Carol Asher

Direct inhibition of epithelial Na+ channels by a pH-dependent interaction with calcium, and by other divalent ions

SummaryDirect inhibitory effects of Ca2+ and other ions on the epithelial Na+ channels were investigated by measuring the amiloride-blockable22Na+ fluxes in toad bladder vesicles containing defined amounts of mono- and divalent ions. In agreement with a previous report (H.S. Chase, Jr., and Q. Al-Awqati,J. Gen. Physiol.81:643–666, 1983) we found that the presence of micromolar concentrations of Ca2+ in the internal (cytoplasmic) compartment of the vesicles substantially lowered the channel-mediated fluxes. This inhibition, however, was incomplete and at least 30% of the amiloride-sensitive22Na+ uptake could not be blocked by Ca2+ (up to 1mm). Inhibition of channels could also be induced by millimolar concentrations of Ba2+, Sr2+, or VO2+, but not by Mg2+. The Ca2+ inhibition constant was a strong function of pH, and varied from 0.04 μm at pH 7.8 to >10 μm at pH 7.0 Strong pH effects were also demonstrated by measuring the pH dependence of22Na+ uptake in vesicles that contained 0.5 μm Ca2+. This Ca2+ activity produced a maximal inhibition of22Na+ uptake at pH≥7.4 but had no effect at pH≤7.0. The tracer fluxes measured in the absence of Ca2+ were pH independent over this range. The data is compatible with the model that Ca2+ blocks channels by binding to a site composed of several deprotonated groups. The protonation of any one of these groups prevents Ca2+ from binding to this site but does not by itself inhibit transport. The fact that the apical Na+ conductance in vesicles, can effectively be modulated by minor variations of the internal pH near the physiological value, raises the possibility that channels are being regulated by pH changes which alter their apparent affinity to cytoplasmic Ca2+, rather than, or in addition to changes in the cytoplasmic level of free Ca2+.

Characterization of the interactions between Nedd4-2, ENaC, and sgk-1 using surface plasmon resonance

Previous studies have characterized interactions between the ubiquitin ligase Nedd4-1 and the epithelial Na(+) channel (ENaC). Such interactions control the channel cell surface expression and activity. Recently, evidence has been provided that a related protein, termed Nedd4-2, is likely to be the true physiological regulator of the channel. Unlike Nedd4-1, Nedd4-2 also interacts with the aldosterone-induced channel activating kinase sgk-1. The current study uses surface plasmon resonance to quantify the binding of the four WW domains of Nedd4-2 to synthetic peptides corresponding to the PY motifs of ENaC and sgk-1. The measurements demonstrate that WW3 and WW4 are the only Nedd4-2 domains interacting with both ENaC and sgk-1 and that their binding constants are in the 1-6 microM range.

Expression of amiloride-sensitive Na+ channels of hen lower intestine in Xenopus oocytes: electrophysiological studies on the dependence of varying NaCl intake.

Epithelial Na+ channels were incorporated into the plasma membrane of Xenopus laevis oocytes after micro-injection of RNA from hen lower intestinal epithelium (colon and coprodeum). The animals were fed either a normal poultry food which contained NaCl (HS), or a similar food devoid of NaCl (LS). Oocytes were monitored for the expression of amiloride-sensitive sodium channels by measuring membrane potentials and currents. Oocytes injected with poly(A)+RNA prepared from HS animals or non-injected control oocytes showed no detectable sodium currents, whereas oocytes injected with LS-poly(A)+RNA had large amiloride-blockable sodium currents. These currents were almost completely saturated by sodium concentrations of 20 mM with a Km of about 2.6 mM sodium. Amiloride (10 microM) inhibits the expressed sodium channels entirely and examination of dose response relationships yielded a half-maximal inhibition concentration (Ki) of 120 nM amiloride. I-V difference curves in the presence or absence of sodium or amiloride (10 microM) indicate a potential dependence of the sodium transport which can be described by the Goldman equation. When Na+ is replaced by K+, no amiloride response was detected indicating a high selectivity for Na+ over K+. These results provide strong evidence that intestinal Na+ channels are regulated by dietary salt intake on the RNA level.

FXYD5 (dysadherin) regulates the paracellular permeability in cultured kidney collecting duct cells.

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).

A Specific Functional Interaction between CHIF and Na,K-ATPase

Abstract: CHIF (corticosteroid hormone‐induced factor) is a member of the FXYD family that shares ∼50% homology with the γ subunit of Na,K‐ATPase. It is expressed in renal collecting duct and distal colon, and is upregulated by Na+ deprivation and high K+ diet. Both CHIF and γ are coimmunoprecipitated by an anti‐α subunit antibody, and α is immunoprecipitated by anti‐γ and anti‐CHIF antibodies. 86Rb+ flux experiments in CHIF‐transfected HeLa cells demonstrate that CHIF increases the affinity for cytoplasmic Na+, but does not affect the affinity for extracellular K(Rb). A physiological role of CHIF in kidney function is further elucidated by the phenotypic analysis of CHIF knockout mice. Taken together with data by others, it appears that FXYD proteins are tissue‐specific subunits or regulators of the Na,K‐ATPase whose function is to adjust the pump kinetics to particular physiological needs.

