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B C Rossier

Publications and source records attributed to B C Rossier.

At least 73 records · Page 4Linked to original sources
PubMed1995

Relative expression of the human epithelial Na+ channel subunits in normal and cystic fibrosis airways.

The availability of the newly cloned subunits (alpha, beta, gamma) of the epithelial Na+ channel (ENaC) permits molecular studies of the pathogenesis of the abnormal Na+ transport rates of cystic fibrosis (CF) airway epithelia. Northern analyses of airway epithelia showed that both normal and CF airway epithelia express ENaC subunit mRNAs in a ratio of alpha > beta > gamma. In situ hybridization studies revealed expression of all three ENaC subunits in the superficial epithelium and the alpha- and beta-subunits in the gland ductular and acinar epithelium of both normal and CF airways. Ribonuclease protection assays revealed that the steady-state levels of alpha-, beta-, and gamma-ENaC mRNAs were similar in CF and normal airway superficial epithelia. These findings indicate that 1) Na+ transport defects in CF airways disease may be expressed in glandular acinar and ductal epithelium as well as superficial epithelium, and 2) the molecular pathogenesis of Na+ hyperabsorption in CF airways does not reflect increased levels of Na+ channel mRNAs, and probably number, but reflects an absence of the normal inhibitory regulation of Na+ channels by CF transmembrane conductance regulator proteins.

Base Sequence↗
PubMed1995

Cloning of a bovine renal epithelial Na+ channel subunit.

A bovine homologue of the rat and human epithelial Na+ channel subunits, alpha-rENaC and alpha-hENaC, was cloned. The cDNA clone, termed alpha-bENaC, was isolated from a bovine renal papillary collecting duct cDNA expression library. The bovine cDNA is 3,584 base pairs (bp) long, has an open reading frame of 2,094 bp encoding a 697-amino acid protein, and is 75-85% homologous to its rat and human counterparts. In vitro translation of the transcribed cRNA yields an 80-kDa polypeptide and one at 92 kDa in the presence of pancreatic microsomes. The clone exhibits consensus sequences for N-linked glycosylation and for phosphorylation by protein kinase C, but not for protein kinase A. After expression in Xenopus laevis oocytes, a small amiloride-sensitive Na+ conductance that exhibited inward rectification and a reversal potential greater than +30 mV, consistent with the predicted equilibrium potential for Na+, was identified. The expressed alpha-bENaC-associated Na+ current was not responsive to elevations in adenosine 3',5'-cyclic monophosphate but could be stimulated by phorbol 12-myristate 13-acetate, an activator of protein kinase C. alpha-bENaC also formed amiloride-sensitive chimeric channels when coexpressed with the rat beta- and gamma-ENaC subunits in Xenopus oocytes. alpha-bENaC therefore represents a novel isoform of a growing family of epithelial Na+ channels.

Amino Acid Sequence↗
PubMed1995

Lung epithelial Na channel subunits are differentially regulated during development and by steroids.

Because the alpha-subunit of the rat lung epithelial Na channel (rENaC) is not expressed until late fetal gestation, the developmental immaturity of alpha-rENaC may be involved in the premature fetal lung's inability to mount a Na-absorptive response to appropriate agonists. As previous work has shown that the beta- and gamma-rENaC subunits of the Na channel are required for maximal alpha-rENaC activity, we determined their developmental expression in the fetal lung. In addition, because thyroid and corticosteroid therapy can mature the in vivo fetal lamb lung's ability to transport Na, we wished to determine whether such treatment increased the expression of alpha-, beta-, and gamma-rENaC. Lungs were harvested from normal rat fetuses of 17 through 22 days gestation (term = 22 days), normal rat pups during the first week of life, and adult rats. Initial expression of alpha-rENaC was detected at 19 days gestation and progressively increased in utero. beta- and gamma-rENaC mRNA were not detected until 21 and 22 days gestation, and then only at very low levels. During the first week after birth, the levels of alpha-rENaC declined, whereas beta- and gamma-rENaC mRNA levels increased. This pre- and postnatal pattern of alpha-rENaC expression correlates with the endogenous glucocorticosteroid levels in the fetus and the rat pup's early postnatal corticosteroid resistance. Combined or separate treatment of pregnant rats (16 through 22 days gestational age) with thyroid-releasing hormone (TRH) and/or dexamethasone (Dex) for 48 h showed that Dex, but not TRH, could increase fetal lung alpha-rENaC mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Cortex Hormones↗
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↗
PubMed1994

SCNN1, an epithelial cell sodium channel gene in the conserved linkage group on mouse chromosome 6 and human chromosome 12.

