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J D Horisberger

Publications and source records attributed to J D Horisberger.

At least 19 recordsLinked to original sources
PubMed1998

Protease modulation of the activity of the epithelial sodium channel expressed in Xenopus oocytes.

We have investigated the effect of extracellular proteases on the amiloride-sensitive Na+ current (INa) in Xenopus oocytes expressing the three subunits alpha, beta, and gamma of the rat or Xenopus epithelial Na+ channel (ENaC). Low concentrations of trypsin (2 microg/ml) induced a large increase of INa within a few minutes, an effect that was fully prevented by soybean trypsin inhibitor, but not by amiloride. A similar effect was observed with chymotrypsin, but not with kallikrein. The trypsin-induced increase of INa was observed with Xenopus and rat ENaC, and was very large (approximately 20-fold) with the channel obtained by coexpression of the alpha subunit of Xenopus ENaC with the beta and gamma subunits of rat ENaC. The effect of trypsin was selective for ENaC, as shown by the absence of effect on the current due to expression of the K+ channel ROMK2. The effect of trypsin was not prevented by intracellular injection of EGTA nor by pretreatment with GTP-gammaS, suggesting that this effect was not mediated by G proteins. Measurement of the channel protein expression at the oocyte surface by antibody binding to a FLAG epitope showed that the effect of trypsin was not accompanied by an increase in the channel protein density, indicating that proteolysis modified the activity of the channel present at the oocyte surface rather than the cell surface expression. At the single channel level, in the cell-attached mode, more active channels were observed in the patch when trypsin was present in the pipette, while no change in channel activity could be detected when trypsin was added to the bath solution around the patch pipette. We conclude that extracellular proteases are able to increase the open probability of the epithelial sodium channel by an effect that does not occur through activation of a G protein-coupled receptor, but rather through proteolysis of a protein that is either a constitutive part of the channel itself or closely associated with it.

Amiloride↗
PubMed1997

A mouse model for the renal salt-wasting syndrome pseudohypoaldosteronism.

Aldosterone-dependent epithelial sodium transport in the distal nephron is mediated by the absorption of sodium through the highly selective, amiloride-sensitive epithelial sodium channel (ENaC) made of three homologous subunits (alpha, beta, and gamma). In human, autosomal recessive mutations of alpha, beta, or gammaENaC subunits cause pseudohypoaldosteronism type 1 (PHA-1), a renal salt-wasting syndrome characterized by severe hypovolemia, high plasma aldosterone, hyponatremia, life-threatening hyperkaliemia, and metabolic acidosis. In the mouse, inactivation of alphaENaC results in failure to clear fetal lung liquid at birth and in early neonatal death, preventing the observation of a PHA-1 renal phenotype. Transgenic expression of alphaENaC driven by a cytomegalovirus promoter in alphaENaC(-/-) knockout mice [alphaENaC(-/-)Tg] rescued the perinatal lethal pulmonary phenotype and partially restored Na+ transport in renal, colonic, and pulmonary epithelia. At days 5-9, however, alphaENaC(-/-)Tg mice showed clinical features of severe PHA-1 with metabolic acidosis, urinary salt-wasting, growth retardation, and 50% mortality. Adult alphaENaC(-/-)Tg survivors exhibited a compensated PHA-1 with normal acid/base and electrolyte values but 6-fold elevation of plasma aldosterone compared with wild-type littermate controls. We conclude that partial restoration of ENaC-mediated Na+ absorption in this transgenic mouse results in a mouse model for PHA-1.

Amiloride↗
PubMed1997

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 the protease 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 sodium channel 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.

Amino Acid Sequence↗
PubMed1997

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 gamma peptide which co-purifies with Na,K-ATPase. Immuno-radiolabeling of epitope-tagged gamma subunits in intact oocytes and protease protection assays show that the gamma 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 gamma subunit with various proteins in the oocyte reveals that it specifically associates only with isozymes of Na,K-ATPase. The gamma peptide does not influence the formation and cell surface expression of functional Na,K-ATPase alpha-beta complexes. On the other hand, the gamma 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. Gamma subunits do not associate with individual alpha or beta subunits but only interact with assembled, transport-competent alpha-beta complexes. Finally, electrophysiological measurements indicate that the gamma 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 gamma subunit of Na,K-ATPase.

Amino Acid Sequence↗
PubMed1997

Palytoxin effects through interaction with the Na,K-ATPase in Xenopus oocyte.

