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The physiological implications of interaction of human [alpha]1 and [alpha]2 isoforms of Na,K-ATPase with phospholipids and FXYD1 are discussed.

FXYD5 is a member of a family of tissue-specific regulators of the [Na.sup.+]-[K.sup.+]-ATPase expressed in kidney tubules. Previously, we have shown that FXYD5 interacts with the [alpha][beta]-subunits of the [Na.sup.+]-[K.sup.+]-ATPase and increases its [V.sub.max] (Lubarski I, Pihakaski-Maunsbach K, Karlish SJ, Maunsbach AB, Garty H. J Biol Chem 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 [V.sub.max] are mediated by the transmembrane domain of FXYD5. Several amino acids that participate in the high-affinity interaction between FXYD5 and the [alpha]-subunit of the [Na.sup.+]-[K.sup.+]-ATPase have been identified. The data suggest that different FXYD proteins interact similarly with the [Na.sup.+]-[K.sup.+]-ATPase and their transmembrane domains play a key role in both the structural interactions and functional effects. Other experiments have identified at least one splice variant of FXYD5 with 10 additional amino acids at the COOH terminus, suggesting the possibility 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. RIC; FXYD; glycosylation; sodium pump

The [gamma]-subunit of Na-K-ATPase (FXYD2) and corticosteroid hormone-induced factor (CHIF; FXYD4) are considered pump regulators in kidney tubules. The aim of this study was to expand the information about their locations in the kidney medulla and to evaluate their importance for electrolyte excretion in an animal model. The cellular and subcellular locations and abundances of [gamma] and CHIF in the medulla of control and sodium-depleted rats were analyzed by immunofluorescence and immunoelectron microscopy and semiquantitative Western blotting. The results showed that antibodies against the [gamma]-subunit COOH terminus and splice variant [[gamma].sub.a], but not splice variant [[gamma].sub.b], labeled intercalated cells, but not principal cells, in the initial part of the inner medullary collecting duct (IMCD1). In subsequent segments (IMCD2 and IMCD3), all principal cells exhibited distinct basolateral labeling for both the [gamma]-subunit COOH terminus, splice variant [[gamma].sub.a], and CHIF. Splice variant [[gamma].sub.b] was abundant in the inner stripe of the outer medulla but absent in the inner medulla (IM). Double labeling by high-resolution immunoelectron microscopy showed close structural association between CHIF and the Na-K-ATPase [[alpha].sub.1]-subunit in basolateral membranes. The present observations provide new information about the cellular and subcellular locations of [gamma] and CHIF in the renal medulla and show a new [gamma] variant in the IM. Extensive NaCl depletion did not induce significant changes in the locations or abundances of the [gamma]-subunit COOH terminus and CHIF in different kidney zones. We conclude that the unchanged levels of these two FXYD proteins suggest that they are not primary determinants for urine electrolyte composition during NaCl depletion. Na-K-ATPase [gamma]-subunit; CHIF; kidney medulla

Like the [gamma]-subunit of Na-K-ATPase, the corticosteroid hormone-induced factor (CHIF) is a member of the FXYD family of one-transmembrane-segment proteins. Both CHIF and two splice variants of [gamma], [[gamma].sub.a] and [[gamma].sub.b], are expressed in the kidney. Immunolocalization experiments demonstrate mutually exclusive expression of CHIF and [gamma] in different nephron segments. Specific coimmunoprecipitation experiments demonstrate the existence in kidney membranes of the complexes [alpha]/[beta]/[[gamma].sub.a], [alpha]/[beta]/[[gamma].sub.b], and [alpha]/[beta]/CHIF and exclude mixed complexes such as [alpha]/[beta]/[[gamma].sub.a]/[[gamma].sub.b] and [alpha]/[beta]/[gamma]/CHIF. CHIF has been expressed in HeLa cells harboring the rat [[alpha].sub.1]-subunit of Na-K-ATPase. [sup.86]Rb flux experiments demonstrate that CHIF induces a two-to threefold increase in apparent affinity for cytoplasmic Na ([K'.sub.Na]) but does not affect affinity for extracellular K (Rb) ions ([K'.sub.K]) or [V.sub.max]. Measurements of Na-K-ATPase using isolated membranes show similar but smaller effects of CHIF on [K'.sub.Na], whereas [K'.sub.K] and [K'.sub.ATP] are unaffected. The functional effects of CHIF differ from those of [gamma]. An implication of these findings is that other FXYD proteins could act as tissue-specific modulators of Na-K-ATPase. corticosteroid hormone-induced factor; sodium-potassium-adenosine 5'-triphosphatase; tissue-specific modulator

