In many epithelial tissues in the body, the rate of Na ؉ reabsorption is governed by the activity of the epithelial sodium channel (ENaC). The assembly, trafficking, and turnover of the three ENaC subunits (␣, ␤, and ␥) is complex and not well understood. Recent experiments suggest that ENaC must be proteolytically cleaved for maximal activity and may explain the discrepancies reported in prior biochemical approaches focused on quantitating the trafficking and half-life of full-length subunits. As an alternative approach to examining the dynamics of ENaC subunits, we have generated doxycycline-repressible replication-defective recombinant adenoviruses encoding individual epitope-tagged mouse ENaC subunits and expressed these in polarized MDCK I cells. Co-infection with these viruses encoding all three subunits generates robust amiloride-sensitive currents in polarized MDCK cells. Significant current was also observed in cells expressing ␣-and ␥-mENaC in the absence of ␤-mENaC. These currents did not appear to result from association with endogenous canine ␤-ENaC. Treatment of ␣␤␥-expressing cells with cycloheximide (CHX) resulted in the rapid inhibition (within 3 h) of ϳ50 -80% of the initial current; however, a sizable fraction of the initial current remained even after 6 h of CHX. By contrast, CHX addition to cells expressing only ␣-and ␥-mENaC resulted in rapid decay in current with no residual fraction. Our data suggest that ENaC channels of differing stoichiometries are differentially trafficked and degraded and provide support for the possibility that noncoordinate trafficking of ENaC subunits may function in vivo as a mechanism to modulate ENaC activity.The epithelial sodium channel (ENaC) 1 is an apical heterotetramer containing ␣, ␤, and ␥ subunits that serves as the rate-limiting step in sodium reabsorption in a number of tissues (reviewed in Ref. 1). Given the clinical importance of ENaC activity, the assembly, trafficking, and response to hormonal stimulation of ENaC subunits have been subjects of considerable study and debate. While biochemical studies from several laboratories universally report a short half-life for the total pool of newly synthesized ENaC subunits (typically 1-2 h) (2-7), there is considerable dispute regarding the stability of ENaC subunits that have reached the plasma membrane. Some studies report rapid decay of all three cell surface subunits in polarized MDCK and A6 cells (4, 5), whereas we and others have observed long half-lives of ␣-and ␥-ENaC that were biotinylated at the apical plasma membrane of A6 cells (6,8). We use the term noncoordinate regulation to describe this differential turnover of ENaC subunits and have proposed possible molecular models for this observation based on experimental results from many laboratories (6, 9).Considerable data suggest that activation of ENaC channels is regulated by proteolytic enzymes (10 -12). Recently, new evidence has emerged that suggests that activation of ENaC involves direct furin-mediated cleavage of ␣-and ␥-ENaC subunits wi...