J Biol Chem, Vol. 274, Issue 47, 33183-33185, November 19, 1999

COMMUNICATION
The  Subunit Modulates Na⁺ and K⁺ Affinity of the Renal Na,K-ATPase^*

Elena Arystarkhova, Randall K. Wetzel, Natalya K. Asinovski, and Kathleen J. Sweadner

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

	ABSTRACT

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The Na⁺,K⁺-ATPase catalyzes the active transport of ions. It has two necessary subunits,  and , but in kidney it is also associated with a 7.4-kDa protein, the  subunit. Stable transfection was used to determine the effect of  on Na,K-ATPase properties. When isolated from either kidney or transfected cells, had lower affinities for both Na⁺ and K⁺ than . A post-translational modification of  selectively eliminated the effect on Na⁺ affinity, suggesting three configurations (, , and *) conferring different stable properties to Na,K-ATPase. In the nephron, segment-specific differences in Na⁺ affinity have been reported that cannot be explained by the known  and  subunit isoforms of Na,K-ATPase. Immunofluorescence was used to detect  in rat renal cortex. Cortical ascending limb and some cortical collecting tubules lacked , correlating with higher Na⁺ affinities in those segments reported in the literature. Selective expression in different segments of the nephron is consistent with a modulatory role for the  subunit in renal physiology.

	INTRODUCTION

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The renal control of Na⁺ and K⁺ balance is complex and entails ensembles of apical and basolateral transporters that play specialized roles in different segments of the nephron. One of the most physiologically important transporters is the Na,K-ATPase, or sodium pump, which is crucial for the absorptive, secretory, and concentrating capacity of the kidney. Small changes in Na,K-ATPase ion affinities can have important physiological effects both directly on transepithelial ion and fluid transport and indirectly through the ion gradients used by other transporters (1, 2). In permeabilized microdissected nephron segments, Na,K-ATPase has increased affinity for Na⁺ in thick ascending limb compared with proximal convoluted tubule and has still higher affinity in cortical collecting tubule (3-6). Although isoforms of  and  can have different ion affinities (7), isoforms unique to the nephron segments with higher affinity have not been detected (8-12), leaving the mechanism unexplained.

A small single-span membrane protein, member of a protein family that has ion channel activity in oocytes, copurifies with renal Na,K-ATPase  and  subunits and appears as a doublet on gels (13, 14). Known as the  subunit, it is not present in all tissues, and its role in the Na,K-ATPase has long been an enigma (15-17). As an ancillary subunit it is likely to play a modulatory role. It has been reported to increase ATP affinity when expressed in mammalian cells (18, 19) and to affect the voltage sensitivity of K⁺ activation when expressed in oocytes (20). Here evidence is presented for its role in the control of the most basic property of Na,K-ATPase, the affinity for Na⁺ and K⁺. Most significantly, analysis of independent clones points to a functional role for post-translational modification of the  subunit.

	EXPERIMENTAL PROCEDURES

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Enzyme Preparations-- Na,K-ATPase was purified from rat, pig, and dog kidney by SDS extraction (21), which produces membrane bound, active enzyme. Crude membranes from renal cell lines and transfectants were treated with SDS at 0.56 mg/mg protein and sedimented on 7-30% sucrose gradients. Specific activities of the Na,K-ATPase from rat kidney and NRK-52E cells were 1200-1500 and 120-150 µmol of P_i/mg/hr, respectively.

Gels-- Electrophoresis was in SDS-Tricine¹ gels (22). Proteins were transferred to nitrocellulose, incubated with antibodies, and detection was with chemiluminescence (23).

Antibody to -- A rabbit antiserum (RCT-G1) was raised against the peptide corresponding to the last 14 amino acids (CGGSKKHRQVNEDEL) of rat , conjugated to keyhole limpet hemocyanin (KLH).

ATP Hydrolysis-- Na,K-ATPase activity was measured in media containing 3 mM Tris-ATP, 3 mM MgCl₂, 30 mM histidine, pH 7.3, and various concentrations of Na⁺ (0-20 mM) with [K⁺] fixed at 20 mM, or various concentrations of K⁺ (0-4 mM) with [Na⁺] fixed at 140 mM. Reactions were performed for 30 min at 37 °C with and without 3 mM ouabain, and ouabain-sensitive P_i release was measured colorimetrically.