Corticosteroid hormone-induced factor (CHIF) is a short epithelial-specific protein that is independently induced by aldosterone and a high-[K.sup.+] diet. It is a member of the FXYD family of single-span transmembrane proteins that include phospholemman, Mat-8, and the [gamma]-subunit of [Na.sup.+]-[K.sup.+]-ATPase. A number of studies have suggested that these proteins are involved in the regulation of ion transport and, in particular, functionally interact with the [Na.sup.+]- [K.sup.+]-ATPase. The present study describes the characterization, targeted disruption, and phenotypic analysis of the mouse CHIF gene. The CHIF knockout mice are viable and not distinguishable from wild-type littermates under normal conditions. Under [K.sup.+] loading, they have a twofold higher urine volume and an increased glomerular filtration rate. Similar but smaller effects are observed in mice fed a low-[Na.sup.+] diet. Treating [K.sup.+]-loaded mice for 10 days with furosemide resulted in lethality in the knockout mice (17 of 39) but not in the wild-type group (1 of 39). The data are consistent with an effect of CHIF on the [Na.sup.+]- [K.sup.+]-ATPase that is specific to the outer and inner medullary duct, its major expression site. FXYD proteins; sodium-potassium-adenosine-5'-triphosphatase; [gamma]-subunit; furosemide; corticosteroid hormone-induced factor; aldosterone

The role of the channel-inducing factor or corticosteroid hormone-induced factor (CHIF) in the maintenance of K+ balance was analyzed in adrenal-intact rats that were fed on high-K+ diets. Adrenal-intact and rats fed on high-K+ diets exhibited upregulated CHIF messenger RNA (mRNA) which was mediated by intact adrenal function. Furthermore, the expression of the CHIF was dependent on the secretion of K+.

I. INTRODUCTION The epithelial [Na.sup.+] channel is the constituent of the apical or outward-facing membranes of many salt-reabsorbing epithelia which facilitates [Na.sup.+] movement across this membrane as a first step [...]

The amino acid sequence and structure of the alpha-subunit of the highly Na+-selective amiloride-blockable channel (ENaC) was analyzed by utilizing alpha-subunit ENaC clones (alpha-cENaC) from chicken intestine. The amino acid sequence of the avian alpha-cENaC was similar by 61.7% to rat alpha-ENaC. The avian alpha-cENaC is also composed of a large extracellular loop located between two transmembrane domains with four extracellular N-glycosylation sites.

The effects of isothiouronium derivatives on the epithelial sodium (Na+) channel were investigated by monitoring the initial rate of the channel-mediated Na+ uptake into rat membrane vesicles by 22Na+ flux assay. The results revealed that isothiouronium derivatives inhibit amiloride-sensitive Na+ channels by blocking amiloride binding. The inhibition was attenuated by amiloride but not by Na+.

Gene expression and localization of the channel inducing factor (CHIF) was investigated in rat tissues by in situ hybridization. CHIF mRNA was detected in cells at the distal parts of the nephron and in the epithelial cells of the distal colon. Exposure of the cells in the colon to corticosteroid hormones, sodium restriction, low-potassium diet and metabolic acidosis markedly increased CHIF mRNA expression, whereas metabolic acidosis increased CHIF expression in the kidney.

