Intracellular sodium regulates proteolytic activation of the epithelial sodium channel

Na+ transport across epithelia is mediated in part by the epithelial Na+ channel ENaC. Previous work indicates that Na+ is an important regulator of ENaC, providing a negative feedback mechanism to maintain Na+ homeostasis. ENaC is synthesized as an inactive precursor, which is activated by proteolytic cleavage of the extracellular domains of the alpha and gamma subunits. Here we found that Na+ regulates ENaC in part by altering proteolytic activation of the channel. When the Na+ concentration was low, we found that the majority of ENaC at the cell surface was in the cleaved/active state. As Na+ increased, there was a dose-dependent decrease in ENaC cleavage and, hence, ENaC activity. This Na+ effect was dependent on Na+ permeation; cleavage was increased by the ENaC blocker amiloride and by a mutation that decreases ENaC activity (alpha(H69A)) and was reduced by a mutation that activates ENaC (beta(S520K)). Moreover, the Na+ ionophore monensin reversed the effect of the inactivating mutation (alpha(H69A)) on ENaC cleavage, suggesting that intracellular Na+ regulates cleavage. Na+ did not alter activity of Nedd4-2, an E3 ubiquitin ligase that modulates ENaC cleavage, but Na+ reduced ENaC cleavage by exogenous trypsin. Our findings support a model in which intracellular Na+ regulates cleavage by altering accessibility of ENaC cleavage sites to proteases and provide a molecular explanation for the earlier observation that intracellular Na+ inhibits Na+ transport via ENaC (Na+ feedback inhibition).