Biosynthesis and cell surface delivery of the NHE1 isoform of Na+/H+ exchanger in A6 cells

Coupaye-Gerard, Brigitte; Bookstein, Crescence; Duncan, Pamela G.; Chen, Xi Yin; Smith, Peter; Musch, Mark W.; Ernst, Stephen A.; Chang, Eugene B.; Kleyman, Thomas R.

doi:10.1152/ajpcell.1996.271.5.c1639

Cited by 38 publications

(26 citation statements)

References 19 publications

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“…The Ϸ115-kD band likely corresponds to the mature processed form of NHE1 (91 kD). The Ϸ90-kD (and perhaps also the Ϸ76 kD) band may represent a precursor form of NHE1, as previously reported (Coupaye-Gerard et al 1996). Immunoblotting of plasma membranes with a polyclonal anti-NHE4 antibody showed an immunoreactive band at Ϸ103 kD, which is in agreement with the results of previous studies on fibroblasts transfected with NHE4 cDNA (Bookstein et al 1994) and probably represents glycosylated NHE4.…”

Section: Nhe1 and Nhe4supporting

confidence: 91%

Immunolocalization of Anion Exchanger AE2, Na⁺/H⁺ Exchangers NHE1 and NHE4, and Vacuolar Type H⁺-ATPase in Rat Pancreas

Roussa

Alper

Thévenod

2001

J Histochem Cytochem.

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S U M M A R YWe have studied the expression and localization of several H ϩ and HCO 3 Ϫ transporters, whose presence in the rat pancreas is still unclear. The Cl Ϫ /HCO 3 Ϫ exchanger AE2, the Na ϩ /H ϩ exchangers NHE1 and NHE4, and the 31-kD and 70-kD vacuolar H ϩ -ATPase (V-ATPase) subunits were detected by immunoblotting and immunocytochemical techniques. Immunoblotting of plasma membranes with transporter-specific antibodies revealed protein bands at Ϸ 160 kD for AE2, at Ϸ 90 kD and Ϸ 103 kD for NHE1 and NHE4, respectively, and at 31 kD and 70 kD for V-ATPase. NHE1 and NHE4 were further identified by amplification of isoform-specific cDNA using RT-PCR. Immunohistochemistry revealed a basolateral location of AE2, NHE1, and NHE4 in acinar cells. In ducts, NHE1 and NHE4 were basolaterally located but no AE2 expression was detected. V-ATPase was detected in zymogen granules (ZGs) by immunogold labeling, and basolaterally in duct cells by immunohistochemistry. The data indicate that NHE1 and NHE4 are co-expressed in rat pancreatic acini and ducts. Basolateral acinar AE2 could contribute to Cl Ϫ uptake and/or pH regulation. V-ATPase may be involved in ZG fusion/exocytosis and ductal HCO 3 Ϫ secretion. The molecular identity of the ductal Cl Ϫ /HCO 3 Ϫ exchanger remains unclear.

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Section: Nhe1 and Nhe4supporting

confidence: 91%

Immunolocalization of Anion Exchanger AE2, Na⁺/H⁺ Exchangers NHE1 and NHE4, and Vacuolar Type H⁺-ATPase in Rat Pancreas

Roussa

Alper

Thévenod

2001

J Histochem Cytochem.

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“…35 S]cysteine added to the basolateral surface as described previously (23,24). Cells were then washed once and incubated for 1 h at 28°C in A6 medium supplemented with 2.5 mM methionine, 2.5 mM cysteine, 300 nM aldosterone, and 0, 0.5, or 5 g/ml tunicamycin.…”

Section: The Nucleotide Sequence(s) Reported In This Paper Has Been Smentioning

confidence: 99%

Antiidiotypic Antibody Recognizes an Amiloride Binding Domain within the α Subunit of the Epithelial Na+ Channel

