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B C Rossier

Publications and source records attributed to B C Rossier.

At least 19 recordsLinked to original sources
PubMed1997

A reappraisal of aldosterone effects on the kidney: new insights provided by epithelial sodium channel cloning.

Sodium homeostasis is crucial for the control of extra-cellular volume and blood pressure. Regulation of sodium reabsorption is mainly achieved in the distal nephron by the mineralocorticoid aldosterone, but the molecular pathway of aldosterone action has largely remained unclear. Molecular genetic analysis of inherited diseases disturbing sodium homeostasis has now demonstrated that the amiloride-sensitive epithelial sodium channel is a major effector of aldosterone action. Mechanisms by which aldosterone regulates the epithelial sodium channel activity are beginning to emerge and will be of great importance for a better understanding of salt-sensitive hypertension.

Aldosterone↗
PubMed1996

Cell surface expression of the epithelial Na channel and a mutant causing Liddle syndrome: a quantitative approach.

The epithelial amiloride-sensitive sodium channel (ENaC) controls transepithelial Na+ movement in Na(+)-transporting epithelia and is associated with Liddle syndrome, an autosomal dominant form of salt-sensitive hypertension. Detailed analysis of ENaC channel properties and the functional consequences of mutations causing Liddle syndrome has been, so far, limited by lack of a method allowing specific and quantitative detection of cell-surface-expressed ENaC. We have developed a quantitative assay based on the binding of 125I-labeled M2 anti-FLAG monoclonal antibody (M2Ab*) directed against a FLAG reporter epitope introduced in the extracellular loop of each of the alpha, beta, and gamma ENaC subunits. Insertion of the FLAG epitope into ENaC sequences did not change its functional and pharmacological properties. The binding specificity and affinity (Kd = 3 nM) allowed us to correlate in individual Xenopus oocytes the macroscopic amiloride-sensitive sodium current (INa) with the number of ENaC wild-type and mutant subunits expressed at the cell surface. These experiments demonstrate that: (i) only heteromultimeric channels made of alpha, beta, and gamma ENaC subunits are maximally and efficiently expressed at the cell surface; (ii) the overall ENaC open probability is one order of magnitude lower than previously observed in single-channel recordings; (iii) the mutation causing Liddle syndrome (beta R564stop) enhances channel activity by two mechanisms, i.e., by increasing ENaC cell surface expression and by changing channel open probability. This quantitative approach provides new insights on the molecular mechanisms underlying one form of salt-sensitive hypertension.

Amino Acid Sequence↗
PubMed1996

Identification of a PY motif in the epithelial Na channel subunits as a target sequence for mutations causing channel activation found in Liddle syndrome.

Liddle syndrome is an autosomal dominant form of hypertension, resulting from mutations in the cytoplasmic C-terminus of either the beta or gamma subunits of the amiloride-sensitive epithelial Na channel (ENaC) which lead to constitutively increased channel activity. Most mutations reported to date result in the elimination of 45-75 normal amino acids from these segments, leaving open the question of the identity of the precise amino acids in which mutation can lead to an enhanced channel activity. To address this question, we have performed a systematic mutagenesis study of the C-termini of the alpha, beta and gamma ENaC subunits of the rat channel and have analyzed their function by expression in Xenopus oocytes. The results demonstrate that a short proline-rich segment present in the cytoplasmic C-terminus of each subunit is required for the normal regulation of channel activity. Missense mutations altering a consensus PPPXY sequence of the alpha, beta or gamma subunits reproduced the increase in channel activity found in mutants in which the entire cytoplasmic C-termini are deleted. This proline-rich sequence, referred to as the PY motif, is known to be a site of binding by proteins bearing a WW domain. These findings show that the three PY motifs in the C-termini of ENaC are involved in the regulation of channel activity, probably via protein-protein interactions. This new regulatory mechanism of channel function is critical for the maintenance of normal Na reabsorption in the kidney and of Na+ balance and blood pressure.

Amino Acid Sequence↗
PubMed1996

Liddle disease caused by a missense mutation of beta subunit of the epithelial sodium channel gene.

