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The Na+-K+-ATPase is specifically inhibited by cardiac glycosides, some of which may also function as endogenous mammalian hormones. Previous studies using Xenopus oocytes, yeast cells, or purified isoforms demonstrated that affinities of various cardiac glycosides for three isoforms of the Na+-K+-ATPase (α1-α3 α1) may differ, a finding with potential clinical implication. The present study investigates isoform selectivity and effects of cardiac glycosides on cultured mammalian cells under more physiological conditions. H1299 cells (non-small cell lung carcinoma) were engineered to express only one α-isoform (α1, α2, or α3) by combining stable transfection of isoforms and silencing endogenous α1. Cardiac glycoside binding was measured by displacement of bound ³H-ouabain. The experiments confirm moderate α1/α3: α2 selectivity of ouabain, moderate α2: α1 selectivity of digoxin, and enhanced α2: α1 selectivity of synthetic derivatives (Katz A, Tal DM, Heller D, Haviv H, Rabah B, Barkana Y, Marcovich AL, Karlish SJD. J Biol Chem 289: 21153-21162, 2014). Relative α2: α1 selectivity of digoxin vs. ouabain was also manifested by enhanced internalization of α2 in response to digoxin. Cellular proliferation assays of H1299 cells confirmed the patterns of α2: α1 selectivity for ouabain, digoxin, and a synthetic derivative and reveal a crucial role of surface pump density on sensitivity to cardiac glycosides. Because cardiac glycosides are being considered as drugs for treatment of cancer, effects of ouabain on proliferation of 12 cancer and noncancer cell lines, with variable plasma membrane expression of α1, have been tested. These demonstrated that sensitivity to ouabain indeed depends linearly on the plasma membrane surface density of Na α-K α-ATPase irrespective of status, malignant or nonmalignant. [ABSTRACT FROM AUTHOR]

FXYD5 (dysadherin or also called a related to ion channel, RIC) is a transmembrane auxiliary subunit of the Na+-K+-ATPase shown to increase its maximal velocity (Vmax). FXYD5 has also been identified as a cancer-associated protein whose expression in tumor-derived cell lines impairs cytoskeletal organization and increases cell motility. Previously, we have demonstrated that the expression of FXYD5 in M1 cells derived from mouse kidney collecting duct impairs the formation of tight and adherence junctions. The current study aimed to further explore effects of FXYD5 at a single cell level. It was found that in M1, as well as three other cell lines, FXYD5 inhibits transformation of adhered single cells from the initial radial shape to a flattened, elongated shape in the first stage of monolayer formation. This is also correlated to less ordered actin cables and fewer focal points. Structure- function analysis has demonstrated that the transmembrane domain of FXYD5, and not its unique extracellular segment, mediates the inhibition of change in cell shape. This domain has been shown before to be involved in the association of FXYD5 with the Na+-K+- ATPase, which leads to the increase in Vmax. Furthermore, specific transmembrane point mutations in FXYD5 that either increase or decrease its effect on cell elongation had a corresponding effect on the coimmunoprecipitation of FXYD5 with ɑ Na+-K+-ATPase. These findings lend support to the possibility that FXYD5 affects cell polarization through its transmembrane domain interaction with the Na+-K+-ATPase. Yet interaction of FXYD5 with other proteins cannot be excluded. [ABSTRACT FROM AUTHOR]

FXYD5 (dysadherin or RIC) is a member of the FXYD family of single-span transmembrane proteins associated with the Na+-K+-ATPase. Several studies have demonstrated enhanced expression of FXYD5 during metastasis and effects on cell adhesion and motility. The current study examines effects of FXYD5 on the paracellular permeability in the mouse kidney collecting duct cell line M1. Expressing FXYD5 in these cells leads to a large decrease in amiloride-insensitive transepithelial electrical resistance as well as increased permeability to 4-kDa dextran. Impairment of cell-cell contact was also demonstrated by staining cells for the tight and adherence junction markers zonula occludens-1 and β-catenin, respectively. This is further supported by large expansions of the interstitial spaces, visualized in electron microscope images. Expressing FXYD5 in M1 cells resulted in a decrease in N-glycosylation of β1 Na+-K+-ATPase, while silencing it in H1299 cells had an opposite effect. This may provide a mechanism for the above effects, since normal glycosylation of β1 plays an important role in cell-cell contact formation (Vagin O, Tokhtaeva E, Sachs G. J Biol Chem 281: 39573-39587, 2006). [ABSTRACT FROM AUTHOR]