Palytoxin (PTX) is known to bind to Na,K-ATPase, to inhibit its activity, and to induce cation conductance, but the mechanism of these effects is still poorly understood. In Xenopus oocytes, PTX induced a large cation conductance, an effect that could be prevented or reversed by ouabain for oocytes expressing Xenopus Na,K-pumps but not with those expressing Bufo Na,K-pumps. In both cases patch-clamp experiments demonstrated a 7-8 pS channel in the presence of PTX. A large PTX-induced conductance could be observed with minimal Na,K-pump inhibition. From the single PTX-induced channel and macroscopic whole oocyte conductance, and the number of Na,K-pumps, we can conclude that PTX-induced conductance occurs through a direct interaction of PTX with a small number of Na,K-pumps.

Acrylamides↗
PubMed1997

Novel isoforms of the beta and gamma subunits of the Xenopus epithelial Na channel provide information about the amiloride binding site and extracellular sodium sensing.

We have previously identified three homologous subunits alpha, beta, and gamma of the highly selective amiloride-sensitive Na channel from the Xenopus laevis kidney A6 cell line, which forms a tight epithelium in culture. We report here two novel genes, termed beta2 and gamma2, which share 90 and 92% sequence identity with the previously characterized beta and gamma XENaC, respectively. beta2 and gamma2 transcripts were detected in lung, kidney, and A6 cells grown on porous substrate. The physiological and pharmacological profile of the Na channel expressed after alphabeta2gamma XENaC cRNA injection in Xenopus oocyte did not differ from alphabetagamma XENaC. By contrast, the channel expressed after alphabetagamma2 injection showed: (i) a lower maximal amiloride-sensitive sodium current, (ii) a higher apparent affinity for external sodium and inactivation of the sodium current by high sodium concentrations, and (iii) a lower apparent affinity for amiloride (KI alphabetagamma2; 1.34 microM versus alphabetagamma 0.35 microM). These data indicate that the gamma (and/or gamma2) subunit participates in amiloride binding and the sensing of the extracellular sodium concentration. The close homology between gamma and gamma2 will help to define the domains involved in sensing external sodium and in the structure of this important drug receptor.

Amiloride↗
PubMed1997

Early effect of aldosterone on the rate of synthesis of the epithelial sodium channel alpha subunit in A6 renal cells.

Transepithelial Na+ reabsorption across tight epithelia is regulated by aldosterone. The amiloride-sensitive epithelial sodium channel (ENaC) is a major target for the natriferic action of aldosterone. In this study, the effect of aldosterone on ENaC mRNA abundance and the rate of protein synthesis for each of the three ENaC subunits (alpha, beta and gamma) in the A6 kidney cell line were examined. In cells grown on plastic, aldosterone induced a large and rapid increase in epithelial sodium channel (ENaC) beta and gamma subunit mRNA abundance, but this effect is not translated into the synthesis of the corresponding proteins. In cells grown on a porous substrate, amiloride-sensitive electrogenic sodium transport was expressed and was upregulated by aldosterone (300 nM) as early as 1 h after the addition of the hormone. The alpha, beta, and gamma mRNA abundance was not changed by aldosterone during the first 3 h of stimulation, whereas a fourfold increase over control was observed after 24 h. The rate of synthesis of alpha subunit was significantly increased above control already 60 min after aldosterone addition, whereas beta subunit synthesis increased only 6 h after hormone addition, with no significant change for the gamma subunit. The half-lives of each subunit as assessed by 35S methionine pulse-chase experiments were short (between 40 and 50 min) and were not modified by aldosterone. Taking into account the short half-life of ENaC protein and assuming that the synthesis of the alpha subunit is a limiting factor in the assembly and expression of new channels at the cell surface, it is proposed that the aldosterone regulation of sodium transport might be, in part, mediated by de novo synthesis of the channel protein.

Aldosterone↗
PubMed1996

Role in cation translocation of the N-terminus of the alpha-subunit of the Na(+)-K+ pump of Bufo.

