Distinct regulatory effects of the Na,K-ATPase gamma subunit

The two variants of the gamma subunit of the rat renal sodium pump, gamma(a) and gamma(b), have similar effects on the Na,K-ATPase. Both increase the affinity for ATP due to a shift in the enzyme's E(1) <--> E(2) conformational equilibrium toward E(1). 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 gamma were expressed in rat alpha1-transfected HeLa cells. At the N terminus, the variant-distinct region was deleted (gammaNDelta7) or replaced by alanine residues (gammaN7A). At the C terminus, four (gamma(a)CDelta4) or ten (gamma(a)CDelta10) 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 (gammaNDelta7, gamma(a)CDelta4, and gamma(a)CDelta10) abolished the decrease in K'(ATP) seen with wild-type gamma(a) or gamma(b). It is concluded that different regions of the gamma chain mediate the distinct functional effects of gamma, 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 Mg(2+)-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 gamma but not alphabeta pumps to the cell surface and abrogates functional effects of gamma on alphabeta pumps. These findings underscore a potentially important role of gamma in affecting solute transport, in this instance Mg(2+) reabsorption, consequent to its primary effect on the sodium pump.     

Functional role and immunocytochemical localization of the gamma a and gamma b forms of the Na,K-ATPase gamma subunit

The gamma subunit of the Na,K-ATPase is a member of the FXYD family of type 2 transmembrane proteins that probably function as regulators of ion transport. Rat gamma is present primarily in the kidney as two main splice variants, gamma(a) and gamma(b), which differ only at their extracellular N termini (TELSANH and MDRWYL, respectively; Kuster, B., Shainskaya, A., Pu, H. X., Goldshleger, R., Blostein, R., Mann, M., and Karlish, S. J. D. (2000) J. Biol. Chem. 275, 18441-18446). Expression in cultured cells indicates that both variants affect catalytic properties, without a detectable difference between gamma(a) and gamma(b). At least two singular effects are seen, irrespective of whether the variants are expressed in HeLa or rat alpha1-transfected HeLa cells, i.e. (i) an increase in apparent affinity for ATP, probably secondary to a left shift in E(1) <--> E(2) conformational equilibrium and (ii) an increase in K(+) antagonism of cytoplasmic Na(+) activation. Antibodies against the C terminus common to both variants (anti-gamma) abrogate the first effect but not the second. In contrast, gamma(a) and gamma(b) show differences in their localization along the kidney tubule. Using anti-gamma (C-terminal) and antibodies to the rat alpha subunit as well as antibodies to identify cell types, double immunofluorescence showed gamma in the basolateral membrane of several tubular segments. Highest expression is in the medullary portion of the thick ascending limb (TAL), which contains both gamma(a) and gamma(b). In fact, TAL is the only positive tubular segment in the medulla. In the cortex, most tubules express gamma but at lower levels. Antibodies specific for gamma(a) and gamma(b) showed differences in their cortical location; gamma(a) is specific for cells in the macula densa and principal cells of the cortical collecting duct but not cortical TAL. In contrast, gamma(b) but not gamma(a) is present in the cortical TAL only. Thus, the importance of gamma(a) and gamma(b) may be related to their partially overlapping but distinct expression patterns and tissue-specific functions of the pump that these serve.     

The gamma subunit modulates Na(+) and K(+) affinity of the renal Na,K-ATPase

The Na(+),K(+)-ATPase catalyzes the active transport of ions. It has two necessary subunits, alpha and beta, but in kidney it is also associated with a 7.4-kDa protein, the gamma subunit. Stable transfection was used to determine the effect of gamma on Na, K-ATPase properties. When isolated from either kidney or transfected cells, alphabetagamma had lower affinities for both Na(+) and K(+) than alphabeta. A post-translational modification of gamma selectively eliminated the effect on Na(+) affinity, suggesting three configurations (alphabeta, alphabetagamma, and alphabetagamma*) 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 alpha and beta subunit isoforms of Na,K-ATPase. Immunofluorescence was used to detect gamma in rat renal cortex. Cortical ascending limb and some cortical collecting tubules lacked gamma, 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 gamma subunit in renal physiology.     