FXYD5 is a member of a family of tissue-specific regulators of the Na+-K+-ATPase expressed in kidney tubules. Previously, we have shown that FXYD5 interacts with the αβ-subunits of the Na+-K+-ATPase and increases its Vmax, (Lubarski I, Pihakaski-Maunsbach K, Karlish SJ, Maunsbach AB, Garty H. J Biol Chern 280: 37717-37724, 2005). The current study further characterizes structural interaction and structure-function relationships of FXYD5. FXYD5/FXYD4 chimeras expressed in Xenopus laevis oocytes have been used to demonstrate that both the high-affinity association with the pump and the increase in Vmax are mediated by the transmembrane domain of FXYD5. Several amino acids that participate in the high-affinity interaction between FXYD5 and the α-subunit of the Na+-K+-ATPase have been identified. The data suggest that different FXYD proteins interact similarly with the Na+-K+-ATPase and their transmembrane domains play a key role in both the structural interactions and functional effects. Other experi- ments have identified at least one splice variant of FXYD5 with 10 additional amino acids at the COOH terminus, suggesting the possi- bility of other functional effects not mediated by the transmembrane domain. FXYD5 could be specifically bound to wheat germ agglutinin beads, indicating that it is glycosylated. However, unlike previous findings in metastatic cells, such glycosylation does not evoke a large increase in the size of the protein expressed in native epithelia and X. laevis oocytes. [ABSTRACT FROM AUTHOR]

Recently, corticosteroid hormone-induced factor (CHIF) and the ?-subunit, two members of the FXYD family of small proteins, have been identified as regulators of renal Na,K-ATPase. In this study, we have investigated the tissue distribution and the s [ABSTRACT FROM AUTHOR]

The biological role of small membrane proteins of the new FXYD family is largely unknown. The best characterized FXYD protein is the σ-subunit of the Na,K-ATPase (NKA) that modulates the Na,K-pump function in the kidney. Here, we report that, similarly to σa and σb splice variants, the FXYD protein CHIF (corticosteroid-induced factor) is a type I membrane protein which is associated with NKA in renal tissue, and modulates the Na,K-pump transport when expressed in Xenopus oocytes. In contrast to σa and &sigmab, which both decrease the apparent Na+ affinity of the Na,K-pump, CHIF significantly increases the Na+ affinity and decreases the apparent K+ affinity due to an increased Na+ competition at external binding sites. The extracytoplasmic FXYD motif is required for stable σ-subunit and CHIF interaction with NKA, white cytoplasmic, positively charged residues are necessary for the σ-subunit's association efficiency and for CHIF's functional effects. These data document that CHIF is a new tissue-specific regulator of NKA which probably plays a crucial rote in aldosterone- responsive tissues responsible for the maintenance of body Na+ and K+ homeostasis. [ABSTRACT FROM AUTHOR]

Presents a study which discussed the in vitro phosphorylation of the COOH termini of epithelial sodium channel subunits expressed as glutathione S-transferase fusion proteins. Details on the mediation of the active sodium reabsorption in kidney collecting duct by an apical sodium channel; Materials and methods used; Results.

Regulation of the epithelial Na+ channel by aldosterone: Open questions and emerging answers. Aldosterone is the principal adrenal steroid controlling Na+ retention in amphibians and mammalians. It acts primarily by increasing the apical Na+ permeability through activation of the epithelial Na+ channel (ENaC). The cellular events mediating the hormonal action are mostly unknown. Early studies have provided evidence that the hormone functions to activate or translocate pre-existing channels by a yet undefined mechanism. In addition, enhanced de novo channel synthesis appears to take place as well. The molecular cloning of the three ENaC subunits has provided new powerful tools for testing and confirming this hypothesis, as well as for characterizing mechanisms by which ENaC is regulated. Another important development is the recent identification of several cDNAs corresponding to aldosterone-induced and suppressed mRNAs. The study of these genes and their putative interactions with ENaC is likely to provide important clues to the mechanisms by which aldosterone controls the apical Na+ permeability of tight epithelia. This article reviews recent developments in the field that may lead to the elucidation of the mechanisms by which the hormone controls Na+ transport. [ABSTRACT FROM AUTHOR]

Inside-out cell envelopes from Halobacterium halobium R1M1 have been isolated by sonication and subsequent sucrose-density-gradient fractionation. The inverted cell envelopes transport protons from the exterior of the vesicles by a light-dependent, uncoupler-inhibited process. Enzymes usually facing the inside of the cell are exposed to the external medium in this preparation. [ABSTRACT FROM AUTHOR]