Influence of Proteolytic Cleavage of ENaC's Gamma Subunit upon Na + and K + Handling.

The 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 (PO ), in vitro . Cleavage proximal to the tract occurs at a furin recognition sequence (143 RKRR146 , 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 RKRR146 mutation to 143 QQQQ146 (γQ4 ) 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 of γQ4/Q4 mouse kidney lysates confirmed loss of a band consistent in size with the furin-cleaved proteolytic fragment. However, γQ4/Q4 male mice on a low Na+ diet did not exhibit altered ENaC PO or flow-induced JNa , though flow-induced JK modestly decreased. Colonic amiloride-sensitive ISC in γQ4/Q4 mice was not altered. γQ4/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 be used in isolation to evaluate ENaC activity. Further, factors independent of γ subunit cleavage modulate channel PO and the influence of ENaC on Na+ , K+ , and fluid volume homeostasis in 129/Sv mice, in vivo .