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J D Horisberger

Publications and source records attributed to J D Horisberger.

At least 19 recordsLinked to original sources
PubMed2001

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 (alpha, beta and gamma ENaC), 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.

Amino Acid Sequence↗
PubMed2001

Effects of 8-cpt-cAMP on the epithelial sodium channel expressed in Xenopus oocytes.

Vasopressin stimulates the activity of the epithelial Na channel (ENaC) through the cAMP/PKA pathway in the cortical collecting tubule, or in similar amphibian epithelia, but the mechanism of this regulation is not yet understood. This stimulation by cAMP could not be reproduced with the rat or Xenopus ENaC expressed in Xenopus oocyte. Recently, it was shown that the alpha-subunit cloned from the guinea-pig colon (alpha gp) could confer the ability to be activated by the membrane-permeant cAMP analogue 8-chlorophenyl-thio-cAMP (cpt-cAMP) to channels produced by expression of alpha gp, beta rat and gamma rat ENaC subunits. In this study we investigate the mechanism of this activation. Forskolin treatment, endogenous production of cAMP by activation of coexpressed beta adrenergic receptors, or intracellular perfusion with cAMP did not increase the amiloride-sensitive Na current, even though these maneuvers stimulated CFTR (cystic fibrosis transmembrane conductance regulator)-mediated Cl currents. In contrast, extracellular 8-cpt-cAMP increased alpha gp, beta rat and gamma rat ENaC activity but had no effect on CFTR. Swapping intracellular domains between the cpt-cAMP-sensitive alpha gp and the cpt-cAMP-resistant alpha rat-subunit showed that neither the N-terminal nor the C-terminal of alpha ENaC was responsible for the effect of cpt-cAMP. The mechanisms of activation of ENaC by cpt-cAMP and of CFTR by the cAMP/PKA pathway are clearly different. cpt-cAMP seems to increase the activity of ENaC formed by alpha gp and beta gamma rat by interacting with the extracellular part of the protein.

Amiloride↗
PubMed2001

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 approximately 100 microM, 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.

Amino Acid Sequence↗
PubMed2001

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

The biological role of small membrane proteins of the new FXYD family is largely unknown. The best characterized FXYD protein is the gamma-subunit of the Na,K-ATPase (NKA) that modulates the Na,K-pump function in the kidney. Here, we report that, similarly to gamma(a) and gamma(b) 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 gamma(a) and gamma(b), 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 gamma-subunit and CHIF interaction with NKA, while cytoplasmic, positively charged residues are necessary for the gamma-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 role in aldosterone-responsive tissues responsible for the maintenance of body Na+ and K+ homeostasis.

Alternative Splicing↗
PubMed2001

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 (mpkCCD(cl4)) 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 mpkCCD(cl4) cell line either by Northern blot hybridization or reverse transcription-PCR. The hepatocyte nuclear transcription factor HNF-3-alpha (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.

Aldosterone↗
PubMed2001

Structural and functional features of the transmembrane domain of the Na,K-ATPase beta subunit revealed by tryptophan scanning.

In oligomeric P2-ATPases such as Na,K- and H,K-ATPases, beta subunits play a fundamental role in the structural and functional maturation of the catalytic alpha subunit. In the present study we performed a tryptophan scanning analysis on the transmembrane alpha-helix of the Na,K-ATPase beta1 subunit to investigate its role in the stabilization of the alpha subunit, the endoplasmic reticulum exit of alpha-beta complexes, and the acquisition of functional properties of the Na,K-ATPase. Single or multiple tryptophan substitutions in the beta subunits transmembrane domain had no significant effect on the structural maturation of alpha subunits expressed in Xenopus oocytes nor on the level of expression of functional Na,K pumps at the cell surface. Furthermore, tryptophan substitutions in regions of the transmembrane alpha-helix containing two GXXXG transmembrane helix interaction motifs or a cysteine residue, which can be cross-linked to transmembrane helix M8 of the alpha subunit, had no effect on the apparent K(+) affinity of Na,K-ATPase. On the other hand, substitutions by tryptophan, serine, alanine, or cysteine, but not by phenylalanine of two highly conserved tyrosine residues, Tyr(40) and Tyr(44), on another face of the transmembrane helix, perturb the transport kinetics of Na,K pumps in an additive way. These results indicate that at least two faces of the beta subunits transmembrane helix contribute to inter- or intrasubunit interactions and that two tyrosine residues aligned in the beta subunits transmembrane alpha-helix are determinants of intrinsic transport characteristics of Na,K-ATPase.

