Jean-Daniel Horisberger

Phosphorylation of Nedd4‐2 by Sgk1 regulates epithelial Na+ channel cell surface expression

The epithelial Na+ channel (ENaC) plays an essential role in the regulation of whole body Na+ balance and blood pressure. The cell surface expression of this channel, a complex of three subunits (α, β and γENaC), has been shown to be regulated by hormones such as aldosterone and vasopressin and by intracellular signaling, including ubiquitylation and/or phosphorylation. However, the molecular mechanisms involving phosphorylation in the regulation of ENaC are unclear. Here we show by expression studies in Xenopus laevis oocytes that the aldosterone‐induced Sgk1 kinase interacts with the ubiquitin protein ligase Nedd4‐2 in a PY motif‐dependent manner and phosphorylates Nedd4‐2 on Ser444 and, to a lesser extent, Ser338. Such phosphorylation reduces the interaction between Nedd4‐2 and ENaC, leading to elevated ENaC cell surface expression. These data show that phosphorylation of an enzyme involved in the ubiquitylation cascade (Nedd4‐2) controls cell surface density of ENaC and propose a paradigm for the control of ion channels. Moreover, they suggest a novel and complete signaling cascade for aldosterone‐dependent regulation of ENaC.

An epithelial serine protease activates the amiloride-sensitive sodium channel

Sodium balance, and ultimately blood pressure and extracellular fluid volume, is maintained by precise regulation of the activity of the epithelial sodium channel (ENaC). In a Xenopus kidney epithelial cell line (A6), exposure of the apical membrane to theprotease inhibitor aprotinin reduces transepithelial sodium transport. Sodium-channel activity can be restored by subsequent exposure to the nonspecific protease trypsin. Using A6 cells and a functional complementation assay to detect increases in ENaC activity, we have cloned a 329-residue protein belonging to the serine protease family. We show that coexpression of this protein with ENaC in Xenopus oocytes increases the activity of the sodiumchannel by two- to threefold. This channel-activating protease (CAP1) is expressed in kidney, gut, lung, skin and ovary. Sequence analysis predicts that CAP1 is a secreted and/or glycosylphosphatidylinositol-anchored protein: ENaC activity would thus be regulated by the activity of a protease expressed at the surface of the same cell. This previously undiscovered mechanism for autocrine regulation may apply to other ion channels, in particular to members of the ENaC family that are present in neurons and epithelial cells.

Defective regulation of the epithelial Na+ channel by Nedd4 in Liddle's syndrome

Liddles syndrome is an inherited form of hypertension linked to mutations in the epithelial Na+ channel (ENaC). ENaC is composed of three subunits (alpha, beta, gamma), each containing a COOH-terminal PY motif (xPPxY). Mutations causing Liddles syndrome alter or delete the PY motifs of beta- or gamma-ENaC. We recently demonstrated that the ubiquitin-protein ligase Nedd4 binds these PY motifs and that ENaC is regulated by ubiquitination. Here, we investigate, using the Xenopus oocyte system, whether Nedd4 affects ENaC function. Overexpression of wild-type Nedd4, together with ENaC, inhibited channel activity, whereas a catalytically inactive Nedd4 stimulated it, likely by acting as a competitive antagonist to endogenous Nedd4. These effects were dependant on the PY motifs, because no Nedd4-mediated changes in channel activity were observed in ENaC lacking them. The effect of Nedd4 on ENaC missing only one PY motif (of beta-ENaC), as originally described in patients with Liddles syndrome, was intermediate. Changes were due entirely to alterations in ENaC numbers at the plasma membrane, as determined by surface binding and immunofluorescence. Our results demonstrate that Nedd4 is a negative regulator of ENaC and suggest that the loss of Nedd4 binding sites in ENaC observed in Liddles syndrome may explain the increase in channel number at the cell surface, increased Na+ reabsorption by the distal nephron, and hence the hypertension.

The gamma subunit is a specific component of the Na,K-ATPase and modulates its transport function.

