title: Intracellular sodium regulates proteolytic activation of the epithelial sodium channel.
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).
authors.affiliation: Departments of Internal Medicine and Molecular Physiology and Biophysics, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242.
authors.forename: Kristin K
authors.initials: KK
authors.lastname: Knight
authors.affiliation: Departments of Internal Medicine and Molecular Physiology and Biophysics, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242.
authors.forename: Danielle M
authors.initials: DM
authors.lastname: Wentzlaff
authors.affiliation: Departments of Internal Medicine and Molecular Physiology and Biophysics, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242. Electronic address: peter-snyder@uiowa.edu.
authors.forename: Peter M
authors.initials: PM
authors.lastname: Snyder
mesh_terms: D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D056827:Endosomal Sorting Complexes Required for Transport; D062686:Epithelial Sodium Channel Blockers; D053503:Epithelial Sodium Channels / Q000235:genetics / Q000378:metabolism*; D006706:Homeostasis / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D008985:Monensin / Q000494:pharmacology; D009154:Mutation; D000075702:Nedd4 Ubiquitin Protein Ligases; D011498:Protein Precursors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000502:physiology; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344; D012964:Sodium / Q000378:metabolism*; D026941:Sodium Channel Blockers / Q000494:pharmacology; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*
chemical_list: D056827:Endosomal Sorting Complexes Required for Transport; D062686:Epithelial Sodium Channel Blockers; D053503:Epithelial Sodium Channels; D007476:Ionophores; D011498:Protein Precursors; D021122:Protein Subunits; D026941:Sodium Channel Blockers; D000584:Amiloride; D008985:Monensin; D012964:Sodium; C000618479:NEDD4L protein, rat; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C000618484:Nedd4 protein, rat; C000618485:Nedd4L protein, human; D044767:Ubiquitin-Protein Ligases