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

Abstract

ABSTRACTThe 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 (PO),in vitro. Cleavage proximal to the tract occurs at a furin recognition sequence (143RKRR146in mouse). Loss of furin-mediated cleavage preventsin vitroactivation of the channel by proteolysis at distal sites. We hypothesized that143RKRR146mutation to143QQQQ146(Q4) in 129/Sv mice would reduce ENaC PO, impair flow-stimulated flux of Na+(JNa) and K+(JK) in perfused collecting ducts, reduce colonic amiloride-sensitive short circuit current (ISC), and impair Na+, K+, and body fluid homeostasis. Immunoblot ofQ4/Q4mouse kidney lysates confirmed loss of a band consistent in size with the furin-cleaved proteolytic fragment. However,Q4/Q4male mice on a low Na+diet did not exhibit altered ENaC POor flow-induced JNa, though flow-induced JKmodestly decreased. Colonic amiloride-sensitive ISCinQ4/Q4mice was not altered.Q4/Q4males, 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 POand the influence of ENaC on Na+, K+, and fluid volume homeostasis in 129/Sv mice,in vivo.