The role of small, hydrophobic peptides that are associated with ion pumps or channels is still poorly understood. By using the Xenopus oocyte as an expression system, we have characterized the structural and functional properties of the γ peptide which co‐purifies with Na,K‐ATPase. Immuno‐radiolabeling of epitope‐tagged γ subunits in intact oocytes and protease protection assays show that the γ peptide is a type I membrane protein lacking a signal sequence and exposing the N‐terminus to the extracytoplasmic side. Co‐expression of the rat or Xenopus γ subunit with various proteins in the oocyte reveals that it specifically associates only with isozymes of Na,K‐ATPase. The γ peptide does not influence the formation and cell surface expression of functional Na,K‐ATPase α–β complexes. On the other hand, the γ peptide itself needs association with Na,K‐ATPase in order to be stably expressed in the oocyte and to be transported efficiently to the plasma membrane. γ subunits do not associate with individual α or β subunits but only interact with assembled, transport‐competent α–β complexes. Finally, electrophysiological measurements indicate that the γ peptide modulates the K+ activation of Na,K pumps. These data document for the first time the membrane topology, the specificity of association and a potential functional role for the γ subunit of Na,K‐ATPase.

Transcriptome of a mouse kidney cortical collecting duct cell line: effects of aldosterone and vasopressin.

Aldosterone and vasopressin are responsible for the final adjustment of sodium and water reabsorption in the kidney. In principal cells of the kidney cortical collecting duct (CCD), the integral response to aldosterone and the long-term functional effects of vasopressin depend on transcription. In this study, we analyzed the transcriptome of a highly differentiated mouse clonal CCD principal cell line (mpkCCDcl4) and the changes in the transcriptome induced by aldosterone and vasopressin. Serial analysis of gene expression (SAGE) was performed on untreated cells and on cells treated with either aldosterone or vasopressin for 4 h. The transcriptomes in these three experimental conditions were determined by sequencing 169,721 transcript tags from the corresponding SAGE libraries. Limiting the analysis to tags that occurred twice or more in the data set, 14,654 different transcripts were identified, 3,642 of which do not match known mouse sequences. Statistical comparison (at P < 0.05 level) of the three SAGE libraries revealed 34 AITs (aldosterone-induced transcripts), 29 ARTs (aldosterone-repressed transcripts), 48 VITs (vasopressin-induced transcripts) and 11 VRTs (vasopressin-repressed transcripts). A selection of the differentially-expressed, hormone-specific transcripts (5 VITs, 2 AITs and 1 ART) has been validated in the mpkCCDcl4 cell line either by Northern blot hybridization or reverse transcription–PCR. The hepatocyte nuclear transcription factor HNF-3-α (VIT39), the receptor activity modifying protein RAMP3 (VIT48), and the glucocorticoid-induced leucine zipper protein (GILZ) (AIT28) are candidate proteins playing a role in physiological responses of this cell line to vasopressin and aldosterone.

Identification of a mammalian H+-myo-inositol symporter expressed predominantly in the brain

Inositol and its phosphorylated derivatives play a major role in brain function, either as osmolytes, second messengers or regulators of vesicle endo‐ and exocytosis. Here we describe the identification and functional characterization of a novel H+‐myo‐ inositol co‐transporter, HMIT, expressed predominantly in the brain. HMIT cDNA encodes a 618 amino acid polypeptide with 12 predicted transmembrane domains. Functional expression of HMIT in Xenopus oocytes showed that transport activity was specific for myo‐inositol and related stereoisomers with a Michaelis–Menten constant of ∼100 μM, and that transport activity was strongly stimulated by decreasing pH. Electrophysiological measurements revealed that transport was electrogenic with a maximal transport activity reached at pH 5.0. In rat brain membrane preparations, HMIT appeared as a 75–90 kDa protein that could be converted to a 67 kDa band upon enzymatic deglycosylation. Immunofluorescence microscopy analysis showed HMIT expression in glial cells and some neurons. These data provide the first characterization of a mammalian H+‐coupled myo‐ inositol transporter. Predominant central expression of HMIT suggests that it has a key role in the control of myo‐inositol brain metabolism.

Epithelial sodium channels.

The highly selective amiloride-sensitive epithelial sodium channel is expressed in the distal part of the nephron, the distal colon, and the lung. It plays a critical role in the control of sodium balance, extracellular volume, blood pressure, and of fluid reabsorption in the lung. The primary structure of the rat epithelial sodium channel has recently been determined. It is a heteromultimeric protein made up of three homologous subunits (alpha, beta, and gamma). The biophysical properties, the cell distribution, and the regulation of this channel will be reviewed, with emphasis on its expression in the kidney, colon, and lung, where the clinical implications are most relevant. The epithelial sodium channel is a member of a novel gene superfamily that encodes cation channels involved in the control of cellular and extracellular volume and in the control of distinct functions such as taste transduction and mechanotransduction.