Transfections-- cDNA for the  subunit was obtained by RT-PCR from total rat kidney RNA (CLONTECH, Palo Alto, CA). The following primers were based on a nucleotide sequence for rat  in the GenBank^TM dbEST data base (AA801241) plus EcoRI and BamHI restriction sites for unidirectional cloning: forward primer, 5'-GGAATTCGTGGCTGGGGAAATGAC-3'; reverse primer, 5'-CGCGGATCCCAGCTCATCTTCATTGAC-3'. Gel-purified DNA was ligated into pIRES vector (CLONTECH). Several clones containing the full-length cDNA of  were verified by PCR and nucleotide sequencing. The N-terminal protein sequence is predicted to be MTELSANHGGS, corresponding to the corrected sequence reported by Minor et al. (14) and different from that deposited in GenBank^TM (X70062). Transfection was with cationic liposomes (Clonfectin, CLONTECH). Antibiotic (G418) was added after 48 h, and resistant colonies formed in 15-20 days.

Immunofluorescence-- Cryostat sections were made from rat kidney fixed with periodate-lysine-paraformaldehyde. Sections were double-labeled with mouse anti-1 (McK1, 1:4) and rabbit anti- (RCT-G1, 1:500), stained with Cy3-conjugated anti-mouse and fluorescein isothiocyanate-conjugated anti-rabbit secondary antibodies, and examined with a Bio-Rad MRC 1024 Laser Sharp confocal microscope.

	RESULTS

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Properties of  Transfectants-- While Na,K-ATPase from kidney contains , this is not true of mammalian kidney cell lines (18). NRK-52E, an epithelial line of rat kidney cells, expressed the same Na,K-ATPase 1 and 1 isoforms found in rat kidney, but  could not be detected with specific antibodies (Fig. 1) or by RT-PCR (not shown). The Na⁺ and K⁺ affinities of partially purified Na,K-ATPase with and without  were compared (Table I, top). The apparent affinities for both ions were substantially higher (1.5-2-fold) in enzyme from NRK-52E (11) than from renal medulla (11). Two other renal cell lines, LLC-PK1 (pig) and MDCK (dog), also lacked  (not shown), and again cell-derived enzyme without  had higher affinities for Na⁺ than renal enzyme with it (Table I, top).

View larger version (47K): [in this window] [in a new window]   Fig. 1.   Detection of . Western blots stained for 1 and  of Na,K-ATPase partially purified from rat renal medulla (lane 1), NRK-52E control cells (lane 2), NRK-52E transfected with empty vector (lane 3), and two representative clones transfected with  (lanes 4 and 5). The blot was cut so that the high molecular weight region could be stained with antibody McK1 (1) and the peptide region with antiserum RCT-G1 ().

View this table: [in this window] [in a new window]   Table I Ion affinities for ATP hydrolysis by Na,K-ATPase with and without

To determine whether  was responsible for the kinetic difference, stable transfectants of NRK-52E were generated. Expression of  was detected on Western blots (Fig. 1). By densitometric analysis, the level was 15-20% of the level in the kidney enzyme, normalized to the amount of 1. Two groups of clones were distinguished by the mobility of : those expressing a doublet (as in kidney preparations) and those expressing only the band with slower mobility. The distinction between clones proved to be critical for the kinetic properties of the Na,K-ATPase. The clones that expressed a  doublet showed reduced affinity for Na⁺ as well as K⁺, while the clones with only one band showed reduced affinity for K⁺ only (Fig. 2). This relationship held true in three independent clones with doublets and three clones with single bands. The ion affinities for all of the tested clones are compared in Table I, bottom.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Na+ and K+ affinities were altered by  and its posttranslational modification. Hydrolysis of ATP was measured as a function of either Na+ or K+. Partially purified enzyme from mock-transfected NRK-52E () or clones expressing either the  doublet () or the single band () were tested. Data from Na,K-ATPase purified from rat renal medulla (, dashed lines) are shown on each graph for comparison. a, Na+ affinity; b, K+ affinity. Expression of either form of  altered K+ affinity to resemble that of kidney-derived enzyme, while only the clones expressing the  doublet had altered Na+ affinity.

Post-translational Modification of -- In the literature, doublets of mammalian  appeared during in vitro synthesis in the presence of pancreatic microsomes (17), but not in their absence (20), which suggests that there might be post-translational processing in the endoplasmic reticulum. The  subunit does not have a signal sequence, but trypsin treatment of pig kidney right-side-out sealed microsomal vesicles reduced the doublet to a single band, consistent with a cleavage site near the extracellular N terminus (18). We digested right-side-out rat renal medulla vesicles with trypsin (24). Both bands of the  doublet were reduced to one smaller band, from 7.6 and 6.8 kDa to 5.6 kDa (Fig. 3a), predicting cleavage at Lys-13, analogous to the cleavage of pig  in extensively digested purified enzyme (25). The disappearance of the doublet implies that the modification lies extracellularly in the first 13 amino acids (MTELSANHGGSAK).