Amino Acid Sequence↗
PubMed2001

Modular microsystem for epithelial cell culture and electrical characterisation.

We have realised a microsystem for the culture and electrical characterisation of epithelial cell layers for cell-based diagnostic applications. The main goal of this work is to achieve both cell culture and impedimetric and potentiometric characterisation on a single device. The miniaturised cell culture system enables the uses of scarce epithelial cells, as obtained from transgenic mice or from human biopsies. The device is completely modular and offers high flexibility: a polycarbonate membrane used as cell substrate is glued in between two moulded Polydimethylsiloxane (PDMS) layers to form a sandwich, which is placed between two stacks, containing the microfluidic channels and integrated measurement electrodes. The polycarbonate membrane sandwich can be removed, replaced or analysed at any time. We have characterised the impedimetric properties of our microsystem, demonstrated epithelial cell layer growth within it, and have done the initial electrical characterisation of epithelial cell layers.

Animals↗
PubMed2001

Haemolytic onset of Wilson disease in a patient with homozygous truncation of ATP7B at Arg1319.

We describe a 19-year-old woman with haemolytic anaemia and thrombocytopenia as the initial manifestation of Wilson disease (WD). There are two reasons for reporting such an improbable case. First, it emphasizes the importance of recognizing atypical clinical presentations of potentially lethal recessive traits for which therapy is available. Second, it shows that, even in a monogenic disorder like WD, the phenotype cannot be extrapolated from the mutated genotype in a simple fashion; this patient had a relatively late-onset form of WD despite homozygosity for a genetic lesion leading to an apparent complete loss of function of the WD copper transporter.

Adenosine Triphosphatases↗
PubMed2001

Bufo marinus bladder H-K-ATPase carries out electroneutral ion transport.

Bufo marinus bladder H-K-ATPase belongs to the Na-K-ATPase and H-K-ATPase subfamily of oligomeric P-type ATPases and is closely related to rat and human nongastric H-K-ATPases. It has been demonstrated that this ATPase transports K(+) into the cell in exchange for protons and sodium ions, but the stoichiometry of this cation exchange is not yet known. We studied the electrogenic properties of B. marinus bladder H-K-ATPase expressed in Xenopus laevis oocytes. In a HEPES-buffered solution, K(+) activation of the H-K-ATPase induced a slow-onset inward current that reached an amplitude of approximately 20 nA after 1-2 min. When measurements were performed in a solution containing 25 mM HCO at a PCO(2) of 40 Torr, the negative current activated by K(+) was reduced. In noninjected oocytes, intracellular alkalization activated an inward current similar to that due to B. marinus H-K-ATPase. We conclude that the transport activity of the nongastric B. marinus H-K-ATPase is not intrinsically electrogenic but that the inward current observed in oocytes expressing this ion pump is secondary to intracellular alkalization induced by proton transport.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2001

Functional analyses of a N-terminal splice variant of the alpha subunit of the epithelial sodium channel.

The amiloride-sensitive epithelial sodium channel (ENaC) is the limiting step for sodium absorption in epithelial cells of the distal nephron, distal colon, airways and excretory ducts of several glands. In vivo and in vitro studies showed that the alpha subunit of ENaC is necessary for the expression of functional channels. Using RT-PCR strategy, a novel N-terminal splice variant has been identified which deletes 49 amino acids in the N-terminal region of the mouse alphaENaC subunit. In oocytes expressing the alphaENaC splice variant, together with beta and gammaENaC subunits, amiloride-sensitive currents were less than 20% of values obtained with the wild type ENaC. The single channel conductance and the ionic selectivity were similar and there was only a minor decrease in the level of expression of the protein at the oocyte surface. These findings indicate that the deleted sequence in the N-terminal part of the mouse and rat alphaENaC subunit might play a role in the regulation of the activity of expressed ENaC channels.

Amino Acid Sequence↗
PubMed2000

Structure of the 5th transmembrane segment of the Na,K-ATPase alpha subunit: a cysteine-scanning mutagenesis study.

