution in renal cortex and outer stripe of the outer medulla, however, is strongly biased to different segments. a predominated in proximal tubules, whereas b was detected only in distal and connecting tubules and the thick ascending limb in the outer stripe (23). Very recently, a third splice variant of  has been described in mouse (36), although not in human (37). All of the splice variants in these characterized genes have different promoters, which provides a basis for differential regulation of gene expression.

Here we have investigated the functional properties of  subunit splice variants. Others have reported that a and b have similar effects on intrinsic properties of the pump. Both splice variants decreased apparent affinity for Na⁺ without significant alteration of K⁺ affinity in HeLa transfected cells (21) and in Xenopus oocytes (32), and increased K_m for ATP in HeLa transfectants (21). We find, however, that post-translational modifications that occur in some but not all NRK-52E clones stably expressing a or b affect the final enzyme properties; consequently, a can have modulating effects on Na,K-ATPase activity different from that of b. Taken together with the differential distribution of a and b in kidney, the regulatory effects of  on intrinsic properties of the Na,K-ATPase appear to be subject to multiple layers of control. Preliminary reports have been presented (38, 39).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Preparation of Na,K-ATPase from Kidney-- Isolation of membranes and purification of Na,K-ATPase from rat kidney outer medulla and whole mouse kidneys was performed by the Jørgensen procedure (40). Briefly, microsomal fractions were collected by differential centrifugation, followed by SDS treatment and equilibrium centrifugation on sucrose density gradients.

Plasmid Construction and Site-directed Mutagenesis-- cDNA for the b splice variant was obtained by reverse transcriptase-PCR from total rat kidney RNA (CLONTECH). The primers were based on nucleotide sequences for rat b in the dbEST data base (GenBank^TM) plus EcoRI and BamHI restriction sites for unidirectional cloning: forward degenerate primer, 5'-GCGAATTCCACCATGGAYAGGTGGTACYTG-3', where Y = (C/T); reverse primer, 5'-CGCGGATCCCAGCTCATCTTCATTGAC-3'. Gel-purified DNA was ligated into pIRES vector (CLONTECH) containing an internal ribosome binding site and the neomycin resistance gene. Several clones containing the full-length cDNA of b were verified by nucleotide sequencing (GenBank^TM AF233060). Point mutations were introduced into the a cDNA in the pIRES vector by PCR and confirmed by DNA sequencing.

In Vitro Protein Synthesis-- The full-length cDNAs of rat a (wild type and mutated) and b were ligated into pT7 vector (Novagen) and translated in vitro using the TNT-T7 system (Promega) with or without addition of canine pancreatic microsomes according to the manufacturer's procedure. The reaction was for 90 min at 30 °C, followed by centrifugation in an Airfuge (Beckman) (10 min, 4 °C). The samples were washed with 100 mM NaCl and 25 mM HEPES, pH 7.4, and the final pellets were resuspended in 315 mM sucrose, 1 mM EDTA, and 20 mM Tris-Cl, pH 7.5. Portions were dissolved in SDS sample buffer, run on an SDS-Tricine gel (41), and transferred to nitrocellulose. Detection of the newly synthesized proteins was with the RCT-G1 antibodies against the C terminus of  (31). Luciferase-T7 DNA was used as a positive nonradioactive control; samples without DNA added served as negative controls for transcription/translation background. Blots were scanned with a laser densitometer.

Transfectant Enzyme Preparation and Gel Electrophoresis-- NRK-52E is a rat renal cell line ("normal rat kidney") with polarized epithelial morphology. Transfection of NRK-52E cells and selection of b-containing stable clones were performed as before for a (31), and isolated clones were propagated in the presence of G418 antibiotic selection. Purification of the Na,K-ATPase from mock- or -transfected cells was as described (31). Briefly, cells were grown in 75-cm² flasks until confluent in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, washed with Dulbecco's PBS, and frozen. Crude membranes from the scraped cells were obtained by homogenization and differential centrifugation. Final purification of Na,K-ATPase was with SDS extraction, which leaves the protein in the lipid bilayer but removes many contaminating proteins.

Electrophoresis was in SDS-Tricine gels (41). Proteins were transferred to nitrocellulose, and the blots were incubated with specific antibodies. Detection was with chemiluminescence (Pierce). The RCT-G1 polyclonal antibody raised against the C-terminal peptide of  (31) was employed to detect both splice variants of the protein. The RNGB antibody (rat N terminus of b) (23) was used for b detection. Monoclonal antibody McK1 was used to stain 1 (42).

Enzymatic Assays-- Na,K-ATPase activity was measured as a function of Na⁺ concentration in media containing 3 mM Tris-ATP, 3 mM MgCl₂, 30 mM histidine, pH 7.4, and in the presence of various concentrations of K⁺ (5-100 mM). Na,K-ATPase activity was also measured as a function of K⁺ concentration (0-20 mM) with fixed [Na⁺] at 140 mM. ATP activation curves were obtained with 140 mM Na⁺, 20 mM K⁺, and 4 mM Mg²⁺ in the reaction medium. All the reactions were performed at 37 °C for 30-45 min with and without 3 mM ouabain, and ouabain-sensitive P_i release was measured colorimetrically by using either Fiske-Subbarow or Baginski methods, or by release of ³²P from [-³²P]ATP. Data were analyzed by nonlinear regression using Sigma Plot Graph System (Jandel Scientific). Na⁺ and K⁺ activation curves were fitted according to the Hill model for ligand binding. K_m values for ATP stimulation were derived from the Michaelis-Menten equation, also by nonlinear regression. Student's statistical test was used to assess differences.

