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Papers In Press, published online ahead of print August 6, 2002
J. Biol. Chem, 10.1074/jbc.C200309200
Submitted on May 19, 2002
Revised on July 31, 2002
Accepted on August 6, 2002
Renal Division/Department of Medicine, University of Pittsburgh, Pittsburgh, PA 15261
Corresponding Author: whill+{at}pitt.edu
The epithelial sodium channel (ENaC) present in kidney collecting duct, distal colon and the lung is responsible for salt reabsorption and whole body volume regulation. It is composed of three homologous subunits,
,
and
and mutations to these subunits can lead to the salt wasting disease pseudohypoaldosteronism type I, associated with decreased channel density at the plasma membrane; or to the hypertensive disorder, Liddle's syndrome, in which channel residency time at the plasma membrane is enhanced. Regulation of ENaC trafficking and turnover is therefore critical to sodium homeostasis. In this study we examined whether ENaC is present in the cholesterol-enriched microdomains commonly called lipid rafts, in the endogenously expressing A6 cell line. We demonstrate that a fraction of
,
and
-ENaC are present in detergent-insoluble membranes, that subunits exist in membranes which float on discontinuous sucrose density gradients and that methyl-
-cyclodextrin treatment causes a redistribution of ENaC subunits to higher density membranes. Furthermore, chronic aldosterone stimulation results in a shift in the membrane density of all three subunits. Biotinylation of apical membrane proteins revealed that ENaC is present in lipid rafts on the plasma membrane. In conclusion, these results show that ENaC is present in lipid rafts both intracellularly and on the cell surface. Raft association may be important for trafficking and/or function of the channel.
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