SCNN1, a gene encoding a nonvoltage-gated sodium channel, was detected using a rat colon cDNA probe with homology to Caenorhabditis elegans degenerin genes. Human SCNN1 was assigned to chromosome 12 using the NIGMS hybrid mapping panel 2. Mouse SCNN1 was mapped to a conserved linkage group on distal chromosome 6. The observed order of mouse genes was centromere-Raf1-(2.1 +/- 2.1)-Scnn1, Vwf-(1.9 +/- 1.9)-Ntf3, with 0/101 recombinants between Scnn1 and Vwf. No rearrangements of genomic DNA were detected in the linked mouse mutations deaf waddler (dfw) and opisthotonus (opt).

Animals↗
PubMed1994

An SH3 binding region in the epithelial Na+ channel (alpha rENaC) mediates its localization at the apical membrane.

The amiloride-sensitive Na+ channel constitutes the rate-limiting step for Na+ transport in epithelia. Immunolocalization and electrophysiological studies have demonstrated that this channel is localized at the apical membrane of polarized epithelial cells. This localization is essential for proper channel function in Na+ transporting epithelia. In addition, the channel has been shown to associate with the cytoskeletal proteins ankyrin and alpha-spectrin in renal epithelia. However, the molecular mechanisms underlying the cytoskeletal interactions and apical membrane localization of this channel are largely unknown. In this study we show that the putative pore forming subunit of the rat epithelial (amiloride-sensitive) Na+ channel (alpha ENaC) binds to alpha-spectrin in vivo, as determined by co-immunoprecipitation. This binding is mediated by the SH3 domain of alpha-spectrin which binds to a unique proline-rich sequence within the C-terminal region of alpha rENaC. Accordingly, the C-terminal region is sufficient to mediate binding to intact alpha-spectrin from alveolar epithelial cell lysate. When microinjected into the cytoplasm of polarized primary rat alveolar epithelial cells, a recombinant fusion protein containing the C-terminal proline-rich region of alpha rENaC localized exclusively to the apical area of the plasma membrane, as determined by confocal microscopy. This localization paralleled that of alpha-spectrin. In contrast, microinjected fusion protein containing the N-terminal (control) protein of alpha rENaC remained diffuse within the cytoplasm. These results suggest that an SH3 binding region in alpha rENaC mediates the apical localization of the Na+ channel. Thus, cytoskeletal interactions via SH3 domains may provide a novel mechanism for retaining proteins in specific membranes of polarized epithelial cells.

Amino Acid Sequence↗
PubMed1994

Phosphorylation of the Na,K-ATPase alpha-subunit by protein kinase A and C in vitro and in intact cells. Identification of a novel motif for PKC-mediated phosphorylation.

Na,K-ATPase is a potential target for regulatory phosphorylation by protein kinase A and C (PKA and PKC). To identify the phosphorylation sites, we have mutated the alpha 1-subunit of Bufo marinus in a highly conservative PKA and in 20 different PKC consensus sequences. The mutants were expressed in Xenopus oocytes and their phosphorylation capacity tested in homogenates upon stimulation of PKA or PKC. While serine 943 (Ser-943) was identified as a unique target site for PKA, none of the PKC consensus serine or threonine residues are implicated in PKC phosphorylation. Controlled trypsinolysis of phosphorylated alpha-subunits of various purified enzyme preparations and of alpha/beta complexes from oocyte homogenates revealed that PKC phosphorylation was exclusively associated with the N terminus. A fusion protein containing the first 32 amino acids of the Bufo alpha-subunit was phosphorylated in vitro and serine and threonine residues (Thr-15 and Ser-16) in this region were identified by site-directed mutagenesis as the PKC phosphorylation sites. Finally, the Bufo alpha-subunit was phosphorylated by protein kinases in transfected COS-7 cells. In intact cells, PKA stimulation induced phosphorylation exclusively on Ser-943 and PKC stimulation mainly on Thr-15 and Ser-16, which are contained in a novel PKC phosphorylation motif.

Amino Acid Sequence↗
PubMed1994

Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits.

