Influence of proteolytic cleavage of ENaC's γ subunit upon Na + and K + handling.

The epithelial Na + channel (ENaC) γ subunit is essential for homeostasis of Na + , K + , and body fluid. Dual γ subunit cleavage before and after a short inhibitory tract allows dissociation of this tract, increasing channel open probability (P O ), in vitro. Cleavage proximal to the tract occurs at a furin recognition sequence ( 143 RKRR 146 , in the mouse γ subunit). Loss of furin-mediated cleavage prevents in vitro activation of the channel by proteolysis at distal sites. We hypothesized that 143 RKRR 146 mutation to 143 QQQQ 146 (γ Q4 ) in 129/Sv mice would reduce ENaC P O , impair flow-stimulated flux of Na + (J Na ) and K + (J K ) in perfused collecting ducts, reduce colonic amiloride-sensitive short-circuit current (I SC ), and impair Na + , K + , and body fluid homeostasis. Immunoblot of γ Q4/Q4 mouse kidney lysates confirmed loss of a band consistent in size with the furin-cleaved proteolytic fragment. However, γ Q4/Q4 male mice on a low Na + diet did not exhibit altered ENaC P O or flow-induced J Na , though flow-induced J K modestly decreased. Colonic amiloride-sensitive I SC in γ Q4/Q4 mice was not altered. γ Q4/Q4 males, but not females, exhibited mildly impaired fluid volume conservation when challenged with a low Na + diet. Blood Na + and K + were unchanged on a regular, low Na + , or high K + diet. These findings suggest that biochemical evidence of γ subunit cleavage should not be used in isolation to evaluate ENaC activity. Furthermore, factors independent of γ subunit cleavage modulate channel P O and the influence of ENaC on Na + , K + , and fluid volume homeostasis in 129/Sv mice, in vivo. NEW & NOTEWORTHY The epithelial Na + channel (ENaC) is activated in vitro by post-translational proteolysis. In vivo, low Na + or high K + diets enhance ENaC proteolysis, and proteolysis is hypothesized to contribute to channel activation in these settings. Using a mouse expressing ENaC with disruption of a key proteolytic cleavage site, this study demonstrates that impaired proteolytic activation of ENaC's γ subunit has little impact upon channel open probability or the ability of mice to adapt to low Na + or high K + diets.