Mineralocorticoid versus Glucocorticoid Receptor Occupancy Mediating Aldosterone-Stimulated Sodium Transport in a Novel Renal Cell Line

Aldosterone controls sodium balance by regulating an epithelial sodium channel (ENaC)-mediated sodium transport along the aldosterone-sensitive distal nephron, which expresses both mineralocorticoid (MR) and glucocorticoid receptors (GR). Mineralocorticoid specificity is ensured by 11beta-hydroxysteroid dehydrogenase type 2, which metabolizes cortisol or corticosterone into inactive metabolites that are unable to bind MR and/or GR. The fractional occupancy of MR and GR by aldosterone mediating the sodium transport response in the aldosterone-sensitive distal nephron cannot be studied in vivo. For answering this question, a novel mouse cortical collecting duct cell line (mCCD(cl1)), which expresses significant levels of MR and GR and a robust aldosterone sodium transport response, was used. Aldosterone elicited a biphasic response: Low doses (K(1/2) = approximately 0.5 nM) induced a transient and early increase of sodium transport (peaking at 3 h), whereas high doses (K(1/2) = approximately 90 nM) entailed an approximately threefold larger, long-lasting response. At 3 h, the corticosterone dose-response curve was shifted to the right compared with that of aldosterone by more than two log concentrations, an effect that was fully reverted in the presence of the 11beta-hydroxysteroid dehydrogenase type 2 inhibitor carbenoxolone. Low doses of dexamethasone (0.1 to 1 nM) failed to induce an early response, but high doses elicited a long-lasting response (K(1/2) = approximately 8 nM), similar to that observed for high aldosterone concentrations. Equilibrium binding assays showed that both aldosterone and corticosterone bind to a high-affinity, low-capacity site, whereas dexamethasone binds to one site. Within the physiologic range of aldosterone concentrations, sodium transport is predicted to be controlled by MR occupancy during circadian cycles and by MR and GR occupancy during salt restriction or acute stress.

CHIF, a member of the FXYD protein family, is a regulator of Na,K-ATPase distinct from the γ-subunit

The biological role of small membrane proteins of the new FXYD family is largely unknown. The best characterized FXYD protein is the γ‐subunit of the Na,K‐ATPase (NKA) that modulates the Na,K‐pump function in the kidney. Here, we report that, similarly to γa and γb 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 γa and γb, 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 γ‐subunit and CHIF interaction with NKA, while cytoplasmic, positively charged residues are necessary for the γ‐subunits association efficiency and for CHIFs functional effects. These data document that CHIF is a new tissue‐specific regulator of NKA which probably plays a crucial role in aldosterone‐responsive tissues responsible for the maintenance of body Na+ and K+ homeostasis.

Na Self Inhibition of Human Epithelial Na Channel: Temperature Dependence and Effect of Extracellular Proteases

The regulation of the open probability of the epithelial Na+ channel (ENaC) by the extracellular concentration of Na+, a phenomenon called “Na+ self inhibition,” has been well described in several natural tight epithelia, but its molecular mechanism is not known. We have studied the kinetics of Na+ self inhibition on human ENaC expressed in Xenopus oocytes. Rapid removal of amiloride or rapid increase in the extracellular Na+ concentration from 1 to 100 mM resulted in a peak inward current followed by a decline to a lower quasi-steady-state current. The rate of current decline and the steady-state level were temperature dependent and the current transient could be well explained by a two-state (active-inactive) model with a weakly temperature-dependent (Q10act = 1.5) activation rate and a strongly temperature-dependant (Q10inact = 8.0) inactivation rate. The steep temperature dependence of the inactivation rate resulted in the paradoxical decrease in the steady-state amiloride-sensitive current at high temperature. Na+ self inhibition depended only on the extracellular Na+ concentration but not on the amplitude of the inward current, and it was observed as a decrease of the conductance at the reversal potential for Na+ as well as a reduction of Na+ outward current. Self inhibition could be prevented by exposure to extracellular protease, a treatment known to activate ENaC or by treatment with p-CMB. After protease treatment, the amiloride-sensitive current displayed the expected increase with rising temperature. These results indicate that Na+ self inhibition is an intrinsic property of sodium channels resulting from the expression of the α, β, and γ subunits of human ENaC in Xenopus oocyte. The extracellular Na+-dependent inactivation has a large energy of activation and can be abolished by treatment with extracellular proteases.