Mutations in beta or gamma subunit of the epithelial sodium channel (ENaC) have been found to cause a hereditary form of human hypertension, Liddle syndrome. Most of the mutations reported are either nonsense mutations or frame shift mutations which would truncate the cytoplasmic carboxyl terminus of the beta or gamma subunits of the channel, suggesting that these domains are important for the normal regulation of this channel. We sequenced ENaC in a family with Liddle syndrome and found a missense mutation in beta subunit which predicts substitution of Tyr by His at codon 618, 2 bp downstream from a missense mutation (P616L) that has been reported recently. Presence of this mutation correlates with the clinical manifestations (hypertension, hypokalemia, suppressed aldosterone secretion) in this kindred. Functional expression studies in the Xenopus oocytes revealed constitutive activation of the Y618H mutant indistinguishable from that observed for the deletion mutant (R564stop) identified in the original pedigree of Liddle. Our data suggest that the region between Pro616 and Tyr618 is critically important for regulation of ENaC activity.

Adolescent↗
PubMed1996

The rat distal colon P-ATPase alpha subunit encodes a ouabain-sensitive H+, K+-ATPase.

The functional properties and the pharmacological profile of the recently cloned cDNA colonic P-ATPase alpha subunit (Crowson, M.S., and Shull, G.E. (1992) J. Biol. Chem. 267, 13740-13748) were investigated by using the Xenopus oocyte expression system. Xenopus oocytes were injected with alpha subunit cRNAs from Bufo marinus bladder or rat distal colon and/or with beta subunit cRNA from B. marinus bladder. Two days after injection, K+ uptake was measured by using 86 Rb+ as a K+ surrogate, and pH measurements were performed by means of ion-selective microelectrodes. Co-injection of alpha and beta subunit cRNAs led to a large increase in 86Rb+ uptake, an intracellular alkalinization, and an extracellular medium acidification, as compared to alpha or beta injection alone. These results indicate that the colonic P-ATPase alpha subunit, like the bladder alpha subunit, acts as a functional H+,K+-ATPase, and that co-expression of alpha and beta subunits is required for the function. External K+ activation of the 86Rb+ uptake had a K1/2 of approximately 440 microM for the bladder isoform (consistent with the previously reported value (Jaisser, F., Horisberger, J.D., Geering, K., and Rossier, B.C. (1993) J. Cell. Biol. 123, 1421-1431) and a K1/2 of approximately 730 microM for the colonic isoform. Sch28080 was ineffective to reduce 86Rb+ uptake whereas ouabain inhibited the activity expressed from rat colon alpha subunit with a Ki of 970 microM when measured at the Vmax of the enzyme. We conclude that, when expressed in Xenopus oocytes, the rat colon P-ATPase alpha subunit encodes a ouabain-sensitive H+,K+-ATPase.

Animals↗
PubMed1996

Mutations in subunits of the epithelial sodium channel cause salt wasting with hyperkalaemic acidosis, pseudohypoaldosteronism type 1.

Autosomal recessive pseudohypoaldosteronism type I is a rare life-threatening disease characterized by severe neonatal salt wasting, hyperkalaemia, metabolic acidosis, and unresponsiveness to mineralocorticoid hormones. Investigation of affected offspring of consanguineous union reveals mutations in either the alpha or beta subunits of the amiloride-sensitive epithelial sodium channel in five kindreds. These mutations are homozygous in affected subjects, co-segregate with the disease, and introduce frameshift, premature termination or missense mutations that result in loss of channel activity. These findings demonstrate the molecular basis and explain the pathophysiology of this disease.

Animals↗
PubMed1996

Early death due to defective neonatal lung liquid clearance in alpha-ENaC-deficient mice.

The amiloride-sensitive epithelial sodium channel, ENaC, is a heteromultimeric protein made up of three homologous subunits (alpha, beta and gamma) (1,2). In vitro, assembly and expression of functional active sodium channels in the Xenopus oocyte is strictly dependent on alpha-ENaC--the beta and gamma subunits by themselves are unable to induce an amiloride-sensitive sodium current in this heterologous expression system (2). In vivo, ENaC constitutes the limiting step for sodium absorption in epithelial cells that line the distal renal tubule, distal colon and the duct of several exocrine glands. The adult lung expresses alpha, beta and gamma ENaC (3,4), and an amiloride-sensitive electrogenic sodium reabsorption has been documented in upper and lower airways (3-7), but it is not established whether this sodium transport is mediated by ENaC in vivo. We inactivated the mouse alpha-ENaC gene by gene targeting. Amiloride-sensitive electrogenic Na+ transport was abolished in airway epithelia from alpha-ENaC(-/-) mice. Alpha-ENaC(-/-) neonates developed respiratory distress and died within 40 h of birth from failure to clear their lungs of liquid. This study shows that ENaC plays a critical role in the adaptation of the newborn lung to air breathing.

Animals↗
PubMed1996

Aldosterone-induced increase in the abundance of Na+ channel subunits.