An exception to the above methods applies to data replotted from Ref. 43. There, activity was assayed continuously in a spectrophotometric coupled assay performed in a thermostatted cuvette. Assays were performed with different starting K⁺ concentrations and nominally zero Na⁺, and aliquots of NaCl were added successively, allowing determination of a new slope with each addition.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Gel Mobility and Biosynthesis of the  Structural Variants-- Although it is well accepted that there are two  splice variants in the rat (20, 21, 23, 32, 34, 35), the nature and role of structural modifications of these variants have been controversial. Here we begin by examining the electrophoretic mobilities of  forms found in kidney, and then use in vitro translation of a and b to detect post-translational modification of both splice variants.

Fig. 1A demonstrates  band resolution in gels of typical preparations of Na,K-ATPase from rat and mouse kidney. The blot was stained with the RCT-G1 antibody that recognizes the C terminus of , a site that is highly conserved between species and identical in the splice variants.  was seen as a clear doublet in both species, although the resolution was significantly better for mouse.² A similar slower migration of a was observed in Na,K-ATPase preparations from sheep kidney (not shown). Based on gel mobility, we estimated the differences in apparent molecular masses for splice variants as 1.5 and 2.5 kDa for rat and mouse, respectively, with a appearing larger. For the rat, the calculated molecular masses based on amino acid sequence are almost identical, however: 7245.7 for a and 7234.8 for b, assuming no modification or oxidation. Determination of the molecular masses by mass spectrometry revealed 7184.0 ± 1 for rat a (with carbamidomethyl cysteine and without initiator methionine) and 7337.9 ± 1 for rat b (with carbamidomethyl cysteine and with acetylated methionine) (35), indicating that the larger species actually migrates faster. The sequence of mouse a is four amino acids longer (Fig. 1B), and the calculated difference between molecular masses of unmodified mouse a and b is about 300 (7515.9 for a versus 7204.7 for b). For both species, the calculated masses are close enough that the proteins could co-migrate on SDS gels, but splice variants of  are generally seen as a doublet (23, 32, 35). Thus, the apparent difference in electrophoretic mobility must be influenced by something else, such as net charge or a labile post-translational modification.

View larger version (33K): [in this window] [in a new window]   Fig. 1.   Species-specific difference in electrophoretic mobility of  on SDS gels. A, purified preparations of Na,K-ATPase from rat kidney outer medulla and whole mouse kidneys were run on a 12.5% Tricine gel and transferred to nitrocellulose, and the blot was stained with the RCT-G1 antibody. Migration of a in mouse was significantly slower than in rat. B, alignment of the N-terminal amino acid sequences of a and b from rat and mouse.

We investigated the electrophoretic mobilities by analyzing proteins translated in vitro from synthetic mRNA. First we examined proteins made in reticulocyte lysates without addition of any microsomes. Newly synthesized proteins were analyzed on SDS-Tricine gels and stained with the RCT-G1 antibody, the blot was scanned with a laser densitometer, and the scans were superimposed. As shown in Fig. 2A, the splice variants of rat  migrated at different rates, a slower than b, as shown with tissue-derived samples (23, 35).

View larger version (12K): [in this window] [in a new window]   Fig. 2.   In vitro synthesis of the rat  subunit. cDNAs for rat a and b were transcribed and translated in a reticulocyte system in the absence (A) or presence (B and C) of canine pancreatic microsomes (PM). Newly synthesized proteins were run on Tricine-SDS gels. The blots were stained with the RCT-G1 antibody and scanned with a laser densitometer from top to bottom. Staining of  is expressed in arbitrary units, and its relative position on the gel is marked with arrows.

Addition of endoplasmic reticulum to the reaction mixture permits ribosome attachment and cotranslational membrane protein insertion. When canine pancreatic microsomes were added to the reaction mixture, the electrophoretic mobility of both proteins was decreased, implying that both splice variants can be post-translationally modified (Fig. 2, B and C). The shift observed for a was more pronounced than that for b, indicating either different types of modification, or modification of more sites in a. Either way, the difference in the shift between the splice variants is conjectural evidence that the location of the modification is in the spliced N-terminal segment, and that it occurs in the lumen of rough microsomes. Beguin et al. (32) reported similar shifts in electrophoretic mobility for both splice variants of  synthesized in a reticulocyte system supplemented with microsomes. Thus  may exist (at least in vitro) in four different structural forms: a, a', b, and b', where the mark represents post-translational modification(s).

To identify possible sites of modification by acylation or esterification, we mutated the oxygen-containing residues of the N-terminal segment of . Thr² and Ser⁵ residues are conserved in a from all mammalian species characterized so far (20). Site-directed mutagenesis to alanine was followed by in vitro translation and electrophoresis. As shown in Fig. 3, both residues appeared to be important for post-translational modification. Mutations of either Thr² or Ser⁵ almost completely blocked the shift in mobility of a seen with pancreatic microsomes. However, when Ser¹¹, a potential site for modification in the sequence shared by a and b, was replaced by Ala, migration of the mutated protein was reduced to an intermediate position (Fig. 3C). In the absence of microsomes, all mutants migrated at the same position as their unmutated form (data not shown). The data are consistent with a possible cooperative modification of a at two sites within the extracellular N-terminal splice domain of a, and one in shared sequence. Alternatively, structural perturbation of the extreme N terminus of a could prevent recognition by the modifying enzyme(s).

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Effect of mutations on electrophoretic mobility of a. cDNAs for rat wild type or mutated a were transcribed and translated in vitro, and analyzed as in Fig. 2. Electrophoretic positions are shown for wild type a synthesized in the presence (red) or absence (black) of pancreatic microsomes and mutated a synthesized in the presence of pancreatic microsomes (blue), run on the same gel. Replacement of either Thr2 or Ser5 with Ala (T2A and S5A, respectively) significantly blocked the shift in mobility of a. Replacement of Ser11 (S11A) resulted in an intermediate shift of mobility.