View larger version (38K): [in this window] [in a new window]   Fig. 3.   The  doublet results from a post-translational modification near the N terminus. a, right-side-out sealed renal medullary vesicles (lane 1, control) were trypsinized at a trypsin:protein ratio of 1:3 for 60 min at 37 °C (lane 2) (24). The  doublet was reduced to a single band of lower Mr that still stained with the antiserum against the C terminus. b, purified enzyme from rat renal medulla was treated with 1 M Tris, pH 11 (lane 1, control), or 1 M hydroxylamine, pH 11 (lane 2), at 37 °C overnight (23). The upper  band was reduced in size to that of the lower band by hydroxylamine, and no smaller species were generated.

Hydroxylamine treatment was used to determine whether the modification was because of acylation of the protein (23, 26). At basic pH, hydroxylamine shifted the slower-migrating band but left the faster-migrating band unchanged (Fig. 3b). At neutral pH neither band was affected (not shown), and there is no Asn-Gly in the sequence for hydroxylamine to cleave (27). This suggests a hydroxyester linkage, possibly fatty acid acylation, to Ser or Thr. There are three Ser and Thr residues in the N-terminal segment that are conserved in  from rat, human, and mouse. Modification of Lys-13 is unlikely because trypsin still cleaved, but modification of the N terminus was not ruled out. The Na,K-ATPase 1 subunit in kidney is also modified by a group that is labile to basic hydroxylamine, but this modification is intracellular (23).

Localization of  in the Kidney-- We examined the distribution of  in rat kidney by double-label confocal microscopy, using the 1 subunit of the Na,K-ATPase for comparison because Na,K-ATPase is known to be expressed at higher levels in some nephron segments than in others (Fig. 4). The anti- antibodies were raised against the C terminus and should detect  forms with or without the posttranslational modification. Distal convoluted tubule (DCT) stained brightly for both 1 and , and proximal convoluted tubule (PCT) stained weakly for both. Other segments, however, including cortical thick ascending limb (CTAL) and some, but not all, cortical collecting tubules (CCT) (not shown), expressed 1 without any  detected above the background of secondary antibody stain. Thus the expression of  is segment-specific in the rat nephron.

View larger version (39K): [in this window] [in a new window]   Fig. 4.   Renal cortex stained with 1- and -specific antibodies shows nephron segments with 1 but no . a, 1 stain, which was brightest in distal convoluted tubule and cortical thick ascending limb. Proximal tubule was stained, but more lightly. b,  stain, which colocalized with 1 in distal convoluted tubule but not cortical thick ascending limb, where the  stain was at the level of background. c, two-color image, scale bar 50 µm. DCT, distal convoluted tubule; CTAL, cortical thick ascending limb; PCT, proximal convoluted tubule.

	DISCUSSION

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The most notable consequence of  expression was that the affinities of the Na,K-ATPase for its substrates, Na⁺ and K⁺, could be modified independently. In clones expressing the doublet, as well as enzyme purified from renal medulla (which has the doublet), affinities for both ions were reduced in concert. This is not easy to explain by a shift between Na,K-ATPase E1-E2 conformations as suggested by Blostein and co-workers (19) but could be understood as a consequence of physical association of  with portions of  comprising the ion binding sites. The stoichiometry of  relative to  was lower than in renal medullary Na,K-ATPase, however, which raises the question of whether  acts exclusively as a subunit.  has been shown to form an ion channel in the absence of  (14). It has not been ruled out that the observed effects on ion affinities result from an indirect modulatory pathway activated by its presence.

NRK-52E is morphologically heterogeneous, lending credibility to the idea that subclones may have different phenotypes with respect to  modification. It is intriguing that the clones with fully modified  have alteration of K⁺ affinity alone, while the half-modified clones show alteration of both ion affinities. The modification appears to nullify a major functional consequence of association with .

The anatomical distribution of  is consistent with Na⁺ affinities measured in different nephron segments: the presence of  is accompanied by lower affinity for Na⁺. This suggests that expression of  selectively modifies Na,K-ATPase properties in vivo. Whether the post-translational modification demonstrated here has a specific cellular distribution among -positive nephron segments remains to be determined.

Pathways that alter ion affinities of kidney Na,K-ATPase consequent to hormone treatment or electrolyte dietary restriction have been reported (4, 5, 28, 29). Their molecular mechanisms are still obscure, however, and could entail the expression and/or modification of . Regardless of whether  acts as a subunit or a channel, the evidence here indicates that it is an important contributor to the control of renal transport physiology.

	ACKNOWLEDGEMENTS

We would like to thank Robert Mercer, Steven Karlish, and Kaethi Geering for samples of anti- antibodies used in early stages of this work.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants NS27653 and HL36271.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.

	ABBREVIATIONS

The abbreviations used are: Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; RT-PCR, reverse transcriptase polymerase chain reaction.

	REFERENCES

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