The highly selective, amilorideblockable Na+ channel is a major target to the natriferic action of the mineralocorticoid aldosterone. This rat epithelial Na+ channel (rENaC) has been recently cloned from colon and is composed of three homologous subunits denoted alpha-, beta-, and gamma-rENaC (C. M. Canessa, L. Schild, G. Buell, B. Thorens, L. Gautschi, J.-D. Horisberger, and B. C. Rossier. Nature Lond. 367: 463-467, 1994). We have tested the effects of corticosteroids on the abundance of mRNA coding for each subunit in kidney cortex and distal colon. Chronic treatment of rats with aldosterone or dexamethasone evoked in kidney cortex a small induction of alpha-rENaC and no change in beta- and gamma-rENaC. In distal colon, however, beta- and gamma-rENaC were strongly induced by either aldosterone or dexamethasone, whereas alpha-rENaC was constitutively expressed. Most of the aldosterone-induced increase in beta- and gamma-rENaC mRNA took place during 3-24 h after plasma aldosterone was elevated. A similar differential induction of rENaC subunits in kidney and colon was also evoked by a Na(+)-free diet. The effects of salt deprivation were reversed by resalinating rats with a half time of < 2 h, suggesting a high turnover rate of at least beta- and gamma-rENaC. The data are consistent with the possibility that induction of channel subunits contributes to the chronic but not the acute response to aldosterone in the colon. Such a mechanism is not likely to play a major role in cortical collecting ducts.

Aldosterone↗
PubMed1996

Physiological and pathophysiological role of the epithelial sodium channel in the control of blood pressure.

Blood pressure regulation is an integrated physiological phenomenon known to be influenced by many biological processes and by a variety of environmental factors. Epidemiological studies nevertheless suggest that up to 30% of the variation in blood pressure could be due to genetic factors. Thus, mutations in genes that control blood pressure may be the underlying cause of essential hypertension. Arterial blood pressure is critically dependent on the sodium balance and the regulation of renal sodium excretion is one of the most important homeostatic functions of the body. The identification of genes encoding proteins that transport Na+ across cells of the kidney tubules and of those regulating the activity of these sodium-transporting proteins will therefore bring further insights into the pathophysiology of salt-sensitive hypertension.

Animals↗
PubMed1995

A de novo missense mutation of the beta subunit of the epithelial sodium channel causes hypertension and Liddle syndrome, identifying a proline-rich segment critical for regulation of channel activity.

Liddle syndrome is a mendelian form of hypertension characterized by constitutively elevated renal Na reabsorption that can result from activating mutations in the beta or gamma subunit of the epithelial Na channel. All reported mutations have deleted the last 45-76 normal amino acids from the cytoplasmic C terminus of one of these channel subunits. While these findings implicate these terminal segments in the normal negative regulation of channel activity, they do not identify the amino acid residues that are critical targets for these mutations. Potential targets include the short highly conserved Pro-rich segments present in the C terminus of beta and gamma subunits; these segments are similar to SH3-binding domains that mediate protein-protein interaction. We now report a kindred with Liddle syndrome in which affected patients have a mutation in codon 616 of the beta subunit resulting in substitution of a Leu for one of these highly conserved Pro residues. The functional significance of this mutation is demonstrated both by the finding that this is a de novo mutation appearing concordantly with the appearance of Liddle syndrome in the kindred and also by the marked activation of amiloride-sensitive Na channel activity seen in Xenopus oocytes expressing channels containing this mutant subunit (8.8-fold increase compared with control oocytes expressing normal channel subunits; P = 0.003). These findings demonstrate a de novo missense mutation causing Liddle syndrome and identify a critical channel residue important for the normal regulation of Na reabsorption in humans.

Amino Acid Sequence↗
PubMed1995

CFTR as a cAMP-dependent regulator of sodium channels.

Cystic fibrosis transmembrane regulator (CFTR), the gene product that is mutated in cystic fibrosis (CF) patients, has a well-recognized function as a cyclic adenosine 3',5'-monophosphate (cAMP)-regulated chloride channel, but this property does not account for the abnormally high basal rate and cAMP sensitivity of sodium ion absorption in CF airway epithelia. Expression of complementary DNAs for rat epithelial Na+ channel (rENaC) alone in Madin Darby canine kidney (MDCK) epithelial cells generated large amiloride-sensitive sodium currents that were stimulated by cAMP, whereas coexpression of human CFTR with rENaC generated smaller basal sodium currents that were inhibited by cAMP. Parallel studies that measured regulation of sodium permeability in fibroblasts showed similar results. In CF airway epithelia, the absence of this second function of CFTR as a cAMP-dependent regulator likely accounts for abnormal sodium transport.