The amiloride-sensitive epithelial sodium channel constitutes the rate-limiting step for sodium reabsorption in epithelial cells that line the distal part of the renal tubule, the distal colon, the duct of several exocrine glands, and the lung. The activity of this channel is upregulated by vasopressin and aldosterone, hormones involved in the maintenance of sodium balance, blood volume and blood pressure. We have identified the primary structure of the alpha-subunit of the rat epithelial sodium channel by expression cloning in Xenopus laevis oocytes. An identical subunit has recently been reported. Here we identify two other subunits (beta and gamma) by functional complementation of the alpha-subunit of the rat epithelial Na+ channel. The ion-selective permeability, the gating properties and the pharmacological profile of the channel formed by coexpressing the three subunits in oocytes are similar to that of the native channel.

Amiloride↗
PubMed1994

Cell-specific expression of epithelial sodium channel alpha, beta, and gamma subunits in aldosterone-responsive epithelia from the rat: localization by in situ hybridization and immunocytochemistry.

A highly selective, amiloride-sensitive, epithelial sodium channel from rat colon (rENaC), composed of three homologous subunits termed alpha, beta, and gamma rENaC, has been cloned by functional expression and was proposed to mediate electrogenic sodium reabsorption in aldosterone-responsive epithelia. To determine whether rENaC could account for sodium absorption in vivo, we studied the cellular localization of the sodium channel messenger RNA subunits by in situ hybridization and their cellular and subcellular distribution by immunocytochemistry in the kidney, colon, salivary, and sweat glands of the rat. In the kidney, we show that the three subunit mRNAs are specifically co-expressed in the renal distal convoluted tubules (DCT), connecting tubules (CNT), cortical collecting ducts (CCD), and outer medullary collecting ducts (OMCD), but not in the inner medullary collecting ducts (IMCD). We demonstrate co-localization of alpha, beta, and gamma subunit proteins in the apical membrane of a majority of cells of CCD and OMCD. Our data indicate that alpha, beta, and gamma subunit mRNAs and proteins are co-expressed in the distal nephron (excepting IMCD), a localization that correlates with the previously described physiological expression of amiloride-sensitive electrogenic sodium transport. Our data, however, suggest that another sodium transport protein mediates electrogenic amiloride-sensitive sodium reabsorption in IMCD. We also localized rENaC to the surface epithelial cells of the distal colon and to the secretory ducts of the salivary gland and sweat gland, providing further evidence consistent with the hypothesis that the highly selective, amiloride-sensitive sodium channel is physiologically expressed in aldosterone-responsive cells.

Aldosterone↗
PubMed1994

Modulation of the Na,K-pump function by beta subunit isoforms.

To study the role of the Na,K-ATPase beta subunit in the ion transport activity, we have coexpressed the Bufo alpha 1 subunit (alpha 1) with three different isotypes of beta subunits, the Bufo Na,K-ATPase beta 1 (beta 1NaK) or beta 3 (beta 3NaK) subunit or the beta subunit of the rabbit gastric H,K-ATPase (beta HK), by cRNA injection in Xenopus oocyte. We studied the K+ activation kinetics by measuring the Na,K-pump current induced by external K+ under voltage clamp conditions. The endogenous oocyte Na,K-ATPase was selectively inhibited, taking advantage of the large difference in ouabain sensitivity between Xenopus and Bufo Na,K pumps. The K+ half-activation constant (K1/2) was higher in the alpha 1 beta 3NaK than in the alpha 1 beta 1NaK groups in the presence of external Na+, but there was no significant difference in the absence of external Na+. Association of alpha 1 and beta HK subunits produced active Na,K pumps with a much lower apparent affinity for K+ both in the presence and in the absence of external Na+. The voltage dependence of the K1/2 for external K+ was similar with the three beta subunits. Our results indicate that the beta subunit has a significant influence on the ion transport activity of the Na,K pump. The small structural differences between the beta 1NaK and beta 3NaK subunits results in a difference of the apparent affinity for K+ that is measurable only in the presence of external Na+, and thus appears not to be directly related to the K+ binding site. In contrast, association of an alpha 1 subunit with a beta HK subunit results in a Na,K pump in which the K+ binding or translocating mechanisms are altered since the apparent affinity for external K+ is affected even in the absence of external Na+.

Animals↗
PubMed1994

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.

Animals↗
PubMed1994

Polyadenylation of Na(+)-K(+)-ATPase beta 1-subunit during early development of Xenopus laevis.