Kieber‐Emmons¹,

Lin²,

Foster³

et al. 1999

Journal of Biological Chemistry

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We previously raised an antibody (RA6.3) by an antiidiotypic approach which was designed to be directed against an amiloride binding domain on the epithelial Na ؉ channel (ENaC). This antibody mimicked amiloride in that it inhibited transepithelial Na ؉ transport across A6 cell monolayers. RA6.3 recognized a 72-kDa polypeptide in A6 epithelia treated with tunicamycin, consistent with the size of nonglycosylated Xenopus laevis ␣ENaC. RA6.3 specifically recognized an amiloride binding domain within the ␣-subunit of mouse and bovine ENaC. The deduced amino acid sequence of RA6.3 was used to generate a three-dimensional model structure of the antibody. The combining site of RA6.3 was epitope mapped using a novel computer-based strategy. Organic residues that potentially interact with the RA6.3 combining site were identified by data base screening using the program LUDI. Selected residues docked to the antibody in a manner corresponding to the ordered linear array of amino acid residues within an amiloride binding domain on the ␣-subunit of ENaC. A synthetic peptide spanning this domain inhibited the binding of RA6.3 to ␣ENaC. This analysis provided a novel approach to develop models of antibody-antigen interaction as well as a molecular perspective of RA6.3 binding to an amiloride binding domain within ␣ENaC.Epithelial Na ϩ channels (ENaCs) 1 are expressed in a variety of tissues, including the distal nephron of the kidney, airway and alveolar epithelia in the lung, surface cells in the distal colon, urinary bladder epithelia, skin, and ducts within salivary and sweat glands (1). These transporters facilitate the movement of Na ϩ across the apical (or luminal) plasma membrane and have a critical role in extracellular fluid volume homeostasis, control of blood pressure, fetal lung maturation, and maintenance of airway fluids (1). ENaCs consist of at least three homologous subunits, termed ␣-, ␤-, and ␥ENaC, and are thought to form a tetrameric complex consisting of 2␣-, 1␤-, and 1␥-subunits (2-5), although one group has suggested a subunit stoichiometry of 3␣-, 3␤-, and 3␥-subunits (6). cDNAs encoding these Na ϩ channel subunits have been isolated and characterized from a variety of species. Each ENaC subunit has two predicted membrane-spanning domains. The amino-and carboxyl-terminal regions of the ENaCs are cytoplasmic, and each subunit has a large ectodomain (7-9). Domains of largely hydrophobic residues are located immediately following the putative first membrane-spanning domains and immediately preceding the second membrane-spanning domains of ␣-, ␤-and ␥rENaC (8, 10, 11). The hydrophobic domain immediately preceding the second membrane-spanning region of ENaC (referred to as the H2 domain) may insert in the membrane and form part of the channel pore (3,7,12).The diuretic drug amiloride is a prototypic inhibitor of epithelial Na ϩ channels. Several sites have been identified within the epithelial Na ϩ channel which participate in amiloride binding. Mutagenesis studies suggest that residues preceding the second ...

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“…A hNHE1 lacking N-glycosylation sites and expressed in Saccharomyces cerevisiae was recently used to create a 22-Å resolution structure (14). However, because glycosylation is important for NHE1 trafficking (15), it is uncertain whether this structure is representative of the mature NHE1.…”

mentioning

confidence: 99%

Structural Modeling and Electron Paramagnetic Resonance Spectroscopy of the Human Na+/H+ Exchanger Isoform 1, NHE1

