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.