The polarized distribution of Na⁺,K⁺-ATPase plays a paramount physiological role, because either directly or through coupling with co- and countertransporters, it is responsible for the net movement of, for example, glucose, amino acids, Ca²⁺, K⁺, Cl^-, and CO₃H^-across the whole epithelium. We report here that the β-subunit is a key factor in the polarized distribution of this enzyme. 1) Madin-Darby canine kidney (MDCK) cells (epithelial from dog kidney) express the Na⁺,K⁺-ATPase over the lateral side, but not on the basal and apical domains, as if the contact with a neighboring cell were crucial for the specific membrane location of this enzyme. 2) MDCK cells cocultured with other epithelial types (derived from human, cat, dog, pig, monkey, rabbit, mouse, hamster, and rat) express the enzyme in all (100%) homotypic MDCK/MDCK borders but rarely in heterotypic ones. 3) Although MDCK cells never express Na⁺,K⁺-ATPase at contacts with Chinese hamster ovary (CHO) cells, they do when CHO cells are transfected with β₁-subunit from the dog kidney (CHO-β). 4) This may be attributed to the adhesive property of the β₁-subunit, because an aggregation assay using CHO (mock-transfected) and CHO-β cells shows that the expression of dog β₁-subunit in the plasma membrane does increase adhesiveness. 5) This adhesiveness does not involve adherens or tight junctions. 6) Transfection of β₁-subunit forces CHO-β cells to coexpress endogenous α-subunit. Together, our results indicate that MDCK cells express Na⁺,K⁺-ATPase at a given border provided the contacting cell expresses the dog β₁-subunit. The cell–cell interaction thus established would suffice to account for the polarized expression and positioning of Na⁺,K⁺-ATPase in epithelial cells.

The sodium pump, or Na⁺/K⁺-ATPase (NKA), is an essential enzyme found in the plasma membrane of all animal cells. Its primary role is to transport sodium (Na⁺) and potassium (K⁺) ions across the cell membrane, using energy from ATP hydrolysis. This transport creates and maintains an electrochemical gradient, which is crucial for various cellular processes, including cell volume regulation, electrical excitability, and secondary active transport. Although the role of NKA as a pump was discovered and demonstrated several decades ago, it remains the subject of intense research. Current studies aim to delve deeper into several aspects of this molecular entity, such as describing its structure and mode of operation in atomic detail, understanding its molecular and functional diversity, and examining the consequences of its malfunction due to structural alterations. Additionally, researchers are investigating the effects of various substances that amplify or decrease its pumping activity. Beyond its role as a pump, growing evidence indicates that in various cell types, NKA also functions as a receptor for cardiac glycosides like ouabain. This receptor activity triggers the activation of various signaling pathways, producing significant morphological and physiological effects. In this report, we present the results of a comprehensive review of the most outstanding studies of the past five years. We highlight the progress made regarding this new concept of NKA and the various cardiac glycosides that influence it. Furthermore, we emphasize NKA’s role in epithelial physiology, particularly its function as a receptor for cardiac glycosides that trigger intracellular signals regulating cell–cell contacts, proliferation, differentiation, and adhesion. We also analyze the role of NKA β-subunits as cell adhesion molecules in glia and epithelial cells.

The very existence of higher metazoans depends on the vectorial transport of substances across epithelia. A crucial element of this transport is the membrane enzyme Na⁺,K⁺-ATPase. Not only is this enzyme distributed in a polarized manner in a restricted domain of the plasma membrane but also it creates the ionic gradients that drive the net movement of glucose, amino acids, and ions across the entire epithelium. In a previous work, we have shown that Na⁺,K⁺-ATPase polarity depends on interactions between the β subunits of Na⁺,K⁺-ATPases located on neighboring cells and that these interactions anchor the entire enzyme at the borders of the intercellular space. In the present study, we used fluorescence resonance energy transfer and coprecipitation methods to demonstrate that these β subunits have sufficient proximity and affinity to permit a direct interaction, without requiring any additional extracellular molecules to span the distance.