Expression of Two b Forms in NRK-52E Cells-- To test whether b affects function of the pump, stable transfectants of NRK-52E cells were generated as done previously for a (31). Expression of b is shown in Fig. 4. The level of expression in different clones, relative to  subunit, varied between 20 and 80% of the level of b detected in rat kidney membranes (mean, 50-60%). Two groups of clones were identified based on electrophoretic mobility. In some clones (3 of 9 analyzed) b comigrated with b synthesized in vitro (data not shown). In the majority of clones, however, b migrated slower (compare lane 1 with lanes 3 and 4, Fig. 4A). The shift in electrophoretic mobility was similar to that observed in vitro upon the addition of canine pancreatic microsomes (Fig. 2). The difference in migration of b in transfectants was apparent in either crude membrane preparations or partially purified enzyme (data not shown), indicating that difference in mobility was not a purification artifact. Both N and C termini of b were preserved in these preparations because the difference in electrophoretic mobility was seen either with RCT-G1 (Fig. 4A) or RNGB (Fig. 4B) antibodies. This ruled out the possibility of proteolysis, pointing to other post-translational modification as the cause of differences in electrophoretic mobility for b in transfected cells. Because the NRK-52E cell line is morphologically heterogeneous (unpublished observations), cells with different phenotypes might have different cell machinery with respect to b modification similar to the differences already observed in a transfected cells (31).

View larger version (39K): [in this window] [in a new window]   Fig. 4.   Expression of b in NRK-52E cells. A, purified preparations of Na,K-ATPase from rat kidney (lane 2), b (lane 1), and b' (lanes 3 and 4) expressing clones were run on Tricine SDS gels (5.5 inches long). The blots were cut in the middle, and the upper portion was stained with McK1 (1-specific antibody), whereas the bottom panel was stained with RCT-G1 (antibody to the C terminus of ). B, preparations of Na,K-ATPase from rat kidney (lane 1), b (lane 2) and b' (lane 3) expressing clones were electrophoretically separated as in A, and stained with RNGB (antibody to the N terminus of b). The shift in mobility was detected with both -specific antibodies, suggesting a post-translational modification of b. C, purified preparations of Na,K-ATPase from a (partially modified) (lane 1) and b' (lane 2) clones were run separately or together (lane 3). The blot was stained with RCT-G1 antibody. Unmodified a on the gel could not be resolved from b.

The shift in mobility of b in different clones was reliably seen only when electrophoretic separation was performed on long (5.5-inch) Tricine gels (12.5% acrylamide), suggesting that the size of the modifying group was rather small or that it cancelled some other subtle effect on anomalous mobility. Conversely, when a was post-translationally modified in NRK-transfected cells, the shift in electrophoretic mobility was visible even on minigels (2.5-inch) (31). Thus different types of modification (or numbers of residues modified) were apparently exploited in a and b transfectants. The data are in line with our in vitro translation studies, thus providing further support for the structural complexity of .

Fig. 4C shows an experiment in which membrane preparations from clones containing modified b and heterogeneously modified a (31) were mixed together and separated on a long (5.5-inch) Tricine gel. Surprisingly, a doublet (not a triplet as expected) was observed with the RCT-G1 antibodies, which shared a thick band at the lower position. This implies that the electrophoretic mobility of modified b can coincide with that of unmodified a on Tricine gels, complicating the analysis of  structural forms.

Functional Effects of the  Splice Variants on Na,K-ATPase Activity-- Na⁺, K⁺, and ATP ligand dependences of the Na,K-ATPase from b-transfected cells were determined and compared with those from a- and mock-transfected cells. As in our previous study (31), we worked with individually isolated stable clones, and all of the assays were performed on partially purified preparations of Na,K-ATPase. Three clones expressing b and six clones expressing b' were analyzed. The data shown summarize multiple experiments.

Variations in the total specific activity between clones precluded any evaluation of an effect of  expression on V_max. Specific activity was in the range of 50-150 µmol of P_i/mg protein/h. As shown in Fig. 5A, the apparent affinity for Na⁺ was significantly decreased when either b or b' was expressed. Based on nonlinear regression analysis of data fit to the Hill equation, K_0.5 for Na⁺ was shifted from 5.4 mM in wild-type NRK-52E cells to 7.3-7.4 mM for b when measured in the presence of 20 mM K⁺ (Table I). Na,K-ATPase from rat kidney possessed an even lower apparent affinity for sodium (9.5 ± 0.4 mM). Apparently, post-translational modification of b did not influence its effect on Na⁺ affinity because similar changes were observed in both types of b-containing clones (Fig. 5A). The data substantiate the conclusion that the major consequence of  association with the Na,K-ATPase is a decrease of apparent affinity for Na⁺ (21, 31, 32).

