title: Surface expression of epithelial Na channel protein in rat kidney.
abstract: 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 beta and gammaENaC subunits were assayed by immunoblot. Surface alphaENaC was assessed by membrane fractionation. Most of the gammaENaC 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.
authors.affiliation: Department of Physiology and Biophysics, Weill Medical College of Cornell University, New York, NY 10065, USA.
authors.forename: Gustavo
authors.initials: G
authors.lastname: Frindt
authors.forename: Zuhal
authors.initials: Z
authors.lastname: Ergonul
authors.forename: Lawrence G
authors.initials: LG
authors.lastname: Palmer
mesh_terms: D000450:Aldosterone / Q000378:metabolism; D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives / Q000737:chemistry; D002458:Cell Fractionation; D002462:Cell Membrane / Q000502:physiology; D016764:Cell Polarity / Q000502:physiology; D002846:Chromatography, Affinity; D004847:Epithelial Cells / Q000502:physiology; D053503:Epithelial Sodium Channels / Q000096:biosynthesis*; D005260:Female; D015870:Gene Expression / Q000502:physiology*; D015640:Ion Channel Gating / Q000187:drug effects; D007685:Kidney Tubules, Collecting / Q000502:physiology*; D008239:Lysine / Q000737:chemistry; D008297:Male; D009865:Oocytes; D018408:Patch-Clamp Techniques; D021122:Protein Subunits / Q000096:biosynthesis; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology; D013388:Succinimides / Q000737:chemistry; D014357:Trypsin / Q000494:pharmacology; D014981:Xenopus
chemical_list: D053503:Epithelial Sodium Channels; D021122:Protein Subunits; D013388:Succinimides; C033788:biotinyl N-hydroxysuccinimide ester; D000450:Aldosterone; D001710:Biotin; D012964:Sodium; D014357:Trypsin; D008239:Lysine