Aldosterone modulates the association between NCC and ENAC

Distal sodium transport is a final step in the regulation of blood pressure. As such, understanding how the two main sodium transport proteins, the thiazide-sensitive sodium chloride cotransporter (NCC) and the epithelial sodium channel (ENaC), are regulated is paramount. Both are expressed in the late distal nephron; however, no evidence has suggested that these two sodium transport proteins interact. Recently, we established that these two sodium transport proteins functionally interact in the second part of the distal nephron (DCT2). Given their co-localization within the DCT2, we hypothesized that NCC and ENaC interactions might be modulated by aldosterone (Aldo). Aldo treatment increased NCC and αENaC colocalization (electron microscopy) and interaction (coimmunoprecipitation). Finally, with co-expression of the Aldo-induced protein serum- and glucocorticoid-inducible kinase 1 (SGK1), NCC and αENaC interactions were increased. These data demonstrate that Aldo promotes increased interaction of NCC and ENaC, within the DCT2 revealing a novel method of regulation for distal sodium reabsorption

Isoprenylcysteine-O-carboxyl methyltransferase regulates aldosterone- sensitive Na+ reabsorption

The Xenopus laevis distal tubule epithelial cell line A6 was used as a model epithelia to study the role of isoprenylcysteine-O-carboxyl methyltransferase (pcMTase) in aldosterone-mediated stimulation of Na+ transport. Polyclonal antibodies raised against X. laevis pcMTase were immunoreactive with a 33-kDa protein in whole cell lysate. These antibodies were also reactive with a 33-kDa product from in vitro translation of the pcMTase cDNA. Aldosterone application increased pcMTase activity resulting in elevation of total protein methyl esterification in vivo, but pcMTase protein levels were not affected by steroid, suggesting that aldosterone increased activity independent of enzyme number. Inhibition of pcMTase resulted in a reduction of aldosterone-induced Na+ transport demonstrating the necessity of pcMTase-mediated transmethylation for steroid induced Na+ reabsorption. Transfection with an eukaryotic expression construct containing pcMTase cDNA increased pcMTase protein level and activity. This resulted in potentiation of the natriferic actions of aldosterone. However, overexpression did not change Na+ reabsorption in the absence of steroid, suggesting that pcMTase activity is not limiting Na+ transport in the absence of steroid, but that subsequent to aldosterone addition, pcMTase activity becomes limiting. These results suggest that a critical transmethylation is necessary for aldosterone-induction of Na+ transport. It is likely that the protein catalyzing this methylation is isoprenylcysteine-O-carboxyl methyltransferase and that aldosterone activates pcMTase without affecting transferase expression.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

PIP2 Interacts Electrostatically with MARCKS-like Protein-1 and ENaC in Renal Epithelial Cells

We examined the interaction of a membrane-associated protein, MARCKS-like Protein-1 (MLP-1), and an ion channel, Epithelial Sodium Channel (ENaC), with the anionic lipid, phosphatidylinositol 4, 5-bisphosphate (PIP2). We found that PIP2 strongly activates ENaC in excised, inside-out patches with a half-activating concentration of 21 ± 1.17 µM. We have identified 2 PIP2 binding sites in the N-terminus of ENaC β and γ with a high concentration of basic residues. Normal channel activity requires MLP-1’s strongly positively charged effector domain to electrostatically sequester most of the membrane PIP2 and increase the local concentration of PIP2. Our previous data showed that ENaC covalently binds MLP-1 so PIP2 bound to MLP-1 would be near PIP2 binding sites on the cytosolic N terminal regions of ENaC. We have modified the charge structure of the PIP2 –binding domains of MLP-1 and ENaC and showed that the changes affect membrane localization and ENaC activity in a way consistent with electrostatic theory