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Effects of  on Na+, K+, and ATP affinity by Na,K-ATPase in transfected NRK-52E cells. A, activity of partially purified Na,K-ATPase from mock-transfected (red) or b-transfected (blue and green) cells was tested as a function of Na+ concentration at a fixed K+ concentration of 20 mM. Data from Na,K-ATPase purified from rat renal medulla (black) are shown for comparison. Each set of data points is the mean ± S.D. from at least four independent experiments (with duplicate determinations) expressed as percentage of maximal Na,K-ATPase activity. The data were fitted to Equation 1. Na+ affinity of Na,K-ATPase was altered by b regardless of post-translational modification: b (blue) and b' (green) caused a similar decrease in apparent affinity for Na+. B, preparations of Na,K-ATPase from rat kidney (black), mock-transfected (red), or b and b'-transfected cells (blue and green, respectively) were assayed for ATPase activity as a function of K+ concentration. Each set of data points shown is the mean ± S.D. from at least five independent experiments (with duplicate determinations) expressed as percentage of the ATPase activity at 20 mM K+. The data were fitted to Equation 1. Post-translational modification of b resulted in modulation of the apparent affinity for K+. C, purified preparations of Na,K-ATPase from rat kidney (black), mock-transfected (red), a-transfected (purple and cyan), and b-transfected (blue and green) NRK-52E cells were assayed with concentrations of ATP from 50 µM to 1.5 mM. Data were analyzed by the Michaelis-Menten equation. Each set of data is the mean ± S.D. from at least three independent experiments (with duplicate determinations). Affinity for ATP was slightly different in transfected cells expressing different structural forms of .

View this table: [in this window] [in a new window]   Table I Differential effects of structural forms of  on affinity for Na+ and K+ Affinities were determined with partially purified Na,K-ATPase preparations from transfected NRK-52E cells or from rat kidney medulla. Na+ and K+ activation curves were fitted according to a cooperative model for ligand binding. The apparent affinity K0.5 = K1/n, where n is the Hill coefficient, nh. Clones expressing a as a doublet were incompletely post-translationally modified. The data shown for a doublet clones combine data published previously (31) with additional experiments. The data marked with double asterisks are taken from (31). Multiple tests of significance were performed.

As we reported previously (31), the apparent affinity for K⁺ was also higher in Na,K-ATPase from -deficient NRK-52E cells than from rat kidney membranes, and so we evaluated the contribution of b. The two groups of clones expressing b or b' were examined, and data from multiple experiments were statistically analyzed. Fig. 5B shows that expression of unmodified b did not change the apparent affinity for K⁺ compared with mock-transfected NRK cells. However, with b', K_0.5 for K⁺ was significantly increased from 0.45 ± 0.05 mM (control cells) to 0.75 ± 0.08 mM (b' transfectants) (Table I). It should be noted here that the assay was performed in the presence of 100 mM Na⁺, i.e. in conditions in which the kinetics of activation by K⁺ depend on both the intrinsic affinity for K⁺ and on competition between extracellular Na⁺ and K⁺ ions. Beguin et al. (32) have observed that this is affected by both  and CHIF in a voltage-dependent manner. Their data at 0 mV (corresponding to our experiments in open membranes) show a similar tendency to lower K⁺ affinity with , and an opposite tendency to higher affinity when oocytes are hyperpolarized. In our previous work (31), introduction of a caused a similar reduction in the apparent affinity for K⁺ (from 0.44 ± 0.03 mM to 0.70 ± 0.04 mM, in mock- and -transfected enzyme, respectively). However, the post-translational modification of a did not alter the effect on K⁺ affinity, whereas it eliminated the reduction in apparent affinity for Na⁺. Thus, the four structural forms of  differentially influenced the kinetic parameters of the Na,K-ATPase for its major physiological ligands.

Similar to the two other kinetic parameters (apparent affinity for Na⁺ and K⁺), the K_m for ATP (at the low affinity site) was lower in wild type NRK-52E cells compared with rat kidney membranes (Table II). Transfection with  did not make the ATP affinity more like that of the kidney enzyme, however. Instead it further increased the affinity for at least two of the molecular forms (Fig. 5C), b and fully modified a' (Table II). However, we did not see any effects that reached statistical significance on K_m for ATP for either a (partially modified) or b', nor did any of the changes reach statistical significance compared with mock-transfected cells (Table II). Although the difference in K_m ATP displayed by the two b forms was statistically significant from each other (p = 0.021 by Student's test), the effect was nevertheless very modest. As mentioned above, the level of expression in the transfected cells was never as high as in rat kidney membranes. Thus, the possible interpretation is that association of unmodified b or fully modified a with Na,K-ATPase may lead to a decrease in K_m values for ATP. This is roughly consistent with prior reports (21, 30) and does not explain the higher K_m observed in -containing rat kidney membranes.

View this table: [in this window] [in a new window]   Table II Effect of expression of  on affinity for ATP in different cell lines Km values for ATP for Na,K-ATPase isolated from NRK-52E cells (transfected or mock-transfected) were calculated from the Michaelis-Menten equation. Multiple tests of significance were applied to our own data.

Mechanistic Analysis of the K⁺ Effect on Na⁺ Affinity-- Two alternative equations describing Hill (Equation 1) and noninteractive (Equation 2) models were used to fit the Na⁺ affinity data (Fig. 6A).

View larger version (10K): [in this window] [in a new window]   Fig. 6.   Kinetic analysis of Na+/K+ competition. A, in a typical experiment to determine apparent affinity for Na+, Na,K-ATPase activity was measured in the presence of different concentrations of Na+ and 100 mM K+ in preparations from NRK-52E cells (open circles) and a a transfectant (closed circles). Lines represent the best fit to Equation 1. The inset shows the best fit to Equation 1 (continuous line) as well as for Equation 2 (dashes). Curve-fitting was carried out using SigmaPlot software (Jandel Scientific). The concentration variable was assumed to be homoscedastic. The data are displayed as means ± S.E. B, multiple individual experiments similar to the one depicted in A were performed in the presence of fixed concentrations of 5, 20, 50, or 100 mM K+. K'Na were obtained by fitting with Equation 1 and were plotted against K+ concentration. The curves were drawn by splines. C, to assess whether addition of choline to keep ionic strength constant affects the linearity of the plot of K'Na versus [K+], rat renal medulla Na,K-ATPase was assayed with (closed circles, A) and without choline (open circles, B). The other data (triangles, C) are also from an experiment with rat renal medulla Na,K-ATPase, but data are replotted from Ref. 43, where the data were initially interpreted as fitting a straight line. Dashed lines (A, B, and C) are the best fit to Equation 3, and the solid lines are drawn as splines. D, K'Na versus [K+] for the control NRK-52E cell preparation of panel 2 is shown for K'Na determined from either Equation 1 (open circles) or Equation 2 (closed circles). K'Na for Equation 2 is one eighth of maximal activity, whereas for Equation 1 it is one half of maximal, but both plots are nonlinear.

