The Apical Localization of Na+, K+-ATPase in Cultured Human Retinal Pigment Epithelial Cells Depends on Expression of the β2 Subunit

Abstract

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.

Elucidating the mechanism of action of alpha-1-antitrypsin using retinal pigment epithelium cells exposed to high glucose. Potential use in diabetic retinopathy

“…E-Cadherin levels in RPE not only have an impact on the EMT process, but also are important to maintain NKA apical polarity [75]. NKA distribution and stabilization of its localization are essential to normal function of RPE [76]. Furthermore, since NKA activity is also affected by high glucose concentrations on the RPE [77], changes on its expression or localization could affect NKA activity.…”

CrossTalk proposal: Apical NKCC1 of choroid plexus epithelial cells works in the net inward flux mode under basal conditions, maintaining intracellular Cl− and cell volume

“…1991; Joseph & Miller, 1991; Lobato‐Alvarez et al . 2016), the direction of basal net ion fluxes mediated by apical NKCC1 and associated water fluxes in CPECs is controversial, and the cotransporter function unclear. There are two opposing viewpoints: one proposes that under basal conditions apical NKCC1 works in the net efflux mode, cotransporting ions and water, directly contributing to sustained CSF production (Steffensen et al .…”

Apical CLC‐2 in retinal pigment epithelium is crucial for survival of the outer retina

“…For example, Na⁺/K⁺‐ATPase and MCT1 are apical in RPE cells, whereas they are targeted to the basolateral membrane in epithelia of the kidney and small intestine 13,43 . In the case of Na⁺/K⁺‐ATPase, the change in localization appears to depend on the glycosylation of the beta subunit associated with the functional alpha subunit 44 . In the case of MCT1, the change in localization from basolateral in kidney cells to apical in RPE cells depends on the lack of recognition of basolateral signals in basigin, the chaperone protein of MCT transporters 45 .…”

“…13,43 In the case of Na⁺/K⁺-ATPase, the change in localization appears to depend on the glycosylation of the beta subunit associated with the functional alpha subunit. 44 In the case of MCT1, the change in localization from basolateral in kidney cells to apical in RPE cells depends on the lack of recognition of basolateral signals in basigin, the chaperone protein of MCT transporters. 45 The signals that drive CLC-2 to the apical surface of RPE cells are still F I G U R E 7 ERG measurements in lentiviral-transduced and untransduced eyes in CLC-2 −/− mice.…”