Northern hybridization analysis shows that CHIF mRNA is distributed abundantly in the papilla, less in the medulla, and hardly in the cortex of rat kidneys. Primary culture from the inner medullary collecting duct shows the presence of high levels of CHIF, a recently cloned, corticosteroid gene which involves K+ channel activities in oocytes. Perfusing of rats with aldosterone or corticosteroid results in an increase in colonic CHIF, whereas the administration of dexamethasone effects little increase. The results indicate that CHIF is mineralocorticoid induced in the colon but constitutively expressed in kidney.

Aldosterone differentially affects the induction of the alpha, beta, and gamma subunits of the rat epithelial Na+ channel (rENaC) in the kidney cortex and distal colon. Aldosterone or dexamethasone treatment produces a small induction of alpha-rENaC in the kidney cortex, while beta- and gamma-rENaC are unaffected. In contrast, dexamethasone or aldosterone increase inductance of beta- and gamma-rENaC in the distal colon, while alpha-rENaC is unaffected. This suggests that the induction of the subunits is involved in the chronic response of the colon to aldosterone.

A study was conducted to identify the mitochondrial oxidative phosphorylation genes whose expression have the potential to induce changes in ion transport. Differential screening of a rat colon cDNA library with labeled cDNA probes identified several cDNAs whose corresponding mRNA exhibited increased expression after exposure to dexamethasone. Partial sequencing detected the genes that code for subunits of cytochrome-c oxidase and 16S mitochondrial rRNA, which are enzymes responsible for oxidative phosphorylation.

Xenopus oocytes injected with cDNA of the bitopic channel-inducing protein, IsK (channel-inducing factor) show a K+-specific channel activity. The properties of this channel such as its activation by membrane depolarization, 8-bromoadenosine 3',5'-cyclic monophosphate and cytoplasmic Ca+ and its inhibition by clofilium and phorbol ester are similar to that seen for IsK. However the structure of the cDNA is different from that of IsK and is similar to that of phospholemma and the gamma subunit of Na+, K+-ATPase.

Analyses of the occupancies of cytosolic type I and type II receptors and the influence of corticoid agonists RU-28362, RU-38486 and RU-26752 on the apical Na+ permeability of toad bladder reveal that the glucocorticoid receptor mediates at least one-third of the natriferic action of aldosterone estimated in the amphibian urinary bladder. A simple receptor occupancy-response approach cannot explain certain evident influences.

Recent findings have suggested the involvement of protein phosphorylation in the regulation of the epithelial [Na.sup.+] channel (ENaC). This study reports the in vitro phosphorylation of the COOH termini of ENaC subunits expressed as glutathione S-transferase fusion proteins. Channel subunits were specifically phosphorylated by kinase-enriched cytosolic fractions derived from rat colon. The phosphorylation observed was not mediated by the serum- and glucocorticoid-regulated kinase sgk. For the [Gamma]-subunit, phosphorylation occurred on a single, well-conserved threonine residue located in the immediate vicinity of the PY motif (T630). The analogous residue on [Beta](S620) was phosphorylated as well. The possible role of [Gamma]T630 and [Beta]S620 in channel function was studied in Xenopus laevis oocytes. Mutating these residues to alanine had no effect on the basal channel-mediated current. They do, however, inhibit the sgk-induced increase in channel activity but only in oocytes that were preincubated in low [Na.sup.+] and had a high basal [Na.sup.+] current. Thus mutating [Gamma]T630 or [Beta]S620 may limit the maximal channel activity achieved by a combination of sgk and low [Na.sup.+]. epithelial sodium channel; serum- and glucocorticoid-dependent kinase; protein kinase; Nedd4; channel phosphorylation