(Eq. 1)

(Eq. 2)

V_m is the activity obtained at optimum Na⁺ concentration, h is the Hill coefficient, and K'_Na is a measure of the apparent affinity for Na⁺. Equation 1 implies allosteric interaction between the sites for Na⁺. Equation 2 would apply if there is no interaction between three equivalent Na⁺ binding sites, but all three sites must be loaded (44). It should be noticed that K'_Na, a measure of the apparent affinity for Na⁺, has different meanings in each equation, being the Na⁺ concentration that gives the half-maximal velocity in Equation 1, but the concentration giving one eighth of the maximal velocity in Equation 2. It can be seen in Fig. 6A that Equation 1 fit our data with less bias, suggesting that the noninteracting site model may not apply.

The decrease in apparent affinity for Na⁺ observed in -transfected HeLa cells has been analyzed kinetically and concluded to be caused by an increase in K⁺ antagonism of Na⁺ binding at cytoplasmic sites (21). Here, an extensive kinetic analysis was performed to evaluate the influence of K⁺ concentration on apparent affinity for Na⁺ in a-transfected NRK-52E cells. Affinities obtained through fitting the data with Equation 1 were plotted versus K⁺ concentration (Fig. 6B) for enzyme either lacking or containing a. It can be seen that the apparent affinity for Na⁺ decreased as K⁺ concentration rose, loosely in agreement with prior work (21) and that, at all K⁺ concentrations,  reduced the apparent Na⁺ affinity. However, in our hands the shape for the plot of K'_Na versus K⁺ concentration was not linear, suggesting that mechanisms other than K/Na antagonism alone are involved. As shown in Table III, there were technical differences such as in the concentration of free Mg²⁺, which will be discussed below.

A nonlinear plot of K'_Na versus [K⁺] is unexpected, because in earlier studies with various preparations (21, 43-46), K⁺ competition for Na⁺ activation could be described by the simple equation shown below.

(Eq. 3)

K'_Na is the apparent affinity derived from Equation 1 or 2, K_Na is the Na⁺ affinity extrapolated to zero K⁺, and K_K is the affinity for K⁺ as a competitor of Na⁺ binding. Fig. 6C shows, however, that nonlinear plots can also be obtained with rat kidney Na,K-ATPase preparations. We replotted old data from Ref. 43, which were initially interpreted as linear, as well as new data obtained with and without choline added to keep ionic strength constant. Addition of choline shifted the curve to the right at the lower ionic strengths, but did not eliminate the nonlinearity. The solid lines are splines connecting the points, whereas the dashed lines represent the fit to Equation 3, which was poor. Although not as sigmoidal as the data obtained with the NRK-52E transfectants, nonetheless a simple model based on competition between Na⁺ and K⁺ for equivalent, noninteracting sites was not supported in these experimental conditions.

Finally, to rule out any possibility that the method used to calculate K'_Na was responsible for the nonlinearity of the final plot, we recalculated the Na⁺ affinity data for mock-transfected NRK-52E cells using Equation 2 instead of Equation 1. Fig. 6D illustrates the difference in absolute values for K'_Na (1/2 versus 1/8 of V_max) obtained with the two equations, and that the data plotted either way were nonlinear.

Physiological Consequences of  Expression-- We have reported previously that a transfected cells displaying  as a doublet had slower growth rate than cells transfected with empty vector or cells expressing a single band (47). Here we extended our studies with the analysis of the b transfectants. As shown in Fig. 7A, cell growth was reduced compared with the mock-transfected cells, and the delay was somewhat greater than that caused in the partially modified a clones. Practically no disparity in cell growth was observed between the clones containing b or b' (Fig. 7B), suggesting that the alteration in K⁺ affinity did not affect cell proliferation. The 1.5-2-fold greater reduction in the b clones than in the a clone in the second experiment may reflect a higher level of  expression in the b clones. Because all three groups of clones (a doublet, b, and b') possessed lower affinity for Na⁺ than control cells, whereas clones with fully modified a have normal growth rate (47), the interpretation is that  has a deleterious effect on cell proliferation because of a reduction of Na,K-ATPase affinity for Na⁺.

View larger version (14K): [in this window] [in a new window]   Fig. 7.   Expression of  affected NRK-52E growth rate. A and B show two different sets of experiments in which the number of cells per well was determined at 24-h intervals. Mock-transfected cells (open circles), or NRK-5E cells transfected with a (incompletely modified, solid circles), b (solid squares), or b' (solid triangles) were plated at 3 × 104 cells/well in six-well plates, harvested by trypsinization, and counted with a hemacytometer. The data summarize the results of three independent experiments where each data point represents triplicates. The data were fitted to a logarithmic growth curve. Reduction in Na+ but not in K+ affinity correlated with the slower rate of cell proliferation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Regulation of Na,K-ATPase activity in kidney is under several layers of control (48). Although long term alterations involve changes in the number of pump units at the level of gene expression, short term regulators cause rapid modulation of enzyme activity either through recruitment of a latent pool of Na,K-ATPase or through fine-tuning of already existing complexes. The discovery that small single-span membrane proteins (the  subunit and CHIF) regulate the intrinsic properties of the pump through association with provides an additional framework for understanding renal control of Na⁺ and K⁺ balance. A fundamental issue is why different splice variants of  are expressed in kidney and whether they serve distinct functions. The major outcome is the observation that the splice variants may in fact differentially influence the affinities for the major ligands, Na⁺ and K⁺.