3T3 Cells↗
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 mutation in the epithelial sodium channel causing Liddle disease increases channel activity in the Xenopus laevis oocyte expression system.

We have studied the functional consequences of a mutation in the epithelial Na+ channel that causes a heritable form of salt-sensitive hypertension, Liddle disease. This mutation, identified in the original kindred described by Liddle, introduces a premature stop codon in the channel beta subunit, resulting in a deletion of almost all of the C terminus of the encoded protein. Coexpression of the mutant beta subunit with wild-type alpha and gamma subunits in Xenopus laevis oocytes resulted in an approximately 3-fold increase in the macroscopic amiloride-sensitive Na+ current (INa) compared with the wild-type channel. This change in INa reflected an increase in the overall channel activity characterized by a higher number of active channels in membrane patches. The truncation mutation in the beta subunit of epithelial Na+ channel did not alter the biophysical and pharmacological properties of the channel--including unitary conductance, ion selectivity, or sensitivity to amiloride block. These results provide direct physiological evidence that Liddle disease is related to constitutive channel hyperactivity in the cell membrane. Deletions of the C-terminal end of the beta and gamma subunits of rat epithelial Na+ channel were functionally equivalent in increasing INa, suggesting that the cytoplasmic domain of the gamma subunit might be another molecular target for mutations responsible for salt-sensitive forms of hypertension.

Amiloride↗
PubMed1995

Amiloride-sensitive sodium channels in confluent M-1 mouse cortical collecting duct cells.

Confluent M-1 cells show electrogenic Na+ absorption and possess an amiloride-sensitive Na(+)-conductance (Korbmacher et al., J. Gen. Physiol. 102:761-793, 1993). In the present study, we further characterized this conductance and identified the underlying single channels using conventional patch clamp technique. Moreover, we isolated poly(A)+ RNA from M-1 cells to express the channels in Xenopus laevis oocytes, and to check for the presence of transcripts related to the epithelial Na+ channel recently cloned from rat colon (Canessa et al., Nature 361:467-470, 1993). Patch clamp experiments were performed in 6-13-day-old confluent M-1 cells at 37 degrees C. In whole-cell experiments application of 10(-5) M amiloride caused a hyperpolarization of 24.9, SEM +/- 2.2 mV (n = 35) and a reduction of the inward current by 107 +/- 10 pA (n = 51) at a holding potential of -60 mV. Complete removal of bath Na+ had similar effects, indicating that the amiloride-sensitive component of the inward current is a Na+ current. The effect of amiloride was concentration-dependent with half-inhibition at 0.22 microM. The Na+ current saturated with increasing extracellular Na+ concentrations with an apparent Km of 24 mM. Na+ replacement for Li+ demonstrated a higher apical membrane conductance for Li+ than for Na+. In excised inside-out (i/o) or outside-out (o/o) patches from the apical membrane, we observed single-channels which showed slow kinetics and were reversibly inhibited by amiloride. Their average conductance for Na+ was 6.8 +/- 0.5 pS (n = 15) and for Li+ 11.2 +/- 1.0 pS (n = 14). They had no measurable conductance for K+. In o/o patches, channel activity was slightly voltage dependent with an open probability (NPo) of 0.46 +/- 0.14 and 0.16 +/- 0.05 at a holding potential of -100 and 0 mV, respectively (n = 8, P < 0.05). Using the two-microelectrode voltage-clamp technique, we assayed defolliculated stage V-VI Xenopus oocytes for an amiloride-sensitive inward current 1-6 days after injection with H2O or with 20-50 ng of M-1 poly(A)+ RNA. In poly(A)+ RNA-injected oocytes held at -60 or -100 mV application of amiloride (2 microM) reduced the Na-inward current by 25.5 +/- 4.6 nA (n = 25) while it had no effect in H2O-injected oocytes (n = 19). Northern blot analysis of M-1 poly(A+) RNA revealed the presence of transcripts related to the three known subunits of the rat colon Na+ channel (Canessa et al., Nature 367:463-467, 1994). We conclude that the channel in M-1 cells is closely related to the amiloride-sensitive epithelial Na+ channel in the rat colon and that the M-1 cell line provides a useful tool to investigate the biophysical and molecular properties of the corresponding channel in the cortical collecting duct.

Amiloride↗
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↗