1. We have studied the effects on the physiological properties of the Na(+)-K+ pump of both 31- and 40-amino acid N-terminal truncated forms of the alpha-subunit of the Na(+)-K(+)-ATPase. 2. Na(+)-K+ pumps that were moderately ouabain resistant (K1 = 50 microM) were expressed in the Xenopus oocyte by injection of wild-type or truncated variants of the Bufo marinus Na(+)-K(+)-ATPase alpha-subunit cRNA with Bufo beta-subunit cRNA. The function of the Na(+)-K+ pump was studied by electrophysiological methods after Na+ loading and inhibition of the endogenous Xenopus Na(+)-K(+)-ATPase by exposure to a low concentration (0.2 microM) of ouabain. 3. The voltage-dependent potassium activation kinetics of the Na(+)-K+ pump current and the ouabain-sensitive proton-dependent inward current were studied using the two-electrode voltage-clamp technique. A novel technique involving permeabilization of part of the oocyte membrane with digitonin was developed to enable study of the pre-steady-state current following fast voltage perturbation. 4. By comparison with the wild type, the 40-amino acid N-terminal truncation induced a lower level of Na(+)-K+ pump current, a 2- to 3-fold reduction in the apparent external K+ affinity when measured in the presence of extracellular Na+, a relative increase in the proton-dependent inward current, and a reduction in the rate constant of the pre-steady-state current following a voltage step towards a positive membrane potential. The 31-amino acid truncation induced changes that were qualitatively similar but of smaller magnitude. 5. We have analysed these results using a kinetic model of the Na(+)-K+ pump cycle and have shown that all these effects can be explained by the change in a single rate constant in the cycle kinetics, namely a reduction in the rate of the main charge translocating part of the Na(+)-K+ pump cycle, i.e. the forward E1 to E2 conformational change, the deocclusion and release of Na+ to the external side. 6. The highly charged N-terminal segment seems to be directly involved in the mechanism that translocates Na+ ions across the membrane's electrical field.

Amino Acid Sequence↗
PubMed1996

Mercury binding site on Na+/K(+)-ATPase: a cysteine in the first transmembrane segment.

Mercury is an element of great pharmacological and toxicological importance. It reacts with sulfhydryl groups on proteins to form mercaptides. Mercuric mercury (Hg2+), a form that shows primarily epithelial toxicity, can inhibit Na+/K(+)-ATPase at low concentration, but its molecular target site on the protein is not known. To investigate the interaction of Hg2+ with Na+/K(+)-ATPase, we studied the inhibition of Na+/K+ pump activity by inorganic mercury (HgCl2) in Xenopus laevis oocytes expressing wild-type and mutant forms of Na+/K(+)-ATPase. Na+/K+ pump potassium-activated current was inhibited with first-order kinetics (Kon = 7 x 10(3) M-1.sec-1) and an estimated Kd of < or = 170 nM. To study the hypothesis that the cysteine (C113) of the first transmembrane segment of the alpha subunit participates in a Hg2+ binding site, we investigated the inhibition of Na+/K+ pump activity produced by a 1-min exposure to 5 microM HgCl2. Wild-type and C113S and C113Y mutant Na+/K+ pumps were inhibited by 43 +/- 7%, 12 +/- 2%, and 5 +/- 3%, respectively. Because C113 is a component of the cardiac steroid binding site, we studied the interaction of mercury with strophanthidin by exposing oocytes for 2 min to 5 microM HgCl2 in the presence or absence of 50 microM strophanthidin. Strophanthidin reduced the inhibition by mercury from 68 +/- 5% to 30 +/- 7%. Based on the position of C113 in the first transmembrane segment, these results suggest that Hg2+ binding to C113 from the extracellular side is one of the mechanisms by which mercury inhibits Na+/K(+)-ATPase.

Animals↗
PubMed1995

Labeling of a cysteine in the cardiotonic glycoside binding site by the steroid derivative HDMA.

The digoxigenin derivative N-hydroxysuccinimidyl digoxigenin-3-O-methylcarbonyl-epsilon-aminocaproate (HDMA) has been shown to covalently label the ouabain binding site of the Na,K-ATPase epsilon subunit [Antolovic et al. (1995) Eur. J. Biochem. 227, 61-67]. In the present study we observed both, labeling and inactivation of the activity, of wild type Na,K-ATPase overexpressed in Xenopus oocyte. In contrast, no significant inhibition and no labeling could be detected when a Cys-113 of the first transmembrane segment was mutated to serine, although the affinity of this mutant for digoxigenin or HDMA measured in acute inhibition experiments was similar to the wild type. This indicates that after docking of its genin moiety, HDMA can form a thioester bond with Cys-113.

Affinity Labels↗
PubMed1995

A conformation of Na(+)-K+ pump is permeable to proton.

The Na(+)-K+ pump is thought to operate through a two-conformation (E1-E2) transport cycle in which the cation binding sites are accessible only from one side at a time. Using Na(+)-loaded Xenopus oocytes in which Na(+)-K+ pumps were overexpressed by injection of cRNA of the Xenopus Na(+)-K+ pump alpha-and beta-sub units, we observed a Na(+)-K+ pump-mediated (ouabain-sensitive) inward current in the absence of other transportable cations, except H+, in the external solution. This inward current was strongly inwardly rectifying, pH dependent, and larger at acid pH. Under conditions favoring a large ouabain-sensitive inward current, we observed a ouabain-sensitive intracellular acidification, and the amplitude of the acidification was significantly related to the ouabain-sensitive current, indicating that this current was carried by protons. The reversal potential of the ouabain-sensitive current was dependent on external pH as expected for a proton-conductive pathway. We conclude that in the absence of external K+ the Na(+)-K+ pump can mediate a large inward electrogenic transport of proton. This is most easily explained by the hypothesis that the E2 conformation of the Na(+)-K+ pump with cation binding sites exposed to the outside is accessible to protons from both sides and thus provides a channellike pathway for protons.