Structural Forms of -- The appearance of  on SDS gels was a mystery for a long time. The doublet was first interpreted as post-translational modification of the protein based on in vitro biosynthesis studies (14). In papers from 1999 and earlier, interpretations of doublets as modified forms of a single  subunit, as well as interpretations of the significance of their susceptibility to tryptic digestion, were confounded by not knowing about b (29-31). More recently, genetic analysis (20, 36, 37), mass spectroscopy (35), and immunostaining with specific antibodies (23, 35) showed splice variants to form the  doublet. We have confirmed these conclusions with in vitro translation; despite the very close molecular masses of a and b, the proteins migrated differently on SDS gels. The electrophoretic separation was even more pronounced when canine microsomes were added. Evidence was obtained that both splice variants can be post-translationally modified, yielding four structural forms of . Our findings are in line with the recent report that demonstrated that both splice variants of  were susceptible to post-translational modification in vitro, as was CHIF (32). Interestingly, our data showed that, despite the extensive identity between a and b, their susceptibility to modification in vitro appeared to be different. The electrophoretic mobility of b was changed to a lesser extent than that of a, implying involvement of the variable N-terminal segment. This was supported by site-directed mutagenesis in the N-terminal segment of a. When either Thr² or Ser⁵ was replaced by Ala, the change in mobility upon addition of pancreatic microsomes was almost completely blocked. On the other hand, some shift in mobility was retained when Ser¹¹ was replaced. The results suggest that the observed changes in migration were not caused simply by substitution of hydrophobic for oxygen-containing amino acids, and that modification of more than one site may be involved in a, perhaps including Ser¹¹ in the shared sequence. Others have reported that mutations outside of the N terminus (the common FXYD motif and C-terminal domain) had no effect on formation of doublets (32).

The factors that determine the anomalous mobility of  splice variants are not clear, but something similar occurs with the catalytic subunit of the Na,K-ATPase, where 3, the smallest among the four known isoforms, migrates slowest. The N terminus of b has more bulky hydrophobic residues (tryptophan, tyrosine) and less net negative charge than that of a.

Mass spectroscopy of  from rat kidney did not reveal any sign of modified structure other than removal of the N-terminal methionine from a and acetylation of the N-terminal methionine in b (35). Although the size of the acetyl group is rather small to be detected on SDS gels (42 Da), it could have changed the binding of SDS through an effect on charge. Because we found functional differences between b and b', the challenge would be to test whether deacetylation of b could in fact lead to modulation of apparent affinity for K⁺. Interestingly, there is a precedent from the literature that acetylation of the N-terminal methionine in phospholamban, a single-span regulatory protein from a different gene family, significantly changed its inhibition of the sarcoplasmic, endoplasmic reticulum Ca²⁺-ATPase (49).

The findings suggesting no other post-translational modification of  in kidney (35) must be considered carefully. First, the post-translational modification(s) could be chemically unstable in the conditions used for peptide purification. Both  bands had blocked N termini, precluding direct sequencing (29), but this would not be expected for a if it has no initiation methionine and no other added mass. Second, the Na,K-ATPase used for mass spectroscopy was apparently purified from rat kidney outer medulla (based on its high specific activity). We, however, have identified proximal tubules as a potential source for modified a, which migrated slower on SDS gels compared with samples from outer medulla (23). Finally, post-translational modification of the protein could be dependent on the physiological status of the animal, and turned on and off in response to need. The mass spectroscopy studies were performed with hypertensive rats (35). Regulation of Na,K-ATPase is known to be implicated in hypertension (50).

Three lines of evidence demonstrate that  undergoes post-translational modification when expressed in NRK-52E cells. First, shifts in electrophoretic mobility in transfected cells were always toward higher molecular weight species. Second, with specific antibodies, we showed that both N and C termini of b were preserved. (Unfortunately, the available antibodies against the N terminus of a do not recognize the SDS-denatured protein with enough sensitivity to do this experiment.) Third, the changes in the migration rate of a and b from transfectants were similar to that observed during in vitro translation with and without pancreatic microsomes.

Additional bands seen on Western blots of transfected HeLa and HEK cells (21, 30, 35) have led to some confusion, however. In both cell types, a in transfectants appeared to migrate faster than the upper band of the kidney-derived doublet (35). Because the blot was stained for the C terminus, this could be indicative of either proteolysis at the N terminus of a in transfectants or a post-translational modification of a in renal membranes. The lower band seen in HEK-a could conceivably represent a product of proteolysis or, alternatively, an adduct with a qualitatively different effect on mobility. The position of the upper band in HeLa-b (21) was much higher than would be expected for modified b based on in vitro translation studies (this work or Ref. 32). Further identification of cell-specific modifications of  and elucidation of its possible role in the Na,K-ATPase are required.

Functional Effects of : Apparent Affinities for Ions and ATP-- General agreement has finally been reached that a major effect of  is related to Na⁺ affinity (21, 28, 30-32). This is supported by our recent finding that renal Na,K-ATPase from  knock-out mice displayed higher affinity for Na⁺ than that from wild type animals.³ Beguin et al. (32) also showed in Xenopus oocytes that  decreased the apparent affinity for extracellular K⁺, and only in the presence of extracellular Na⁺, although it was not possible to evaluate the functional impact of post-translational modification because oocytes apparently lack the necessary machinery. The consequences of post-translational modification and effects of  on ATP affinity are not well understood, and there are differences in technical details, and in interpretations of results, that should be reconciled.

