We previously showed that ENaC is present in lipid rafts in A6 cells, a Xenopus kidney cell line. We now demonstrate that ENaC can be detected in lipid rafts in mouse cortical collecting duct ( MPK CCD 14 ) cells by detergent insolubility, buoyancy on density gradients using two distinct approaches, and colocalization with caveolin 1. Less than 30% of ENaC subunits were found in raft fractions. The channel subunits also colocalized on sucrose gradients with known vesicle targeting and fusion proteins syntaxin 1A, Vamp 2, and SNAP23. Hormonal stimulation of ENaC activity by either forskolin or aldosterone, short or long term, did not alter the lipid raft distribution of ENaC. Methyl--cyclodextrin added apically to MPK CCD 14 cells resulted in a slow decline in amiloride-sensitive sodium transport with short circuit current reductions of 38.1 ؎ 9.6% after 60 min. The slow decline in ENaC activity in response to apical cyclodextrin was identical to the rate of decline seen when protein synthesis was inhibited by cycloheximide. Apical biotinylation of MPK CCD 14 cells confirmed the loss of ENaC at the cell surface following cyclodextrin treatment. Acute stimulation of the recycling pool of ENaC was unaffected by apical cyclodextrin application. Expression of dominant negative caveolin isoforms (CAV1-eGFP and CAV3-DGV) which disrupt caveolae, reduced basal ENaC currents by 72.3 and 78.2%, respectively; but, as with cyclodextrin, the acute response to forskolin was unaffected. We conclude that ENaC is present in and regulated by lipid rafts. The data are consistent with a model in which rafts mediate the constitutive apical delivery of ENaC.
Page F261: Peter C. Will, Jonathan L. Lebowitz, and Ulrich Hopfer. “Induction of amiloride-sensitive sodium transport in the rat colon by mineralocorticoids.” Page F267: the second equation in the appendix should read (See PDF)
Using the yeast two-hybrid system, we identified a number of proteins that interacted with the carboxyl termini of murine epithelial sodium channel (ENaC) subunits. Initial screens indicated an interaction between the carboxyl terminus of -ENaC and I B kinase- (IKK), the kinase that phosphorylates I  and results in nuclear targeting of NF-B. A true two-hybrid reaction employing full-length IKK and the carboxyl termini of all three subunits confirmed a strong interaction with -ENaC, a weak interaction with ␥-ENaC, and no interaction with ␣-ENaC. Co-immunoprecipitation studies for IKK were performed in a murine cortical collecting duct cell line that endogenously expresses ENaC. Immunoprecipitation with -ENaC, but not ␥-ENaC, resulted in co-immunoprecipitation of IKK. To examine the direct effects of IKK on ENaC activity, co-expression studies were performed using the two-electrode voltage clamp technique in Xenopus oocytes. Oocytes were injected with cRNAs for ␣␥-ENaC with or without cRNA for IKK. Co-injection of IKK significantly increased the amiloride-sensitive current above controls. Using cell surface ENaC labeling, we determined that an increase of ENaC in the plasma membrane accounted for the increase in current. The injection of kinase-dead IKK (K44A) in ENaC-expressing oocytes resulted in a significant decrease in current. Treatment of mpkCCD c14 cells with aldosterone increased whole cell amounts of IKK. Because this result suggested that aldosterone might activate NF-B, mpkCCD c14 cells were transiently transfected with a luciferase reporter gene responsive to NF-B activation. Both aldosterone and tumor necrosis factor-␣ (TNF␣) stimulation caused a similar and significant increase in luciferase activity as compared with controls. We conclude that IKK interacts with ENaC by up-regulating ENaC at the plasma membrane and that the presence of IKK is at very least permissive to ENaC function. These studies also suggest a previously unexpected interaction between the NF-B transcription pathway and steroid regulatory pathways in epithelial cells.
We have previously shown that IB kinase- (IKK) interacts with the epithelial Na ؉ channel (ENaC) -subunit and enhances ENaC activity by increasing its surface expression in Xenopus oocytes. Here, we show that the IKK-ENaC interaction is physiologically relevant in mouse polarized kidney cortical collecting duct (mpkCCD c14 ) cells, as RNA interference-mediated knockdown of endogenous IKK in these cells by ϳ50% resulted in a similar reduction in transepithelial ENaC-dependent equivalent short circuit current. Although IKK binds to ENaC, there was no detectable phosphorylation of ENaC subunits by IKK in vitro. Because IKK stimulation of ENaC activity occurs through enhanced channel surface expression and the ubiquitin-protein ligase Nedd4-2 has emerged as a central locus for ENaC regulation at the plasma membrane, we tested the role of Nedd4-2 in this regulation. IKK-dependent phosphorylation of Xenopus Nedd4-2 expressed in HEK-293 cells occurred both in vitro and in vivo, suggesting a potential mechanism for regulation of Nedd4-2 and thus ENaC activity.32 P labeling studies utilizing wild-type or mutant forms of Xenopus Nedd4-2 demonstrated that Ser-444, a key SGK1 and protein kinase A-phosphorylated residue, is also an important IKK phosphorylation target. ENaC stimulation by IKK was preserved in oocytes expressing wild-type Nedd4-2 but blocked in oocytes expressing either a dominant-negative (C938S) or phospho-deficient (S444A) Nedd4-2 mutant, suggesting that Nedd4-2 function and phosphorylation by IKK are required for IKK regulation of ENaC. In summary, these results suggest a novel mode of ENaC regulation that occurs through IKK-dependent Nedd4-2 phosphorylation at a recognized SGK1 and protein kinase A target site.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.