To study the structure of the pathway of cations across the Na, K-ATPase, we applied the substituted cysteine accessibility method to the putative 5th transmembrane segment of the alpha subunit of the Na,K-ATPase of the toad Bufo marinus. Only the most extracellular amino acid position (A(796)) was accessible from the extracellular side in the native Na,K-pump. After treatment with palytoxin, six other positions (Y(778), L(780), S(782), P(785), E(786) and L(791)), distributed along the whole length of the segment, became readily accessible to a small-size methanethiosulfonate compound (2-aminoethyl methanethiosulfonate). The accessible residues are not located on the same side of an alpha-helical model but the pattern of reactivity would rather suggest a beta-sheet structure for the inner half of the putative transmembrane segment. These results demonstrate the contribution of the 5th transmembrane segment to the palytoxin-induced channel and indicate which amino acid positions are exposed to the pore of this channel.

Acrylamides↗
PubMed2000

Regulation of the cardiac voltage-gated Na+ channel (H1) by the ubiquitin-protein ligase Nedd4.

The cardiac voltage-gated Na+ channel H1, involved in the generation of cardiac action potential, contains a C-terminal PY motif (xPPxY). Since PY motifs are known ligands to WW domains, we investigated their role for H1 regulation and the possible involvement of the WW domain containing ubiquitin-protein ligase Nedd4, taking advantage of the Xenopus oocyte system. Mutation of the PY motif leads to higher peak currents when compared to wild-type channel. Moreover, co-expression of Nedd4 reduced the peak currents, whereas an enzymatically inactive Nedd4 mutant increased them, likely by competing with endogenous Nedd4. The effect of Nedd4 was not observed in the PY motif mutated channel or in the skeletal muscle voltage-gated Na+ channel, which lacks a PY motif. We conclude that H1 may be regulated by Nedd4 depending on WW-PY interaction, and on an active ubiquitination site.

Amino Acid Motifs↗
PubMed2000

Transport and pharmacological properties of nine different human Na, K-ATPase isozymes.

Na,K-ATPase plays a crucial role in cellular ion homeostasis and is the pharmacological receptor for digitalis in man. Nine different human Na,K-ATPase isozymes, composed of 3 alpha and beta isoforms, were expressed in Xenopus oocytes and were analyzed for their transport and pharmacological properties. According to ouabain binding and K(+)-activated pump current measurements, all human isozymes are functional but differ in their turnover rates depending on the alpha isoform. On the other hand, variations in external K(+) activation are determined by a cooperative interaction mechanism between alpha and beta isoforms with alpha2-beta2 complexes having the lowest apparent K(+) affinity. alpha Isoforms influence the apparent internal Na(+) affinity in the order alpha1 > alpha2 > alpha3 and the voltage dependence in the order alpha2 > alpha1 > alpha3. All human Na,K-ATPase isozymes have a similar, high affinity for ouabain. However, alpha2-beta isozymes exhibit more rapid ouabain association as well as dissociation rate constants than alpha1-beta and alpha3-beta isozymes. Finally, isoform-specific differences exist in the K(+)/ouabain antagonism which may protect alpha1 but not alpha2 or alpha3 from digitalis inhibition at physiological K(+) levels. In conclusion, our study reveals several new functional characteristics of human Na,K-ATPase isozymes which help to better understand their role in ion homeostasis in different tissues and in digitalis action and toxicity.

Animals↗
PubMed2000

Regulation of the epithelial Na+ channel by Nedd4 and ubiquitination.

The epithelial Na+ channel (ENaC) is comprised of three subunits, alpha, beta and gamma, and plays an essential role in Na+ and fluid absorption in the kidney, colon and lung. We had identified proline-rich sequences at the C termini of alpha beta gamma ENaC, which include the sequence PPxY, the PY motif. This sequence in beta or gamma ENaC is deleted or mutated in Liddle's syndrome, a hereditary form of arterial hypertension. Our previous work demonstrated that these PY motifs bind to the WW domains of Nedd4, a ubiquitin protein ligase containing a C2 domain, three or four WW domains and a ubiquitin protein ligase Hect domain. Accordingly, we have recently demonstrated that Nedd4 regulates ENaC function by controlling the number of channels at the cell surface, that this regulation is impaired in ENaC bearing Liddle's syndrome mutations, and that ENaC stability and function are regulated by ubiquitination. The C2 domain is responsible for localizing Nedd4 to the plasma membrane in a Ca(2+)-dependent manner, and in polarized epithelial MDCK cells this localization is primarily apical. In accordance, electrophysiological characterization of ENaC expressed in MDCK cells revealed inhibition of channel activity by elevated intracellular Ca2+ levels. Thus, in response to Ca2+, Nedd4 may be mobilized to the apical membrane via its C2 domain, where it binds ENaC via Nedd4-WW:ENaC-PY motifs' interactions, leading to ubiquitination of the channel by the Nedd4-Hect domain and subsequent channel endocytosis and lysosomal degradation. This process may be at least partially impaired in Liddle's syndrome due to reduced Nedd4 binding, leading to increased retention of ENaC at the cell surface.