Synthesis and assembly of functional mammalian Na,K-ATPase in yeast.

The yeast Saccharomyces cerevisiae was investigated as an in vivo protein expression system for mammalian Na,K-ATPase. Unlike animal cells, yeast cells lack endogenous Na,K-ATPase. Expression of high affinity ouabain binding sites, ouabain-sensitive ATPase activity, or ouabain-sensitive p-nitrophenylphosphatase activity in membrane fractions of yeast cells was observed to require the expression of both alpha subunit and beta subunit polypeptides of Na,K-ATPase in the same cell. High affinity ouabain binding sites are also expressed at the cell surface of intact yeast cells containing both the alpha subunit and the beta subunit of Na,K-ATPase. These observations demonstrate that both the alpha subunit and the beta subunit of Na,K-ATPase are required for the expression of functional Na,K-ATPase activity and that yeast cells can correctly assemble this oligomeric membrane protein and transport it to the cell surface.     

Modulation of Na,K-ATPase by the gamma subunit: studies with transfected cells and transmembrane mimetic peptides

The enzymatic activity of the Na,K-ATPase, or sodium pump, is modulated by members of the so-called FXYD family of transmembrane proteins. The best characterized member, FXYD2, also referred to as the gamma subunit, has been shown to decrease the apparent Na+ affinity and increase the apparent ATP affinity of the pump. The effect on ATP affinity had been ascribed to the cytoplasmic C-terminal end of the protein, whereas recent observations suggest that the transmembrane (TM) segment of gamma mediates the Na+ affinity effect. Here we use a novel approach involving synthetic transmembrane mimetic peptides to demonstrate unequivocally that the TM domain of gamma effects the shift in apparent Na+ affinity. Specifically, we show that incubation of these peptides with membranes containing alphabeta pumps modulates Na+ affinity in a manner similar to transfected full-length gamma subunit. Using mutated gamma peptides and transfected proteins, we also show that a specific glycine residue, Gly-41, which is associated with a form of familial renal hypomagnesemia when mutated to Arg, is important for this kinetic effect, whereas Gly-35, located on an alternate face of the transmembrane helix, is not. The peptide approach allows for the analysis of mutants that fail to be expressed in a transfected system.     

Expression of an ouabain-resistant Na,K-ATPase in CV-1 cells after transfection with a cDNA encoding the rat Na,K-ATPase alpha 1 subunit

We have used a gene transfer system to investigate the relationship between expression of the rat Na,K-ATPase alpha 1 subunit gene and ouabain-resistant Na,K-ATPase activity. A cDNA clone encoding the entire rat Na,K-ATPase alpha 1 subunit was inserted into the expression vector pSV2neo. This construct (pSV2 alpha 1) conferred resistance to 100 microM ouabain to ouabain-sensitive CV-1 cells. Hybridization analysis of transfected clones revealed the presence of both rat-specific and endogenous Na,K-ATPase alpha 1 subunit DNA and mRNA sequences. A single form of highly ouabain-sensitive 86Rb+ uptake was detected in CV-1 cells, whereas two distinct classes of ouabain-inhibitable uptake were observed in transfectants. One class exhibited the high ouabain sensitivity of the endogenous monkey Na,K-ATPase, while the second class showed the reduced ouabain sensitivity characteristic of the rodent renal Na,K-ATPase. Examination of the ouabain-sensitive, sodium-dependent ATPase activity of the transfectants also revealed a low affinity component of Na,K-ATPase activity characteristic of the rodent kidney enzyme. These results suggest that expression of the rat alpha 1 subunit gene is directly responsible for ouabain-resistant Na,K-ATPase activity in transfected CV-1 cells.     

