["Acta Physiologica, Volume 242, Issue 6, June 2026. ", "\nABSTRACT\n\nAims\nThe epithelial sodium channel ENaC consists of the subunits α, β, and γ and is activated at an individual channel level by proteolytic processing. Murine γENaC contains a distal polybasic tract 186RKRK mediating proteolytic ENaC activation by serine proteases in vitro. The relevance of ENaC activation at this cleavage site for sodium homeostasis in vivo is unknown.\n\n\nMethods\nMice were generated carrying a mutation of the distal polybasic tract (RKRK186QQQQ or γENaCki/ki) using CRISP/Cas9. Sodium homeostasis and proteolytic processing of γENaC were investigated under a low sodium diet, pharmacological ENaC blockade, and induction of nephrotic syndrome.\n\n\nResults\nUnder control conditions, the response to bolus amiloride was reduced in γENaCki/ki mice compared to γENaCwt/wt mice. Under a low sodium diet for 4 days, urinary sodium excretion was similarly lowered in both genotypes; however, γENaCki/ki mice required significantly higher plasma aldosterone concentrations. Both genotypes were similarly tolerant to amiloride exposure for 4 days and developed similar sodium retention and body weight gain after induction of nephrotic syndrome. Proteolytic processing of γENaC leading to increased expression of distally cleaved γENaC at ~54 kDa was stimulated in both γENaCwt/wt and γENaCki/ki mice under all interventions without an appreciable difference in the migration pattern.\n\n\nConclusion\nMice harboring the RKRK186QQQQ mutation of the distal polybasic tract develop hyperaldosteronism under a low sodium diet, pointing to the relevance of this tract for sodium preservation. However, proteolytical processing of γENaC in these mice appears to be compensated for by the involvement of other adjacent cleavage sites.\n\n"]