Animals↗
PubMed1995

Primary structure and functional expression of the mouse and frog alpha-subunit of the gastric H(+)-K(+)-ATPase.

The H(+)-K(+)-ATPase of the gastric parietal cells is responsible for the acidification of the stomach lumen. This heterodimeric protein belongs to the family of cation-translocating P-type ATPases, which includes the closely related Na(+)-ATPase. We have cloned the alpha-subunit cDNA of the Xenopus and murine gastric H(+)-K(+)-ATPase (alpha H-K). We have expressed Xenopus and murine alpha H-K along with the previously cloned gastric H(+)-K(+)-ATPase beta-subunit of rabbit (beta H-K) in Xenopus oocytes by cRNA injection. An antibody directed against the beta H-K coimmunoprecipitates under nondenaturing conditions the alpha H-K of both species, demonstrating assembly of the alpha/beta complex. Additionally, we demonstrate the presence of K(+)-transporting H(+)-K(+)-ATPase in the plasma membrane of oocytes by 86Rb- uptake. The H(+)-K(+)-ATPase-mediated K+ uptake was inhibited by the gastric H(+)-K(+)-ATPase inhibitor Sch-28080, but not by ouabain, and shows K(+)-dependent activation (K1/2 approximately 2 mM). Furthermore, H(+)-K(+)-ATPase-expressing oocytes show a Sch-28080 inhibitable proton extrusion. Our data indicate that the expressed H(+)-K(+)-ATPase behaves functionally in oocytes as in the gastric gland.

Animals↗
PubMed1995

The highly selective low-conductance epithelial Na channel of Xenopus laevis A6 kidney cells.

In Na-reabsorbing tight epithelia, the rate-limiting step for Na transport is the highly selective low-conductance amiloride-sensitive epithelial Na channel (type 1 ENaC). In rat distal colon, type 1 ENaC is made of three homologous subunits. The aim of this study was to identify the corresponding genes of the renal channel from the kidney-derived A6 cell line of Xenopus laevis. Three homologous subunits were identified and coexpressed in the Xenopus oocyte system. The reconstituted channel had all the characteristics of the native type 1 ENaC described in A6 cells: 1) high selectivity, 2) low single-channel conductance, 3) slow gating kinetics, and 4) high affinity for amiloride. Transcripts for alpha-, beta-, and gamma-subunits of the Xenopus epithelial Na channel (xENaC) were detected in A6 kidney cells, Xenopus kidney, lung, and to a lesser extent in stomach and skin. Each subunit of the xENaC shares approximately 60% overall identity with the corresponding rat homologue (alpha, beta, and gamma rENaC). Our data suggest that the triplication of the ENaC subunits occurred before the divergence between mammalian and amphibian lineages.

Amino Acid Sequence↗
PubMed1995

Human ATP1AL1 gene encodes a ouabain-sensitive H-K-ATPase.

The cDNA for ATP1AL1, the fifth member of the human Na-K-adenosinetriphosphatase (ATPase)/H-K-ATPase gene family, was recently cloned (A. V. Grishin, V. E. Sverdlov, M. B. Kostina, and N. N. Modyanov. FEBS Lett. 349: 144-150, 1994). The encoded protein (ATP1AL1) has all the primary structural features common to the catalytic alpha-subunit of ion-transporting P-type ATPases and is similar (63-64% identity) to the Na-K-ATPase alpha-subunit isoforms and the gastric H-K-ATPase alpha-subunit. In this study, ATP1AL1 was expressed in Xenopus laevis oocytes in combination with the beta-subunit of rabbit gastric H-K-ATPase. The functional properties of the stable alpha/beta-complex were studied by 86Rb+ uptake and demonstrated that ATP1AL1 is a novel human K(+)-dependent ATPase [apparent half-constant activation/(K1/2) for K+ approximately 375 microM)]. ATP1AL1-mediated inward K+ transport was inhibited by ouabain (inhibition constant approximately 13 microM) and was found to be inhibited by high concentrations of SCH-28080 (approximately 70% at 500 microM). ATP1AL1 expression resulted in the alkalinization of the oocytes' cytoplasm and ouabain-sensitive proton extrusion, as measured with pH-sensitive microelectrodes. These data argue that ATP1AL1 is the catalytic alpha-subunit of a human nongastric P-type ATPase capable of exchanging extracellular potassium for intracellular protons.

Animals↗