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 (¹⁴³RKRR¹⁴⁶, 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 ¹⁴³RKRR¹⁴⁶ mutation to ¹⁴³QQQQ¹⁴⁶ (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.