In fully grown Xenopus oocytes, the synthesis of beta-subunits is limiting for the formation of functional Na(+)-K(+)-adenosinetriphosphatase alpha/beta-complexes (Geering, K. FEBS Lett. 285: 189-193, 1991). In the present study, we show that during oocyte growth (from stage I to stage VI) alpha 1-, but not beta 1- or beta 3-isoform, mRNAs accumulate. In addition, beta-mRNAs are apparently sequestered in an untranslated pool in fully grown oocytes (stage VI). From fertilization to morulation, the total pools of alpha 1-, beta 1-, or beta 3-mRNAs vary little. Whereas polyadenylated [poly(A)+] alpha 1- and beta 3-isoform mRNAs did not change significantly, poly(A)+ beta 1-mRNA abundance increased three- to fourfold at morulation, accompanied by a parallel increase in beta 1-protein synthesis. After midblastula transition (i.e., at early gastrula) and during neurulation, poly(A)+ alpha 1- and beta 3-mRNAs accumulated rapidly, whereas poly(A)+ beta 1-mRNA accumulation was delayed by approximately 2 h, beginning only at early neurula. Our results indicate that 1) the abundance of poly(A)+ beta 1-mRNA is rate limiting during embryonic development for the assembly of alpha 1/beta 1-heterodimers, shown to be involved in the vectorial transport of sodium in kidney cells, and 2) the polyadenylation of beta 1-mRNA is a rate-limiting factor during morulation for the synthesis and assembly of new sodium pumps at the time of blastocoel fluid formation. The 3'-untranslated region of beta 1-mRNA (but not of alpha 1-mRNA) expresses cytoplasmic polyadenylation elements (CPEs) with the consensus sequence AXX-AUUUU(A/U)(A/U)(A/U). A role of CPE in the differential polyadenylation of alpha 1- and beta 1-mRNA is proposed.

Aging↗
PubMed1994

Membrane topology of the epithelial sodium channel in intact cells.

The highly selective amiloride-sensitive epithelial sodium channel is formed of three homologous subunits termed alpha-, beta-, and gamma-rENaC. Each subunit has two putative transmembrane domains (M1 and M2), yielding a protein with a large (approximately 50 kDa) hydrophilic loop (between M1 and M2) and short hydrophilic NH2- and COOH-termini (9 and 10 kDa). All three subunits are glycosylated in a cell-free translation assay, demonstrating that they share in vitro a common pattern of membrane insertion. The membrane topology of the alpha-rENaC subunit in intact cells was studied in Xenopus laevis oocytes. We demonstrate that 1) all six potential N-linked glycosylation sites (N190, N259, N320, N339, N424, and N538) of the large hydrophilic loop are used in intact cells; 2) the glycosylation of alpha-rENaC does not play a significant role in the functional expression of the channel; and 3) the two hydrophobic domains M1 (A109-F131) and M2 (S588-L612) serve in intact cells as start- and stop-transfer signals, respectively. We conclude that alpha-rENaC spans the membrane twice with the short NH2- and COOH-terminal ends on the cytoplasmic side and a large hydrophilic loop in the extracellular space.

Amiloride↗
PubMed1993

Mutation of a tyrosine in the H3-H4 ectodomain of Na,K-ATPase alpha subunit confers ouabain resistance.

In a highly ouabain-resistant clone from the Madin-Darby canine kidney cell line (Ki > 4 mM), we have previously identified mutations (C113 to Y or C113 to F) in the first transmembrane helix (H1) of the Na,K-ATPase alpha subunit that increase the Ki of a ouabain-sensitive Na,K pump by 1000-fold. Here we report another mutation (Y317 to C) located in the extracellular segment that joins the third and fourth transmembrane domains (H3-H4 ectodomain) of the alpha subunit that also changes ouabain sensitivity of the Na pump. When this mutation (Y317C) was introduced into the Na,K-ATPase alpha 1 subunit of Xenopus laevis, the ouabain inhibition constant increased by a factor of 5, from 130 (wild type) to 800 nM (mutant). However, the expression of double mutants (C113Y + Y317C) in Xenopus oocytes resulted in highly ouabain-resistant Na,K pumps (Ki approximately 7 mM), reproducing the phenotype of the original Madin-Darby canine kidney cell line. When a more conservative change (Y317F) was introduced into the Na,K-ATPase alpha 1 subunit of X. laevis, the ouabain koff increased and expression of double mutants (C113Y + Y317F) resulted in an intermediate ouabain-resistant Na,K pump (Ki approximately 500 microM). We propose that, in addition to the previously identified H1-H2 ectodomain of Na,K-ATPase alpha subunit, the H3-H4 ectodomain also participates in the structure and/or the function of the ouabain binding site. In this respect, the Y317 plays a critical role since the most conservative change Y313 to F is sufficient to significantly affect ouabain binding.

