Characterization of SGK’s Interaction with the Epithelial Sodium Channel

Jennifer Reth Giampaolo
University of California, Berkeley

ENaC (epithelial sodium channel) membrane localization is the rate-limiting step in sodium reabsorption in tight epithelia such as that found in the cortical collecting duct of the kidney. Regulation begins at the hormonal level with aldosterone binding to a mineralocorticoid receptor and inducing transcription of specific genes within tight epithelia. One of the genes specifically responsible for increased localization of ENaC to the cell surface as well as increased open probability of the channel is the serine/threonine kinase SGK (serum and glucocorticoid-regulated kinase). SGK is activated downstream of the PI3K (phosphatidylinositol-3-kinase) pathway once induced by various factors such as hyperosmotic stress. Various labs, including ours, have found that expression of SGK in A6 kidney cells results in increased cell surface localization of ENaC. Down-regulation of ENaC cell surface localization is engineered via binding of the ubiquitin ligase Nedd4-2 to ENaC. Activated SGK phosphorylates Nedd4-2, probably inducing a conformational change that causes Nedd4-2’s dissociation from ENaC. SGK’s effect on increasing ENaC cell surface localization is thus established by its ability to phosphorylate Nedd4-2.

The main focus of my research is in the binding interaction between SGK and ENaC. It has already been shown in our lab that SGK binds to the C-terminus of the beta-subunit of ENaC. Due to complications with in vitro-translated SGK truncations, a computer-generated theoretical structure of the catalytic domain of SGK was created and manipulated in order to best predict potential binding sites to ENaC. SGK alanine-scanning mutants were cloned using two-step pcr mutagenesis and tested for binding to ENaC using GST-pulldown assays. Further investigation includes using SGK point mutations to create an SGK-ENaC binding mutant (EBM). This EMB will be used to explore the question of whether the inability of SGK to bind to ENaC affects its ability to phosphorylate Nedd4-2, subsequently resulting in the inhibition of increased expression and activity at the cell surface of ENaC.