Abstract
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 (alpha, beta, and gamma) 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 alpha- and gamma-mENaC in the absence of beta-mENaC. These currents did not appear to result from association with endogenous canine beta-ENaC. Treatment of alpha beta gamma-expressing cells with cycloheximide (CHX) resulted in the rapid inhibition (within 3 h) of approximately 50-80% 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 alpha- and gamma-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.
Highlights
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
Because sodium current rather than biochemical levels of ENaC subunits is the ultimate expression of ENaC activity, we have taken an alternative approach to examining ENaC trafficking and dynamics by measuring the stability of ENaC current upon inhibition of new protein synthesis
MDCK I Cells Express Undetectable Levels of Endogenous -ENaC Subunit—We considered the possibility that the Isc we observed in MDCK I cells infected with AVs encoding ␣and ␥-mouse ENaC (mENaC) was due to assembly of these subunits with endogenous canine -ENaC
Summary
The epithelial sodium channel (ENaC) 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). Protein expression in our system is dependent on transcription via the tetracycline operon and can be reversibly inhibited by inclusion of low levels of doxycycline in the growth medium This system offers tremendous flexibility in our ability to regulate the expression of ENaC subunits in a renal epithelial cell line that maintains physiologic regulatory mechanisms. Using this system, we have observed distinct current decay profiles in cycloheximide (CHX)-treated cells infected with different combinations of ENaC subunits. Our results suggest that stoichiometry-dependent trafficking of ENaC subunit combinations could serve as a physiological mechanism to regulate sodium reabsorption in vivo
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