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 ␣ and ␥ 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 dosedependent 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 (␣ H69A ) and was reduced by a mutation that activates ENaC (␤ S520K ). Moreover, the Na ؉ ionophore monensin reversed the effect of the inactivating mutation (␣ 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).Transport of Na ϩ across epithelia is critical to maintain Na ϩ homeostasis. Defects in Na ϩ transport cause inherited forms of hypertension (e.g. Liddle syndrome) and hypotension (pseudohypoaldosteronism type 1) (1). In the distal nephron of the kidney, lung, colon, and sweat duct, transport is mediated by the epithelial Na ϩ channel ENaC, a heterotrimer composed of homologous ␣, ␤, and ␥ subunits (2-5). ENaC is located at the apical membrane, where it functions as a conduit for Na ϩ to enter the cell (reviewed in Refs. 6 and 7). Coupled with Na ϩ exit at the basolateral membrane via the Na ϩ -K ϩ -ATPase, ENaC provides a pathway for Na ϩ reabsorption across these tissues. Although Na ϩ is the permeant ion for this pathway, Na ϩ also regulates its own transport through negative feedback mechanisms (8 -16). Under conditions of Na ϩ /volume excess, the Na ϩ concentration in the distal nephron is high (Ͼ50 mM) (17), which inhibits ENaC in order to minimize Na ϩ reabsorption. Conversely, under conditions of Na ϩ /volume depletion, the Na ϩ concentration is low (ϳ1 mM) (17), which activates ENaC to maximize Na ϩ reabsorption. By countering changes in Na ϩ delivery to the distal nephron, this pathway functions to maintain Na ϩ homeostasis. Previous work indicates that both extracellular Na ϩ ("Na ϩ self-inhibition" (10, 11, 18)) and intracellular Na ϩ ("Na ϩ feedback inhibition" (12-16)) regulate ENaC activity. However, an understanding of the underlying mechanisms has remained elusive.In this work, we tested the hypothesis that N...

Inhibition of Lung Fluid Clearance and Epithelial Na+ Channels by Chlorine, Hypochlorous Acid, and Chloramines

“…13A), in agreement with previous reports (31). The addition of trypsin had no effect on I Na of these mutant channels; perfusion with 1 mM HOCl for 10 min or incubation with 100 M HOCl for 2 h (which reduced I Na of wild type ENaC to zero) had little effect on I Na of these channels either (Fig.…”

Feedback inhibition of ENaC: Acute and chronic mechanisms

“…14 In our experiments, cell-surface gENaC appeared as both intermediately cleaved and maximally cleaved forms (Figs. 2-4).…”

“…9 After ENaC was cloned, 10 the Xenopus oocyte has become a convenient expression system to study feedback inhibition of ENaC. [11][12][13][14] 12 However, decreased currents were still observed when channel P o was increased to 1 with cysteine modification of a degenerin mutant, implicating other mechanisms of inhibition under these conditions.…”

“…Other possible mechanisms include Na C -dependent changes in the activity of proteases, 14 or preferential retrieval of specific cleaved products of ENaC from the cell surface. 3.…”

Regulation of Epithelial Sodium Channel Trafficking by Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9)

“…Proteolytic cleavage of ENaC is a regulated process. For example, cleavage is inhibited by increased intracellular Na ϩ , providing a negative feedback mechanism to regulate Na ϩ absorption (17). Conversely, cleavage is enhanced by Na ϩ depletion and aldosterone infusion (18,19).…”