Abstract
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).
Highlights
In the distal nephron of the kidney, lung, colon, and sweat duct, transport is mediated by the epithelial Naϩ channel ENaC, a heterotrimer composed of homologous ␣, , and ␥ subunits [2,3,4,5]
When the cells were incubated for 1 h in 135 mM Naϩ, two forms of ␣ENaC were present at the cell surface (Fig. 1A); the 90-kDa band corresponds to full-length ␣ENaC, and the 65-kDa band is the C-terminal fragment of the proteolytically cleaved subunit
Naϩ Does Not Alter Nedd4-2 Activity—In previous work, we found that ENaC cleavage was modulated by Nedd4-2, an E3 ubiquitin ligase that targets ENaC for degradation
Summary
Naϩ Decreases ENaC Cleavage—To test the hypothesis that Naϩ alters proteolytic cleavage of ENaC, we incubated HEK 293T cells expressing ␣-, -, and ␥ENaC (␣ or ␥ subunit contained a C-terminal FLAG) in extracellular solutions containing either 135 or 0 mM Naϩ. After 15 min in 0 mM Naϩ, ENaC cleavage increased, resulting in a decrease in the ratio of full-length to cleaved channels (Fig. 2, C and D).
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