Amino Acid Sequence↗
PubMed1993

Epithelial sodium channel related to proteins involved in neurodegeneration.

The epithelial amiloride-sensitive sodium channel constitutes the rate limiting step for sodium reabsorbtion by the epithelial lining the distal part of the kidney tubule, the urinary bladder and the distal colon. Reabsorbtion of sodium through this channel, which is regulated by hormones such as aldosterone and vasopressin, is one of the essential mechanisms involved in the regulation of sodium balance, blood volume and blood pressure. Here we isolate a DNA from epithelial cells of rat distal colon and identify it by functional expression of an amiloride-sensitive sodium current in Xenopus oocyte. The deduced polypeptide (698 amino acids) has at least two putative transmembrane segments. Expression of this protein in Xenopus oocytes reconstitutes the functional properties of the highly selective amiloride-sensitive, epithelial sodium channel. The gene encoding this rat sodium channel subunit shares significant sequence similarity with mec-4 and deg-1, members of a family of Caenorhabditis elegans genes involved in sensory touch transduction and, when mutated, neuronal degeneration. We propose that the gene products of these three genes are members of a gene family coding for cation channels.

Absorption↗
PubMed1993

Primary sequence and functional expression of a novel beta subunit of the P-ATPase gene family.

The cortical collecting tubule (CCT) of the mammalian kidney reabsorbs sodium and potassium, processes that are mediated by Na/K-ATPase and H/K-ATPase. CCT is also an important site for proton secretion, which is driven, in part, by H/K-ATPase. Na/K-ATPase and H/K-ATPase are members of the ion-motive P-ATPase gene family. They are closely related plasma membrane proteins which consist of alpha beta heterodimers. The urinary bladder of the toad Bufo marinus is the amphibian counterpart of mammalian CCT. We have previously characterized a ouabain-resistant Na/K-ATPase [see ref. 17], from TBM cells, a clonal cell line derived from the toad bladder, which expresses transepithelial sodium transport. In the present study, we report the primary sequence and functional expression of a novel beta subunit (beta bladder = beta b1) isolated from a toad bladder epithelial cell cDNA library. The deduced polypeptide is 299 amino acids in length and has a predicted molecular mass of 33 kDa. The beta b1 protein exhibits 35% amino acid identity to the previously characterized beta 1 of B. marinus Na/K-ATPase and 39% identity with beta 3 of B. marinus Na/K-ATPase. It shares 38% identity with the mammalian beta gastric H/K-ATPase and 52% with the mammalian beta 2 Na/K-ATPase. Northern blot analysis shows that a 1.4 x 10(3)-base mRNA is expressed at a high level in bladder epithelial cells and eye and at a trace level in kidney; it is not detectable in significant amounts in the stomach, colon and small intestine.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphatases↗
PubMed1993

Mechanisms of urinary K+ and H+ excretion: primary structure and functional expression of a novel H,K-ATPase.

The kidney plays an essential role in regulating potassium and acid balance. A major site for these regulations is in the collecting tubule. In the present study, we report the primary sequence of a novel alpha subunit of the P-ATPase gene family, which we isolated from the urinary bladder epithelium of the toad Bufo marinus, the amphibian equivalent of the mammalian collecting tubule. The cDNA encodes a protein of 1,042 amino acids which shares approximately 67% identity with the alpha 1 subunit of the ouabain-inhibitable Na,K-ATPase and approximately 69% identity with the alpha subunit of the SCH28080-inhibitable gastric H,K-ATPase. When coexpressed in Xenopus oocytes with a beta subunit isolated from the same cDNA library, the ATPase is able to transport rubidium (a potassium surrogate) inward, and hydrogen outward, leading to alkalization of the intracellular compartment and acidification of the external medium. The novel ATPase has a unique pharmacological profile showing intermediate sensitivity to both ouabain and SCH28080. Our findings indicate that the bladder ATPase is a member of a new ion motive P-ATPase subfamily. The bladder ATPase is expressed in the urinary tract but not in the stomach or the colon. This H,K-ATPase may be one of the molecules involved in H+ and K+ homeostasis, mediating the transport of these ions across urinary epithelia and therefore regulating their urinary excretion.

Amino Acid Sequence↗