Differential current decay profiles of epithelial sodium channel subunit combinations in polarized renal epithelial cells.
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.
Mohan
Savita
S
Laboratory of Epithelial Cell Biology, Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, Pennsylvania 15261, USA.
Bruns
Jennifer R
JR
Weixel
Kelly M
KM
Edinger
Robert S
RS
Bruns
James B
JB
Kleyman
Thomas R
TR
Johnson
John P
JP
Weisz
Ora A
OA
D000256:Adenoviridae / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D003513:Cycloheximide / Q000494:pharmacology; D004285:Dogs; D004318:Doxycycline / Q000737:chemistry / Q000494:pharmacology; D004594:Electrophysiology; D053503:Epithelial Sodium Channels; D004848:Epithelium / Q000378:metabolism*; D000939:Epitopes / Q000737:chemistry; D019084:Fluorescent Antibody Technique, Indirect; D007668:Kidney / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012313:RNA / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000494:pharmacology; D015222:Sodium Channels / Q000737:chemistry*; D013997:Time Factors
D053503:Epithelial Sodium Channels; D000939:Epitopes; D011500:Protein Synthesis Inhibitors; D015222:Sodium Channels; D012313:RNA; D003513:Cycloheximide; D012964:Sodium; D004318:Doxycycline