The observation that Na⁺ and K⁺ affinities could be modified independently of each other through modification of a and b (Table IV) implies that the structural differences are functionally important. The only sequence difference is in the short N-terminal stretch exposed outside the cell (28, 30). It is attractive to hypothesize that this segment, through its interaction with extracellular segments of  and/or , influences the ion-binding sites within the ATPase complex. The independent changes of Na⁺ and K⁺ affinity (Table IV) do not follow either from the proposal that  stabilizes the E1 conformation, or from the proposal that K⁺ affinity as a competitor of Na⁺ activation can somehow be varied without effect on intrinsic Na⁺ affinity or distribution between E1 and E2 forms (51). Furthermore, effects on K_m for ATP that appear to be consistent with  stabilization of E1 are seen in all three transfected cell lines (Table II), but do not explain the different ATP affinities among the cell lines and -containing renal Na,K-ATPase. Experiments with  knock-out animals also suggest that elimination of  does not result in a statistically significant change in ATP affinity in mouse kidney.³

View this table: [in this window] [in a new window]   Table IV Functional consequences of the expression of  in NRK-52E cells

Divergent results and interpretations may be because of differences in expression systems used. We analyzed multiple stable transfectants of rat  in normal rat kidney cells that express rat 11 and no other known Na,K-ATPase subunit isoform. Pu et al. (21) have ruled out that the use of human 1 instead of rat 1 influenced the functional effect in transfections of HeLa cells with rat . However, there is no evidence excluding functional association of  with the glycoprotein  subunit. The human  subunit(s) expressed in HEK (human embryonic kidney) cells has not been determined to our knowledge, but human embryonic kidney expresses 2 in vivo (52). We have found both 1 and 3 to be expressed in HeLa cells (data not shown). Although there is a high degree of homology between human and rat 1, there are some structural variations (17/304 substitutions) that might affect subunit interactions, and 2 and 3 are significantly divergent (32-34% identity to 1).

K⁺/Na⁺ Antagonism-- Na⁺ is normally rate-limiting for Na,K-ATPase enzyme activity, and so changes in effective Na⁺ affinity should have direct physiological consequences by regulating net activity in tissues. The practical issue is that the concentration of intracellular K⁺ exceeds that of Na⁺ by a factor of 30, and the ions are thought to be transported in a ping-pong manner, utilizing the same sites. Of the three cytoplasmic ion binding sites, the first two to be occupied do not have a marked preference for Na⁺, whereas the third site apparently binds Na⁺ exclusively (53). The lack of specificity of two of the sites results in K⁺/Na⁺ competition.

Recently, Pu et al. (21) found linear relationships with different slopes for apparent Na⁺ affinity plotted as a function of concentration of competing K⁺, and concluded that the mechanism of  entailed an increase in K⁺/Na⁺ antagonism at the intracellular Na⁺ binding sites, without effect on intrinsic Na⁺ affinity (21, 51). They used a noninteractive site model to calculate K'_Na (Equation 2 shown above), put forward originally by Garay and Garrahan in 1973 (44), and showed data compatible with simple competition of K⁺ for the Na⁺ sites (21). Sachs (45) reported similar linearity in erythrocyte ghosts, like the original studies on erythrocyte ion fluxes (44). The concept that  changes K⁺/Na⁺ competition has appeal because the Blostein group has previously demonstrated tissue-specific (not isoform-specific) differences in K⁺/Na⁺ antagonism (46, 54), which could conceivably be because of expression of  or other FXYD family members modulating the intrinsic properties of the pump. Our data, in contrast, showed a nonlinear increase in apparent affinity for Na⁺ with increasing concentrations of K⁺ that tended to decline to a plateau. The presence of  shifted the curve to higher values but the shift was not as strongly dependent on K⁺ as described by Pu et al. (21). In addition, the slopes did not have the same intercepts, suggesting intrinsically different Na⁺ affinities in the presence and absence of .

Although the final results and interpretation seem to be different from ours, there are several practical considerations that may reconcile the findings. The kinetic model used may have contributed to the difference, although it cannot explain it alone. The model they used assumes that K⁺ competes with Na⁺ binding to any one of three equivalent sites on the cytoplasmic surface and includes no provision for cooperativity or ordered binding and release. More is known currently about the kinetics and properties of ion binding than in 1973, and it is well documented that binding of the first and second Na⁺ differs substantially from the binding of the third Na⁺ in both character (electroneutral versus electrogenic) and affinity (53, 55). Strictly ordered release of the three Na⁺ ions has been also shown for Na,K-ATPase (56). To analyze our current data with  transfectants, we derived K'_Na from a cooperative model (Equation 1) rather than a noncooperative one (Equation 2). It can be seen that equation 1 showed less bias and better described our experimental results, something that has also been found by others (57). Measurement of pump currents as a function of Na⁺ concentration has also been shown to be well described by a Hill equation (32, 58, 59). For comparison, we re-analyzed our data with the noninteractive model (Equation 2). The data were still nonlinear, but the nonlinearity was less obvious. Therefore, it is plausible that K⁺ competition for Na⁺ binding may be more complex than the simplest model suggests, depending on the specific conditions and occupancy of the sites.

A technical difference between the Pu et al. (21) data and ours is that we did not keep the ionic strength constant, whereas they kept constant ionic strength by addition of choline. Choline is not inert in its effect on Na,K-ATPase, tending to favor the E1 conformation (60, 61). In control experiments performed with renal medulla Na,K-ATPase preparations, we found a rightward shift from choline addition and somewhat less nonlinearity, although the final relation was still nonlinear.