Nygaard

Lagerstedt

Bjerre

et al. 2011

Journal of Biological Chemistry

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We previously presented evidence that transmembrane domain (TM) IV and TM X-XI are important for inhibitor binding and ion transport by the human Na The ubiquitous plasma membrane Na ϩ /H ϩ exchanger isoform 1 (NHE1) 4 plays central roles in cellular pH and volume homeostasis, cell migration, proliferation, and survival, and increased NHE1 activity contributes to ischemia-reperfusion injury as well as tumor growth and proliferation (1, 2). Hence, the ability to selectively block NHE1, although of high clinical relevance, is hampered by a lack of detailed understanding of NHE1 structure and mechanism of ion translocation. Hydropathy analyses and accessibility studies indicate that NHE1 has 12 transmembrane (TM) segments and a large Cterminal cytoplasmic region (3). Cysteine accessibility studies suggest the presence of two small re-entrant loops between TM IV and TM V (intracellular loop (IL) II) and TM VIII and TM IX (IL IV), respectively, and a larger re-entrant loop between TM IX and TM X (extracellular loop V) (1, 3). Portions of IL II and IL IV are located within the membrane and accessible from either side of the membrane, suggesting that they may form structures lining an aqueous pore and could be involved in ion translocation by NHE1 (3). Extracellular loop V is also interesting in this regard, because it resembles the Ploops found in voltage-gated ion channels (1, 3). These putative re-entrant loops are highly conserved among several NHE1 homologs, consistent with the notion that they are critical for NHE1 function (3-5).A number of regions within the NHE1 protein have been implicated in inhibitor binding, e.g. TM IV and TM IX (6 -11); however, the mechanism(s) of interaction between NHE1 and its commonly used inhibitors, amiloride and benzoyl guanidine type compounds, remain to be fully elucidated.Using a comparative approach based on chimeras generated using human NHE1 (hNHE1) and two NHE1 homologs (flounder paNHE1 and Amphiuma tridactylum NHE1) with high sequence homology to hNHE1 yet markedly different inhibitor profiles (4, 5), we previously obtained novel information on the regions of NHE1 important for inhibitor binding and ion transport (12). These studies confirmed that TM IV plays a central role in inhibitor binding (12) as suggested by earlier point mutation studies (6 -11). Moreover, we demonstrated that regions in TM X-XI and/or IL V and extracellular loop VI are important determinants of inhibitor sensitivity (12).The three-dimensional structure of NHE1 is unknown; however, the structure of the distantly related bacterial (Esch-

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Biosynthesis and cell surface delivery of the NHE1 isoform of Na+/H+ exchanger in A6 cells

Cited by 38 publications

References 19 publications

Immunolocalization of Anion Exchanger AE2, Na⁺/H⁺ Exchangers NHE1 and NHE4, and Vacuolar Type H⁺-ATPase in Rat Pancreas

Immunolocalization of Anion Exchanger AE2, Na⁺/H⁺ Exchangers NHE1 and NHE4, and Vacuolar Type H⁺-ATPase in Rat Pancreas

Antiidiotypic Antibody Recognizes an Amiloride Binding Domain within the α Subunit of the Epithelial Na+ Channel

Structural Modeling and Electron Paramagnetic Resonance Spectroscopy of the Human Na+/H+ Exchanger Isoform 1, NHE1

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Biosynthesis and cell surface delivery of the NHE1 isoform of Na+/H+ exchanger in A6 cells

Cited by 38 publications

References 19 publications

Immunolocalization of Anion Exchanger AE2, Na+/H+ Exchangers NHE1 and NHE4, and Vacuolar Type H+-ATPase in Rat Pancreas

Immunolocalization of Anion Exchanger AE2, Na+/H+ Exchangers NHE1 and NHE4, and Vacuolar Type H+-ATPase in Rat Pancreas

Antiidiotypic Antibody Recognizes an Amiloride Binding Domain within the α Subunit of the Epithelial Na+ Channel

Structural Modeling and Electron Paramagnetic Resonance Spectroscopy of the Human Na+/H+ Exchanger Isoform 1, NHE1

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Product

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Immunolocalization of Anion Exchanger AE2, Na⁺/H⁺ Exchangers NHE1 and NHE4, and Vacuolar Type H⁺-ATPase in Rat Pancreas

Immunolocalization of Anion Exchanger AE2, Na⁺/H⁺ Exchangers NHE1 and NHE4, and Vacuolar Type H⁺-ATPase in Rat Pancreas