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.