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

ABSTRACT 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 .