The other relevant feature of competition at the cytoplasmic Na⁺ sites is that it is by no means limited to K⁺. Mg²⁺ is perhaps the best known physiological competitor (62-65), but even Ca²⁺ and organic cations are known to compete (65, 66). It is notable that the free Mg²⁺ concentrations used by our group and Blostein's group (as well as Ref. 45) were different by severalfold, as calculated from association constants with ATP and EDTA, pH, and ionic strength (Table III). In preliminary experiments with 7 mM free Mg²⁺, we observed a much lower overall Na⁺ affinity and a smaller effect of expression of  in Na,K-ATPase from NRK-52E transfectants (data not shown). This is consistent with the concept that either Mg²⁺ or K⁺ can compete for Na⁺ binding. Measurements of free Mg²⁺ in cultured renal cells are in the range we used (67, 68), but free Mg²⁺ in skeletal, cardiac, and smooth muscle cells is as high as 1 mM (69).

Finally, the concentration of ATP may be a factor in determining K⁺/Na⁺ apparent antagonism through its effect on conformation, and this could introduce complexity either through the experimental use of different ATP concentrations, or through effects of  on ATP affinity. However, if  increases ATP affinity, it would be expected to reduce K⁺ affinity as a competitor and thus enhance Na⁺ apparent affinity, but this is the opposite of what is observed in  transfectants. Conceptually, this is consistent with other evidence that effects of  on Na⁺ affinity and on ATP affinity are distinct (21, 51).

Although prior papers have attempted to analyze 's function effects in terms of a shift in Na,K-ATPase E1-E2 equilibrium, we think that this theoretical framework is not helpful. The data suggesting that  favors E1 (or that anti- antibody favors E2) are mostly based on observations in nonphysiological concentrations of ligands (29, 30), and seem to contradict the evidence that  enhances K⁺ competition at intracellular Na⁺ sites (21). K⁺/Na⁺ competition should be inseparable from the loading of E2(K), E1(K), and E1(Na) intermediates and therefore inseparable from a tendency to remain in E2 (46, 53).  has been proposed to increase K⁺ affinity as a competitor without affecting intrinsic Na⁺ affinity (51), but the very concept of an intrinsic Na⁺ affinity is complicated because, by the nature of vectorial transport, Na⁺ is always drained away. Although Na⁺ binding affinities for the three cytoplasmic sites can be estimated in the absence of ATP, a physically meaningful binding affinity cannot be obtained in its presence (at temperatures permissive for enzyme turnover) because of the failure to obtain equilibrium as a result of transport (53). Consequently, all reported Na⁺ affinities derived from enzyme during turnover are "apparent" affinities, regardless of how terms may be defined. These arguments, combined with the current concept that the pump cycle does not have any single "power stroke" that can be uniquely targeted to regulate activity (53), suggest that , as an extra subunit, may impact multiple steps of the Post-Albers scheme. The ability of different forms of  to affect Na⁺, K⁺, and ATP affinities separately argues that a reductionist attempt to understand mechanism as an effect on any one step may be difficult to accomplish.

A final physiological variable that we have not addressed is membrane potential (70). In proteoliposomes containing renal Na,K-ATPase, a potential positive on the cytoplasmic side (equivalent to cell depolarization) increased Na⁺ affinity without altering the affinity for competing K⁺ or organic cations (71). Membrane potential has been shown to affect the magnitude of the effects of both  (28) and CHIF (32) to external Na⁺ and K⁺ measured in Xenopus oocytes. In short, the functional consequences of  expression for Na,K-ATPase properties is potentially complex, and merits further investigation in intact cells.

In conclusion, apparent Na⁺ affinity can be affected by multiple factors: voltage; K⁺, Mg²⁺, and ATP concentrations for any given preparation; and expression of  and CHIF for any given set of conditions. The data suggest that the effect  has on the affinity of K⁺ as a competitor of Na⁺ binding is not quantitatively explained by a simple competition model.

Physiological Relevance of  Expression-- It is notable that the level of  expression in stably transfected clones is lower than that found in the renal medulla (21, 31). This may be because of the fact that  expression can slow growth rates. We routinely observed that empty vector clones appeared several days before -expressing clones, suggesting that there may be selective pressure against clones with even higher levels of expression.

This laboratory has recently shown that  expression can be induced in NRK-52E cells by exposure to hypertonic media for six or more h (72). Others have reported a similar finding in inner medullary collecting duct cells (73). How this relates to conditions in the kidney is a matter of some importance, particularly because the expression of  and each splice variant varies markedly in different nephron segments (22, 23), and the hypertonicity of the nephron varies with depth and physiological status.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant HL27653 (to K. J. S.).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: 149-6118, Massachusetts General Hospital, 149 13th St., Charlestown, MA 02129. Tel.: 617-726-8579; Fax: 617-726-7526; E-mail: sweadner@helix.mgh.harvard.edu.

Published, JBC Papers in Press, December 27, 2001, DOI 10.1074/jbc.M111552200

² A third, larger splice variant, c, has been reported for mouse (36), but if it was present in adult mouse kidney, it was at levels that were not detected here.

³ D. H. Jones, Y. Li, K. J. Barr, E. Arystarkhova, R. K. Wetzel, K. J. Sweadner, G.-H. Fong, and G. M. Kidder, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: Na, K-ATPase, sodium and potassium-exchanging adenosine triphosphatase; HEK, human embryonic kidney; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; FXYD, gene nomenclature based on a conserved motif, Phe-X-Tyr-Asp, pronounced like "fix-it.".

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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