Shi, Haikun, Rivi Levy-Holzman, Francoise Cluzeaud, Nicolette Farman, and Haim Garty. Membrane topology and immunolocalization of CHIF in kidney and intestine. Am J Physiol Renal Physiol 280: F505-F512, 2001.--Corticosteroid hormone-induced factor (CHIF) is an aldosterone-induced gene, the function of which is yet unknown. It is specifically expressed in kidney collecting duct (CD) and distal colon and is upregulated by either [Na.sup.+] deprivation or [K.sup.+] loading. Hence, it may play a role in epithelial electrolyte transport. Previous studies have characterized regulation and tissue distribution of CHIF mRNA but provided no information on the protein itself. The present paper addresses this issue by using Western blotting, immunochemistry, and in vitro translation. CHIF is an ~8-kDa membranal protein, and protease digestion experiments suggest that its COOH tail faces the cell interior. The protein is abundant in distal colon, kidney medulla, and papilla but cannot be detected in a variety of other tissues. Confocal immunocytochemistry demonstrates that CHIF is present in the basolateral membrane of CD principal cells and distal colon surface cells, with occasional intracellular staining. Dexamethasone and low [Na.sup.+] intake increase the abundance of CHIF. Unlike previous Northern data, induction of CHIF protein by low-[Na.sup.+] intake was apparent not only in the distal colon but also in the kidney. corticosteroid hormone-induced factor; phospholemman; gamma-sodium-potassium-adenosine 5'-triphosphatase; collecting duct; membrane topology; aldosterone

Shigaev, Alexander, Carol Asher, Hedva Latter, Haim Garty, and Eitan Reuveny. Regulation of sgk by aldosterone and its effects on the epithelial [Na.sup.+] channel. Am J Physiol Renal Physiol 278: F613-F619, 2000.--Aldosterone is the major corticosteroid regulating [Na.sup.+] absorption in tight epithelia and acts primarily by activating the epithelial [Na.sup.+] channel (ENaC) through unknown induced proteins. Recently, it has been reported that aldosterone induces the serum- and glucocorticoid-dependent kinase sgk and that coexpressing ENaC with this kinase in Xenopus laevis oocytes increases the amiloride-sensitive [Na.sup.+] current (Chen SY, Bhargava A, Mastroberardino L, Meijer OC, Wang J, Buse P, Firestone GL, Verrey F, and Pearce D. Proc Natl Acad Sci USA 96: 2514-2519, 1999). The present study was done to further characterize regulation of sgk by aldosterone in native mammalian epithelia and to examine its effect on ENaC. With both in vivo and in vitro protocols, an almost fivefold increase in the abundance of sgk mRNA has been demonstrated in rat kidney and colon but not in lung. Induction of sgk by aldosterone was detected in kidney cortex and medulla, whereas the papilla expressed a constitutively high level of the kinase. The increase in sgk mRNA was detected as early as 30 min after the hormonal application and was independent of de novo protein synthesis. The observed aldosterone dose-response relationships suggest that the response is mediated, at least in part, by occupancy of the mineralocorticoid receptor. Coexpressing sgk and ENaC in Xenopus oocytes evoked a fourfold increase in the amiloride-blockable [Na.sup.+] channel activity. A point mutation in the [Beta]-subunit known to impair regulation of the channel by Nedd4 (Y618A) had no significant effect on the response to sgk. epithelial sodium channel; Xenopus laevis oocytes; kidney collecting duct

FXYD5 is a Na+-K+-ATPase regulator, expressed in a variety of normal epithelia. In parallel, it has been found to be associated with several types of cancer and effect lethal outcome by promoting metastasis. However, the molecular mechanism underlying FXYD5 mediated invasion has not yet been identified. In this study, using in vivo 4T1 murine breast cancer model, we found that FXYD5-specific shRNA significantly inhibited lung cancer metastasis, without having a substantial effect on primary tumor growth. Our study reveals that FXYD5 participates in multiple stages of metastatic development and exhibits more than one mode of E-cadherin regulation. We provide the first evidence that FXYD5-related morphological changes are mediated through its interaction with Na+-K+-ATPase. Experiments in cultured 4T1 cells have indicated that FXYD5 expression may downregulate the β1 isoform of the pump. This behavior could have implications on both transcellular interactions and intracellular events. Further studies suggest that differential localization of the adaptor protein Annexin A2 in FXYD5-expressing cells may correlate with matrix metalloproteinase 9 secretion and adhesion changes in 4T1 wild-type cells. [ABSTRACT FROM AUTHOR]