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Stability of functional ENaC at the apical membrane of A6 cells.
Author(s) -
Yu Ling,
Yue Q,
Yue G,
Eaton Douglas C,
Malik Bela
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a796-c
Subject(s) - epithelial sodium channel , apical membrane , microbiology and biotechnology , brefeldin a , chemistry , protein subunit , xenopus , golgi apparatus , biology , biochemistry , endoplasmic reticulum , sodium , membrane , organic chemistry , gene
Epithelial sodium channel (ENaC) play an essential role in maintaining total body fluid and electrolyte homeostasis. ENaC protein consisting of three subunits alpha, beta and gamma; each ENaC subunit consists of a large extracellular domain and a small intracellular N‐ and C‐terminal domain. Stability of functional ENaC at the apical membrane is unresolved because ENaC maturation involves not only glycosylation and disulfide bonds but also proteolytic cleavage of ENaC subunits, and therefore, the molecular size of functional ENaC subunits remains unclear. Some investigators analyzed stability of functional ENaC at the plasma membrane either by measuring trans‐epithelial or whole cell current, and therefore, could not reach a consensus. In this study we measured stability of ENaC in Xenopus laevis distal nephron epithelial cell line (A6) by blocking protein synthesis and also by disrupting golgi apparatus and then measuring single channel ENaC activity by patch clamp and also membrane resident ENaC subunits by western blot analysis. ENaC function and membrane resident ENaC protein levels were measured for several hrs after A6 cells were incubated with either inhibitors of protein translation, Cycloheximide or Puromycine, or disruption of golgi apparatus by either Brefeldin A or Nocodazole. Our electrophysiological data suggests that inhibition of protein synthesis or disruption of golgi apparatus decrease the number of ENaC at the apical membrane with no significant change in channel open probability. This decrease in number of channels reached half maximum in 2–6 hrs after the drug treatment. The biochemical data also suggest a decrease in number of ENaC at the apical membrane. Supported by DK‐37963, DK‐061521 and DK‐065080‐01A1