Turnover of the catalytic subunit of Na,K-ATPase in HTC cells

A polyclonal antibody to the catalytic subunit of rat kidney Na,K-ATPase has been raised in rabbits and used to analyze the turnover of the subunit in the rat hepatoma cell line HTC. It had been shown previously (Baumann, H., and Doyle, D. (1978) J. Biol. Chem. 253, 4408-4418) that the membrane proteins of these cells displayed multicomponent turnover kinetics, the minority of the surface proteins turning over with a half-time of about 20 h and the remainder with a half-time of about 100 h. That the antibody precipitated both the alpha (catalytic) and beta (glycosylated) subunits of the Na,K-ATPase from Triton extracts of HTC cells could be demonstrated following metabolic labeling of the cells with either [3H]leucine or a mixture of [3H] mannose and [3H]fucose, but following labeling with [35S]methionine radioactivity was found only in the alpha subunit of the precipitates. Incorporation of [35S]methionine into the alpha subunit could be detected 2 min after addition of the isotope to the cell suspension. Then and at all times thereafter the label was recoverable only from the particulate fraction of a 150,000 X g 60-min centrifugation; no labeled alpha subunit was ever detected in the supernatant fraction. By quantitative densitometry of radioautographs of sodium dodecyl sulfate-polyacrylamide gels of labeled antibody precipitates, it could be shown in pulse-chase experiments that the specific activity of the alpha subunit remained unchanged for 3-4 h (transit time) after the pulse was initiated and that the activity subsequently decayed exponentially with a half-time of 18 h. In a population growing with a generation time (tG) of 33 h, this decay corresponds to a turnover rate constant of 0.49/tG. The catalytic subunit is among those membrane proteins with a rapid turnover rate.     

Expression of Na,K-ATPase alpha subunit isoforms in the human ciliary body and cultured ciliary epithelial cells

We have analyzed the expression of Na,K-ATPase alpha subunit isoforms in the transporting ciliary processes of the human eye and in cultured cells derived from non-pigmented (NPE) and pigmented (PE) ciliary epithelium. Northern hybridization analysis shows that the mRNAs encoding all the three distinct forms of Na,K-ATPase alpha subunit [alpha 1, alpha 2, and alpha 3] are expressed in the human ciliary processes in vivo. Immunohistochemical analysis using antibodies specific for each of the three alpha subunit isoforms confirms that these polypeptides are present in the microsomal fraction from the human ciliary processes. The monoclonal antibody McB2, which is specific to the Na,K-ATPase alpha 2 subunit isoform, has been found to decorate specifically the basolateral membrane domains of NPE cells but not of the PE cells, suggesting its expression in vivo only in the ocular NPE ciliary epithelium. However, cultured cells derived from the NPE and PE layers exhibit a different pattern of expression of mRNA and protein for the Na,K-ATPase alpha subunit isoforms when compared to the tissue. Both the NPE and PE cells express alpha 1 and alpha 3 mRNA and polypeptide, whereas alpha 2 mRNA and polypeptide are undetectable in these cells. The established cell lines derived from the NPE layer express comparable levels of the alpha 1 and alpha 3 isoforms of Na,K-ATPase as detected in the primary culture. However, the established NPE cell lines are also distinguishable from the normal PE cells when analyzed by Western blot analysis with A x 2 antibodies. The results presented here clearly show that the NPE and PE cells in the ciliary body have a distinct expression of Na,K-ATPase alpha subunit isoforms as compared to cultured cells.     

Structure-function relations of interactions between Na,K-ATPase,the gamma subunit,and corticosteroid hormone-induced factor

Corticosteroid hormone-induced factor (CHIF) and the gamma subunit of the Na,K-ATPase (gamma) are two members of the FXYD family whose function has been elucidated recently. CHIF and gamma interact with the Na+ pump and alter its kinetic properties, in different ways, which appear to serve their specific physiological roles. Although functional interactions with the Na,K-ATPase have been clearly demonstrated, it is not known which domains and which residues interact with the alpha and/or beta subunits and affect the pump kinetics. The current study provides the first systematic analysis of structure-function relations of CHIF and gamma. It is demonstrated that the stability of detergent-solubilized complexes of CHIF and gamma with alpha and/or beta subunits is determined by the trans-membrane segments, especially three residues that may be involved in hydrophobic interactions. The transmembrane segments also determine the opposite effects of CHIF and gamma on the Na+ affinity of the pump, but the amino acids involved in this functional effect are different from those responsible for stable interactions with alpha.     