Na+, K+-ATPase, or the Na+ pump, is a key component in the maintenance of the epithelial phenotype. In most epithelia, the pump is located in the basolateral domain. Studies from our laboratory have shown that the β1 subunit of Na+, K+-ATPase plays an important role in this mechanism because homotypic β1-β1 interactions between neighboring cells stabilize the pump in the lateral membrane. However, in the retinal pigment epithelium (RPE), the Na+ pump is located in the apical domain. The mechanism of polarization in this epithelium is unclear. We hypothesized that the apical polarization of the pump in RPE cells depends on the expression of its β2 subunit. ARPE-19 cells cultured for up to 8 weeks on inserts did not polarize, and Na+, K+-ATPase was expressed in the basolateral membrane. In the presence of insulin, transferrin and selenic acid (ITS), ARPE-19 cells cultured for 4 weeks acquired an RPE phenotype, and the Na+ pump was visible in the apical domain. Under these conditions, Western blot analysis was employed to detect the β2 isoform and immunofluorescence analysis revealed an apparent apical distribution of the β2 subunit. qPCR results showed a time-dependent increase in the level of β2 isoform mRNA, suggesting regulation at the transcriptional level. Moreover, silencing the expression of the β2 isoform in ARPE-19 cells resulted in a decrease in the apical localization of the pump, as assessed by the mislocalization of the α2 subunit in that domain. Our results demonstrate that the apical polarization of Na+, K+-ATPase in RPE cells depends on the expression of the β2 subunit.

Adhesion is a crucial characteristic of epithelial cells to form barriers to pathogens and toxic substances from the environment. Epithelial cells attach to each other using intercellular junctions on the lateral membrane, including tight and adherent junctions, as well as the Na⁺,K⁺-ATPase. Our group has shown that non-adherent chinese hamster ovary (CHO) cells transfected with the canine β₁ subunit become adhesive, and those homotypic interactions amongst β₁ subunits of the Na⁺,K⁺-ATPase occur between neighboring epithelial cells. Ouabain, a cardiotonic steroid, binds to the α subunit of the Na⁺,K⁺-ATPase, inhibits the pump activity and induces the detachment of epithelial cells when used at concentrations above 300 nM. At nanomolar non-inhibiting concentrations, ouabain affects the adhesive properties of epithelial cells by inducing the expression of cell adhesion molecules through the activation of signaling pathways associated with the α subunit. In this study, we investigated whether the adhesion between β₁ subunits was also affected by ouabain. We used CHO fibroblasts stably expressing the β₁ subunit of the Na⁺,K⁺-ATPase (CHO β₁), and studied the effect of ouabain on cell adhesion. Aggregation assays showed that ouabain increased the adhesion between CHO β₁ cells. Immunofluorescence and biotinylation assays showed that ouabain (50 nM) increases the expression of the β₁ subunit of the Na⁺,K⁺-ATPase at the cell membrane. We also examined the effect of ouabain on the activation of signaling pathways in CHO β₁ cells, and their subsequent effect on cell adhesion. We found that cSrc is activated by ouabain and, therefore, that it likely regulates the adhesive properties of CHO β₁ cells. Collectively, our findings suggest that the β₁ subunit adhesion is modulated by the expression levels of the Na⁺,K⁺-ATPase at the plasma membrane, which is regulated by ouabain.

The β₂ subunit of Na⁺, K⁺-ATPase was originally identified as the adhesion molecule on glia (AMOG) that mediates the adhesion of astrocytes to neurons in the central nervous system and that is implicated in the regulation of neurite outgrowth and neuronal migration. While β₁ isoform have been shown to trans-interact in a species-specific mode with the β₁ subunit on the epithelial neighboring cell, the β₂ subunit has been shown to act as a recognition molecule on the glia. Nevertheless, none of the works have identified the binding partner of β₂ or described its adhesion mechanism. Until now, the interactions pronounced for β₂/AMOG are heterophilic cis-interactions. In the present report we designed experiments that would clarify whether β₂ is a cell–cell homophilic adhesion molecule. For this purpose, we performed protein docking analysis, cell–cell aggregation, and protein–protein interaction assays. We observed that the glycosylated extracellular domain of β₂/AMOG can make an energetically stable trans-interacting dimer. We show that CHO (Chinese Hamster Ovary) fibroblasts transfected with the human β₂ subunit become more adhesive and make large aggregates. The treatment with Tunicamycin in vivo reduced cell aggregation, suggesting the participation of N-glycans in that process. Protein–protein interaction assay in vivo with MDCK (Madin-Darby canine kidney) or CHO cells expressing a recombinant β₂ subunit show that the β₂ subunits on the cell surface of the transfected cell lines interact with each other. Overall, our results suggest that the human β₂ subunit can form trans-dimers between neighboring cells when expressed in non-astrocytic cells, such as fibroblasts (CHO) and epithelial cells (MDCK).