Early Aldosterone-Induced Gene Product Regulates the Epithelial Sodium Channel by Deubiquitylation

Abstract

The mineralocorticoid hormone aldosterone controls sodium reabsorption and BP largely by regulating the cell-surface expression and function of the epithelial sodium channel (ENaC) in target kidney tubules. Part of the stimulatory effect of aldosterone on ENaC is mediated by the induction of serum-and glucocorticoid-regulated kinase 1 (Sgk1), a kinase that interferes with the ubiquitylation of ENaC by ubiquitin-protein ligase Nedd4-2. In vivo early aldosterone-regulated mRNA now has been identified in microselected mouse distal nephron by microarray. From 22 mRNA that displayed a two-fold or more change, 13 were downregulated and nine were upregulated. Besides Sgk1, the induced mRNA include Grem2 (protein related to DAN and cerebrus [PRDC]), activating transcription factor 3, cAMP responsive element modulator, and the ubiquitin-specific protease Usp2-45. The induction of this last enzyme isoform was verified in mouse distal nephron tubule at the protein level. With the use of Hek293 cells, Xenopus oocytes, and mpkCCD c14 cells as expression systems, it was shown that Usp2-45 deubiquitylates ENaC and stimulates ENaC-mediated sodium transport, an effect that is not additive to that of Sgk1. A deubiquitylating enzyme that targets ENaC in vitro and thus may play a role in sodium transport regulation was identified within a series of new in vivo early aldosterone-regulated gene products.

Intracellular Ubiquitylation of the Epithelial Na+ Channel Controls Extracellular Proteolytic Channel Activation via Conformational Change

“…We also observed that ENaC channels composed of lysine-free ␣␥ combined with wt ␤ (lanes 14 -16) were stronger expressed at the cell surface than wild-type channels or channels mutated on the lysines of all three subunits (lanes 11-13). This suggests that 1) ubiquitylation of ␣ and ␥ENaC has an impact on ENaC cell surface expression, and 2) some of the cytoplasmic lysine residues in ␤ are important for proper assembly and export of the channel to the plasma membrane consistent with our previous findings (22,24).…”

“…ENaC channels containing Liddle's mutations, and hence lacking the binding sites for Nedd4-2 have been shown to be preferentially cleaved (19) and to display increased open probability Po (33), compatible with the idea that cleavage of ENaC increases Po (12). Aldosterone induces the expression of GILZ, Sgk1, and Usp2-45, which all are involved in the suppression of Nedd4-2-dependent ENaC ubiquitylation (22,34,35); accordingly they are also expected to induce ENaC cleavage, as shown for Usp2-45 in this paper. Indeed, Fejes-Toth et al (36) presented recently evidence that the proteolytic processing of ␥ENaC is reduced in mice lacking Sgk1.…”

“…␤ENaC was tagged with a C-terminal Myc tag, and ␥ENaC with a C-terminal VSV tag. The ENaC lysine to arginine mutants were generated by PCR as described (22). Furin sites on ␣ENaC (R205A, R231A) or ␥ENaC (R138A) were mutated either in wild-type ENaC or in the lysine mutant.…”

“…To obtain additional evidence that it is indeed the ubiquitylation of ENaC, which affects the proteolytic cleavage, we expressed in Hek293 cells either wild-type ENaC, or a previously described ENaC mutant (22), in which all cytoplasmic lysines of the 3 subunits were mutated to arginine (ENaC-KR), hence representing a channel complex that cannot become modified by ubiquitin on these sites. We immunoprecipitated the ENaC subunits individually, and blotted the precipitated material with either anti-ENaC or anti-ubiquitin antibodies (Fig.…”

Toward Quantitative Proteomics of Organ Substructures: Implications for Renal Physiology

“…A similar problem is often encountered in preparing subcellular fractions such as brush border or basolateral membranes. Hand dissection of nephron segments has been performed for transcriptome analysis of human and rodent nephron segments 2,[19][20][21] . It often requires the perfusion and incubation of the tissue with collagenases before dissection 22,23 .…”