Differential regulation of renal Na,K-ATPase by splice variants of the gamma subunit.

Sodium and potassium-exchanging adenosine triphosphatase (Na,K-ATPase) in the kidney is associated with the gamma subunit (gamma, FXYD2), a single-span membrane protein that modulates ATPase properties. Rat and human gamma occur in two splice variants, gamma(a) and gamma(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 gamma. Site-directed mutagenesis revealed Thr(2) and Ser(5) 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 gamma(a) into NRK-52E cells resulted in reduction of apparent affinities for Na(+) and K(+). Individual clones differed in gamma post-translational modification, however, and the effect on Na(+) affinity was absent in clones with full modification. Here, transfection of gamma(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 gamma(a), gamma(b), and gamma(b') transfectants in culture. The differential influence of the four structural variants of gamma on affinities of the Na,K-ATPase for Na(+) and K(+), together with our previous finding of different distributions of gamma(a) and gamma(b) along the rat nephron, suggests a highly specific mode of regulation of sodium pump properties in kidney.     

Three differentially expressed Na,K-ATPase alpha subunit isoforms: structural and functional implications

We have characterized cDNAs coding for three Na,K-ATPase alpha subunit isoforms from the rat, a species resistant to ouabain. Northern blot and S1-nuclease mapping analyses revealed that these alpha subunit mRNAs are expressed in a tissue-specific and developmentally regulated fashion. The mRNA for the alpha 1 isoform, approximately equal to 4.5 kb long, is expressed in all fetal and adult rat tissues examined. The alpha 2 mRNA, also approximately equal to 4.5 kb long, is expressed predominantly in brain and fetal heart. The alpha 3 cDNA detected two mRNA species: a approximately equal to 4.5 kb mRNA present in most tissues and a approximately equal to 6 kb mRNA, found only in fetal brain, adult brain, heart, and skeletal muscle. The deduced amino acid sequences of these isoforms are highly conserved. However, significant differences in codon usage and patterns of genomic DNA hybridization indicate that the alpha subunits are encoded by a multigene family. Structural analysis of the alpha subunits from rat and other species predicts a polytopic protein with seven membrane-spanning regions. Isoform diversity of the alpha subunit may provide a biochemical basis for Na,K-ATPase functional diversity.     

Cyclic AMP increases cell surface expression of functional Na,K-ATPase units in mammalian cortical collecting duct principal cells

Cyclic AMP (cAMP) stimulates the transport of Na(+) and Na,K-ATPase activity in the renal cortical collecting duct (CCD). The aim of this study was to investigate the mechanism whereby cAMP stimulates the Na,K-ATPase activity in microdissected rat CCDs and cultured mouse mpkCCD(c14) collecting duct cells. db-cAMP (10(-3) M) stimulated by 2-fold the activity of Na,K-ATPase from rat CCDs as well as the ouabain-sensitive component of (86)Rb(+) uptake by rat CCDs (1.7-fold) and cultured mouse CCD cells (1.5-fold). Pretreatment of rat CCDs with saponin increased the total Na,K-ATPase activity without further stimulation by db-cAMP. Western blotting performed after a biotinylation procedure revealed that db-cAMP increased the amount of Na,K-ATPase at the cell surface in both intact rat CCDs (1.7-fold) and cultured cells (1.3-fold), and that this increase was not related to changes in Na,K-ATPase internalization. Brefeldin A and low temperature (20 degrees C) prevented both the db-cAMP-dependent increase in cell surface expression and activity of Na,K-ATPase in both intact rat CCDs and cultured cells. Pretreatment with the intracellular Ca(2+) chelator bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid also blunted the increment in cell surface expression and activity of Na,K-ATPase caused by db-cAMP. In conclusion, these results strongly suggest that the cAMP-dependent stimulation of Na,K-ATPase activity in CCD results from the translocation of active pump units from an intracellular compartment to the plasma membrane.     

Expression of functional Na,K-ATPase isozymes in normal human cardiac biopsies.