Induction of FKBP51 by aldosterone in intestinal epithelium.

Screening female rat distal colon preparations for aldosterone-induced genes identified the Hsp90-binding immunophilin FKBP51 as a major aldosterone-induced mRNA and protein. Limited induction of FKBP51 was observed also in other aldosterone-responsive tissues such as kidney medulla and heart. Ex vivo measurements in colonic tissue have characterized time course, dose response and receptor specificity of the induction of FKBP51. FKBP51 mRNA and protein were strongly up regulated by physiological concentrations of aldosterone in a late (greater than 2.5h) response to the hormone. Maximal increase in FKBP51 mRNA requires aldosterone concentrations that are higher than those needed to fully occupy the mineralocorticoid receptor (MR). Yet, the response is fully inhibited by the MR antagonist spironolactone and not inhibited and even stimulated by the glucocorticoid receptor (GR) antagonist RU486. These and related findings cannot be explained by a simple activation and dimerization of either MR or GR but are in agreement with response mediated by an MR-GR heterodimer. Overexpression or silencing FKBP51 in the kidney collecting duct cell line M1 had little or no effect on the aldosterone-induced increase in transepithelial Na(+) transport.

Intracellular Trafficking of FXYD1 (Phospholemman) and FXYD7 Proteins in Xenopus Oocytes and Mammalian Cells

Background: FXYD proteins are auxiliary subunits of the Na+/K+ ATPase that modulate its kinetic properties. Results: FXYD1 can be expressed in the plasma membrane only when coexpressed with Na+/K+ ATPase whereasFXYD7 may reach the membrane without the Na+/K+ ATPase. Conclusion: O-glycosylation of FXYD7 in the Golgi is required for its surface expression. Significance: FXYD7 and FXYD1 have very different trafficking behavior. FXYD proteins are a group of short single-span transmembrane proteins that interact with the Na+/K+ ATPase and modulate its kinetic properties. This study characterizes intracellular trafficking of two FXYD family members, FXYD1 (phospholemman (PLM)) and FXYD7. Surface expression of PLM in Xenopus oocytes requires coexpression with the Na+/K+ ATPase. On the other hand, the Na+/Ca2+ exchanger, another PLM-interacting protein could not drive it to the cell surface. The Na+/K+ ATPase-dependent surface expression of PLM could be facilitated by either a phosphorylation-mimicking mutation at Thr-69 or a truncation of three terminal arginine residues. Unlike PLM, FXYD7 could translocate to the cell surface of Xenopus oocytes independently of the coexpression of α1β1 Na+/K+ ATPase. The Na+/K+ ATPase-independent membrane translocation of FXYD7 requires O-glycosylation of at least two of three conserved threonines in its ectodomain. Subsequent experiments in mammalian cells confirmed the role of conserved extracellular threonine residues and demonstrated that FXYD7 protein, in which these have been mutated to alanine, is trapped in the endoplasmic reticulum and Golgi apparatus.

Modulation of cell polarization by the Na+-K+-ATPase-associated protein FXYD5 (dysadherin)

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 α 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.

FXYD5 Protein Has a Pro-inflammatory Role in Epithelial Cells.

The FXYD proteins are a family of small membrane proteins that share an invariant four amino acid signature motif F-X-Y-D and act as tissue-specific regulatory subunits of the Na,K-ATPase. FXYD5 (also termed dysadherin or RIC) is a structurally and functionally unique member of the FXYD family. As other FXYD proteins, FXYD5 specifically interacts with the Na,K-ATPase and alters its kinetics by increasing Vmax. However, unlike other family members FXYD5 appears to have additional functions, which cannot be readily explained by modulation of transport kinetics. Knockdown of FXYD5 in MDA-MB-231 breast cancer cells largely decreases expression and secretion of the chemokine CCL2 (MCP-1). A related effect has also been observed in renal cell carcinoma cells. The current study aims to further characterize the relationship between the expression of FXYD5 and CCL2 secretion. We demonstrate that transfection of M1 epithelial cell line with FXYD5 largely increases lipopolysaccharide (LPS) stimulated CCL2 mRNA and secretion of the translated protein. We have completed a detailed analysis of the molecular events leading to the above response. Our key findings indicate that FXYD5 generates a late response by increasing the surface expression of the TNFα receptor, without affecting its total protein level, or mRNA transcription. LPS administration to mice demonstrates induced secretion of CCL2 and TNFα in FXYD5-expressing lung peripheral tissue, which suggests a possible role for FXYD5 in normal epithelia during inflammation.

FXYD5 (dysadherin) may mediate metastatic progression through regulation of the β-Na+-K+-ATPase subunit in the 4T1 mouse breast cancer model

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.