Sodium absorption in the distal nephron through the epithelial Na+ channel, ENaC, is an important regulator of extracellular fluid volume and blood pressure. Mutations that delete the cytoplasmic C-teminus of the beta and gamma hENaC subunits cause increased Na+ absorption and hypertension (Liddle's syndrome). Loss of function mutations in hENaC cause Na+ wasting (pseudohypoaldosteronism type 1). In preliminary studies, mutations associated with Liddle's syndrome increase Na+ current by increasing the number of channels in the plasma membrane. Mutation of the C-terminal sequence PPPXYXXL in each hENaC subunit reproduced these findings. Interestingly, this sequence is similar to internalization motifs found in a number of proteins. The goal of this application is to understand the function and regulation of hENaC to provide insight into basic mechanisms of Na+ transport and blood pressure control. There are three Specific Aims: 1) To investigate the mechanism(s) of increased surface expression of hENaC caused by Liddle's mutations. The hypothesis to be tested is that the PPPXYXXL motif is important for the internalization of hENaC, and that mutation or deletion of this motif, decreases the rate of channel internalization. An alternate hypothesis, that Liddle's mutations increase the rate of insertion of hENaC into the plasma membrane, will also be tested; 2) Preliminary results show that hENaC function is regulated by the second-messengers cAMP and PKC. The hypothesis to be tested is that these second-messengers regulate hENaC function by altering cell surface expression. An alternate hypothesis, that they alter channel gating, will also be tested; and 3) An important property of ENaC is its high degree of selectivity for Na+ over K+. This is determined by amino aid residues that line the channel pore. Which of the three subunits contribute to the pore will be determined and specific residues that line the pore will be identified, by covalent modification of cysteine residues. Their preliminary results found that two cysteines in the second membrane-spanning segment of gamma ENaC line the channel pore. Using site-directed mutagenesis, they will determine whether analogous residues in the alpha and beta subunits also line the pore. They will also use this strategy to identify other residues in ENaC that line the pore in order to begin to define the structure of the pore, and the molecular basis of its ion selectivity.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29HL058812-02
Application #
2771609
Study Section
Special Emphasis Panel (ZRG4-GMB (04))
Project Start
1997-09-01
Project End
2002-08-31
Budget Start
1998-09-01
Budget End
1999-08-31
Support Year
2
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Iowa
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
041294109
City
Iowa City
State
IA
Country
United States
Zip Code
52242
Butler, Phillip L; Staruschenko, Alexander; Snyder, Peter M (2015) Acetylation stimulates the epithelial sodium channel by reducing its ubiquitination and degradation. J Biol Chem 290:12497-503
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Zhou, Ruifeng; Tomkovicz, Vivian R; Butler, Phillip L et al. (2013) Ubiquitin-specific peptidase 8 (USP8) regulates endosomal trafficking of the epithelial Na+ channel. J Biol Chem 288:5389-97
Sharotri, Vikas; Collier, Daniel M; Olson, Diane R et al. (2012) Regulation of epithelial sodium channel trafficking by proprotein convertase subtilisin/kexin type 9 (PCSK9). J Biol Chem 287:19266-74
Snyder, Peter M (2012) Intoxicated Na(+) channels. Focus on ""ethanol stimulates epithelial sodium channels by elevating reactive oxygen species"". Am J Physiol Cell Physiol 303:C1125-6
Zhou, Ruifeng; Kabra, Rajesh; Olson, Diane R et al. (2010) Hrs controls sorting of the epithelial Na+ channel between endosomal degradation and recycling pathways. J Biol Chem 285:30523-30
Collier, Daniel M; Snyder, Peter M (2009) Extracellular chloride regulates the epithelial sodium channel. J Biol Chem 284:29320-5