In human heart failure, disturbances in Ca2+ homeostasis are well known but the fate of the Na,K-ATPase isoforms (alpha1beta1, alpha2beta1 and alpha3beta1), the receptors for cardiac glycosides, still remains under study. Microsomes have been purified from non-failing human hearts. As judged by the sensitivities of Na,K-ATPase activity to ouabain (IC50 values: 7.0 +/- 2.5 and 81 +/- 11 nM), 3H-ouabain-binding measurements at equilibrium with and without 10 mM K+ and by a biphasic ouabain dissociation process, at least two finctionally active Na,K-ATPase isozymes coexist in normal human hearts. These are demonstrated as a very high- and a high affinity ouabain-binding site. The KD values are 3.6 +/- 1.6 nM and 17 +/- 6 nM, respectively. The two dissociation rate constants are 42 x 10(4) min(-1) and 360 x 10(-4) min(-1). Addition of 10 mM K+ ions shifted the respective KD values for ouabain from 3.6 +/- 1.6 to 20 +/- 5 nM and from 17 +/- 6 nM to 125 +/- 25 nM, respectively. The isozymes involved are identified by comparing these three pharmacological parameters to those of each alpha/beta-isozyme separately expressed in Xenopus oocytes (9). In human heart, the very high affinity site for ouabain is the alpha1beta1 dimer and the high affinity site is alpha2beta1.     

The gamma subunit is a specific component of the Na,K-ATPase and modulates its transport function.

The role of small, hydrophobic peptides that are associated with ion pumps or channels is still poorly understood. By using the Xenopus oocyte as an expression system, we have characterized the structural and functional properties of the gamma peptide which co-purifies with Na,K-ATPase. Immuno-radiolabeling of epitope-tagged gamma subunits in intact oocytes and protease protection assays show that the gamma peptide is a type I membrane protein lacking a signal sequence and exposing the N-terminus to the extracytoplasmic side. Co-expression of the rat or Xenopus gamma subunit with various proteins in the oocyte reveals that it specifically associates only with isozymes of Na,K-ATPase. The gamma peptide does not influence the formation and cell surface expression of functional Na,K-ATPase alpha-beta complexes. On the other hand, the gamma peptide itself needs association with Na,K-ATPase in order to be stably expressed in the oocyte and to be transported efficiently to the plasma membrane. Gamma subunits do not associate with individual alpha or beta subunits but only interact with assembled, transport-competent alpha-beta complexes. Finally, electrophysiological measurements indicate that the gamma peptide modulates the K+ activation of Na,K pumps. These data document for the first time the membrane topology, the specificity of association and a potential functional role for the gamma subunit of Na,K-ATPase.     

Stress-induced expression of the gamma subunit (FXYD2) modulates Na,K-ATPase activity and cell growth

In kidney, the Na,K-ATPase is associated with a single span protein, the gamma subunit (FXYD2). Two splice variants are differentially expressed along the nephron and have a differential influence on Na,K-ATPase when stably expressed in mammalian cells in culture. Here we used a combination of gene induction and gene silencing techniques to test the functional impact of gamma by means other than transfection. NRK-52E cells (of proximal tubule origin) do not express gamma as a protein under regular tissue culture conditions. However, when they were exposed to hyperosmotic medium, induction of only the gammaa splice variant was observed, which was accompanied by a reduction in the rate of cell division. Kinetic analysis of stable enzyme properties from control (alpha1beta1) and hypertonicity-treated cultures (alpha1beta1gammaa) revealed a significant reduction (up to 60%) of Na,K-ATPase activity measured under V(max) conditions with little or no change in the amounts of alpha1beta1. This effect as well as the reduction in cell growth rate was practically abolished when gamma expression was knocked down using specific small interfering RNA duplexes. Surprisingly, a similar induction of endogenous gammaa because of hypertonicity was seen in rat cell lines of other than renal origin: C6 (glioma), PC12 (pheochromocytoma), and L6 (myoblasts). Furthermore, exposure of NRK-52E cells to other stress inducers such as heat shock, exogenous oxidation, and chemical stress also resulted in a selective induction of gammaa. Taken together, the data imply that induction of gammaa may have adaptive value by being a part of a general cellular response to genotoxic stress.     

