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Surface Expression of Epithelial Na Channel Protein in Rat Kidney

Expression of epithelial Na channel (ENaC) protein in the apical membrane of rat kidney tubules was assessed by biotinylation of the extracellular surfaces of renal cells and by membrane fractionation. Rat kidneys were perfused in situ with solutions containing NHS-biotin, a cell-impermeant biotin d...

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Detalles Bibliográficos
Autores principales: Frindt, Gustavo, Ergonul, Zuhal, Palmer, Lawrence G.
Formato: Texto
Lenguaje:English
Publicado: The Rockefeller University Press 2008
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2391254/
https://www.ncbi.nlm.nih.gov/pubmed/18504317
http://dx.doi.org/10.1085/jgp.200809989
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author Frindt, Gustavo
Ergonul, Zuhal
Palmer, Lawrence G.
author_facet Frindt, Gustavo
Ergonul, Zuhal
Palmer, Lawrence G.
author_sort Frindt, Gustavo
collection PubMed
description Expression of epithelial Na channel (ENaC) protein in the apical membrane of rat kidney tubules was assessed by biotinylation of the extracellular surfaces of renal cells and by membrane fractionation. Rat kidneys were perfused in situ with solutions containing NHS-biotin, a cell-impermeant biotin derivative that attaches covalently to free amino groups on lysines. Membranes were solubilized and labeled proteins were isolated using neutravidin beads, and surface β and γENaC subunits were assayed by immunoblot. Surface αENaC was assessed by membrane fractionation. Most of the γENaC at the surface was smaller in molecular mass than the full-length subunit, consistent with cleavage of this subunit in the extracellular moiety close to the first transmembrane domains. Insensitivity of the channels to trypsin, measured in principal cells of the cortical collecting duct by whole-cell patch-clamp recording, corroborated this finding. ENaC subunits could be detected at the surface under all physiological conditions. However increasing the levels of aldosterone in the animals by feeding a low-Na diet or infusing them directly with hormone via osmotic minipumps for 1 wk before surface labeling increased the expression of the subunits at the surface by two- to fivefold. Salt repletion of Na-deprived animals for 5 h decreased surface expression. Changes in the surface density of ENaC subunits contribute significantly to the regulation of Na transport in renal cells by mineralocorticoid hormone, but do not fully account for increased channel activity.
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spelling pubmed-23912542008-12-01 Surface Expression of Epithelial Na Channel Protein in Rat Kidney Frindt, Gustavo Ergonul, Zuhal Palmer, Lawrence G. J Gen Physiol Articles Expression of epithelial Na channel (ENaC) protein in the apical membrane of rat kidney tubules was assessed by biotinylation of the extracellular surfaces of renal cells and by membrane fractionation. Rat kidneys were perfused in situ with solutions containing NHS-biotin, a cell-impermeant biotin derivative that attaches covalently to free amino groups on lysines. Membranes were solubilized and labeled proteins were isolated using neutravidin beads, and surface β and γENaC subunits were assayed by immunoblot. Surface αENaC was assessed by membrane fractionation. Most of the γENaC at the surface was smaller in molecular mass than the full-length subunit, consistent with cleavage of this subunit in the extracellular moiety close to the first transmembrane domains. Insensitivity of the channels to trypsin, measured in principal cells of the cortical collecting duct by whole-cell patch-clamp recording, corroborated this finding. ENaC subunits could be detected at the surface under all physiological conditions. However increasing the levels of aldosterone in the animals by feeding a low-Na diet or infusing them directly with hormone via osmotic minipumps for 1 wk before surface labeling increased the expression of the subunits at the surface by two- to fivefold. Salt repletion of Na-deprived animals for 5 h decreased surface expression. Changes in the surface density of ENaC subunits contribute significantly to the regulation of Na transport in renal cells by mineralocorticoid hormone, but do not fully account for increased channel activity. The Rockefeller University Press 2008-06 /pmc/articles/PMC2391254/ /pubmed/18504317 http://dx.doi.org/10.1085/jgp.200809989 Text en © 2008 Frindt et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jgp.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
spellingShingle Articles
Frindt, Gustavo
Ergonul, Zuhal
Palmer, Lawrence G.
Surface Expression of Epithelial Na Channel Protein in Rat Kidney
title Surface Expression of Epithelial Na Channel Protein in Rat Kidney
title_full Surface Expression of Epithelial Na Channel Protein in Rat Kidney
title_fullStr Surface Expression of Epithelial Na Channel Protein in Rat Kidney
title_full_unstemmed Surface Expression of Epithelial Na Channel Protein in Rat Kidney
title_short Surface Expression of Epithelial Na Channel Protein in Rat Kidney
title_sort surface expression of epithelial na channel protein in rat kidney
topic Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2391254/
https://www.ncbi.nlm.nih.gov/pubmed/18504317
http://dx.doi.org/10.1085/jgp.200809989
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