Endogenously expressed epithelial sodium channel is present in lipid rafts in A6 cells

The epithelial sodium channel (ENaC) present in the 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, alpha, beta, and gamma, 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 alpha, beta, and gamma ENaC is present in detergent-insoluble membranes, that subunits exist in membranes that float on discontinuous sucrose density gradients, and that methyl-beta-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.