Animals↗
PubMed1999

Role of the intracellular domain of the beta subunit in Na,K pump function.

The catalytic alpha subunit of the (Na,K)- and (H,K)-ATPases needs to be coexpressed with a beta subunit in order to produce cation transport activity. Although the isoform of the beta subunit is known to influence the functional characteristics of the Na,K pump, the role of the different domains of the beta subunit is not fully understood. We have studied the function of a Na,K pump resulting from the expression of a wild-type alpha subunit with a N-terminally truncated mutant of the beta subunit using the two-electrode voltage clamp and the cut-open oocyte techniques. While the maximal activity, measured as the K+-activated outward current, was not significantly altered, the beta N-terminal truncation induced an ouabain-sensitive conductance in the absence of extracellular K+. The voltage dependence of the ouabain-sensitive charge distribution indicated that in the Na/Na exchange conditions, the E1-E2 conformation equilibrium was shifted towards the E2 conformation, a change resulting from alteration of both the forward and the backward reaction rate. Removal of the intracellular domain of the beta subunit modifies several aspects of the whole enzyme function by a mechanism that must imply the state of the extracellular and/or transmembrane parts of the alpha/beta subunit complex.

Animals↗
PubMed1999

Feedback inhibition of rat amiloride-sensitive epithelial sodium channels expressed in Xenopus laevis oocytes.

1. Regulation of the amiloride-sensitive epithelial sodium channel (ENaC) is essential for the control of body sodium homeostasis. The downregulation of the activity of this Na+ channel that occurs when the intracellular Na+ concentration ([Na+]i) is increased is known as feedback inhibition. Although intracellular Na+ is the trigger for this phenomenon, its cellular and molecular mediators are unknown. 2. We used the 'cut-open oocyte' technique to control the composition of the intracellular milieu of Xenopus oocytes expressing rat ENaCs to enable us to test several factors potentially involved in feedback inhibition. 3. The effects of perfusion of the intracellular space were demonstrated by an electromicrographic study and the time course of the intracellular solution exchange was established by observing the effect of intracellular pH: a decrease from pH 7.4 to 6.5 reduced the amiloride-sensitive current by about 40 % within 2 min. 4. Feedback inhibition was observed in non-perfused oocytes when Na+ entry induced a large increase in [Na+]i. Intracellular perfusion prevented feedback regulation even though the [Na+]i was allowed to increase to values above 50 mM. 5. No effects on the amiloride-sensitive current were observed after changes in the concentration of Na+ (from 1 to 50 mM), Ca2+ (from 10 to 1000 nM) or ATP (from nominally free to 1 or 5 mM) in the intracellular perfusate. 6. We conclude that feedback inhibition requires intracellular factors that can be removed by intracellular perfusion. Although a rise in [Na+]i may be the trigger for the feedback inhibition of the ENaC, this effect is not mediated by a direct effect of Na+, Ca2+ or ATP on the ENaC protein.

Adenosine Triphosphate↗
PubMed1999

Genetic disorders of membrane transport. V. The epithelial sodium channel and its implication in human diseases.

The epithelial Na+ channel (ENaC) controls the rate-limiting step in the process of transepithelial Na+ reabsorption in the distal nephron, the distal colon, and the airways. Hereditary salt-losing syndromes have been ascribed to loss of function mutations in the alpha-, beta-, or gamma-ENaC subunit genes, whereas gain of function mutations (located in the COOH terminus of the beta- or gamma-subunit) result in hypertension due to Na+ retention (Liddle's syndrome). In mice, gene-targeting experiments have shown that, in addition to the kidney salt-wasting phenotype, ENaC was essential for lung fluid clearance in newborn mice. Disruption of the alpha-subunit resulted in a complete abolition of ENaC-mediated Na+ transport, whereas knockout of the beta- or gamma-subunit had only minor effects on fluid clearance in lung. Disruption of each of the three subunits resulted in a salt-wasting syndrome similar to that observed in humans.

Animals↗