Proteolytic Cleavage of the ENaC γ Subunit - Impact Upon Na ⁺ and K ⁺ Handling

The ENaC gamma subunit is essential for homeostasis of Na ⁺ , K ⁺ , and body fluid. Dual 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 mouse). 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 ( ^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 ^Q4/Q4 mouse kidney lysates confirmed loss of a band consistent in size with the furin-cleaved proteolytic fragment. However, ^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 ^Q4/Q4 mice was not altered. ^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 gamma subunit cleavage should not be used in isolation to evaluate ENaC activity. Further, factors independent of gamma subunit cleavage modulate channel P _O and the influence of ENaC on Na ⁺ , K ⁺ , and fluid volume homeostasis in 129/Sv mice, in vivo .