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.

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