Functional role of oxygen-containing residues in the fifth transmembrane segment of the Na,K-ATPase alpha subunit

The functional roles of Tyr771, Thr772, and Asn776 in the fifth transmembrane segment of the Na, K-ATPase alpha subunit were studied using site-directed mutagenesis, expression, and kinetics analysis. Nonconservative replacements Thr772Tyr and Asn776Ala led to reduced Na,K-ATPase turnover. Replacements at these positions (Asn776Ala, Thr772Leu, and Thr772Tyr) also led to high Na-ATPase activity (in the absence of K+). However, Thr772- and Asn776-substituted enzymes showed only small alterations in the apparent Na+ and K+ affinities (K1/2 for Na,K-ATPase activation). Thus, the high Na-ATPase activity does not appear related to cation-binding alterations. It is probably associated with conformational alterations which lead to an acceleration of enzyme dephosphorylation by Na+ acting at the extracellular space (Argüello et al. J. Biol. Chem. 271, 24610-24616, 1996). Nonconservative substitutions at position 771 (Tyr771Ala and Tyr771Ser) produced a significant decrease of enzyme turnover. Enzyme-Na+ interaction was greatly changed in these enzymes, while their activation by K+ did not appear affected. Although the Na+ K1/2 for Na,K-ATPase stimulation was unchanged (Tyr771Ala, Tyr771Ser), the activation by this cation showed no cooperativity (Tyr771Ala, nHill = 0.75; Tyr771Ser, nHill = 0.92; Control, nHill = 2.28). Substitution Tyr771Phe did not lead to a significant reduction in the cooperativity of the ATPase Na+ dependence (nHill = 1.91). All Tyr771-substituted enzymes showed low steady-state levels of phosphoenzyme during Na-activated phosphorylation by ATP. Phosphorylation levels were not increased by oligomycin, although the drug bound and inactivated Tyr771-substituted enzymes. No E1 left and right arrow E2 equilibrium alterations were detected using inhibition by vanadate as a probe. The data suggest that Tyr771 might play a central role in Na+ binding and occlusion without participating in K+-enzyme interactions.     

The role of the beta1 subunit of the Na,K-ATPase and its glycosylation in cell-cell adhesion

Based on recent data showing that overexpression of the Na,K-ATPase beta(1) subunit increased cell-cell adhesion of nonpolarized cells, we hypothesized that the beta(1) subunit can also be involved in the formation of cell-cell contacts in highly polarized epithelial cells. In support of this hypothesis, in Madin-Darby canine kidney (MDCK) cells, the Na,K-ATPase alpha(1) and beta(1) subunits were detected as precisely co-localized with adherens junctions in all stages of the monolayer formation starting from the initiation of cell-cell contact. The Na,K-ATPase and adherens junction protein, beta-catenin, stayed partially co-localized even after their internalization upon disruption of intercellular contacts by Ca(2+) depletion of the medium. The Na,K-ATPase subunits remained co-localized with the adherens junctions after detergent treatment of the cells. In contrast, the heterodimer formed by expressed unglycosylated Na,K-ATPase beta(1) subunit and the endogenous alpha(1) subunit was easily dissociated from the adherens junctions and cytoskeleton by the detergent extraction. The MDCK cell line in which half of the endogenous beta(1) subunits in the lateral membrane were substituted by unglycosylated beta(1) subunits displayed a decreased ability to form cell-to-cell contacts. Incubation of surface-attached MDCK cells with an antibody against the extracellular domain of the Na,K-ATPase beta(1) subunit specifically inhibited cell-cell contact formation. We conclude that the Na,K-ATPase beta(1) subunit is involved in the process of intercellular adhesion and is necessary for association of the heterodimeric Na,K-ATPase with the adherens junctions. Further, normal glycosylation of the Na,K-ATPase beta(1) subunit is essential for the stable association of the pump with the adherens junctions and plays an important role in cell-cell contact formation.