Regulation of ENaC Trafficking and Activity in the Kidney

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Summary The kidneys control extracellular fluid volume and blood pressure by adjusting the excretion of Na to match the dietary Na intake and the overall physiological needs of the organism. Aldosterone is a key hormone that helps to mediate this process. In response to a reduction in extracellular fluid volume the adrenals increase secretion of this steroid, which in turn signals parts of the renal tubule (the so-called aldosterone-sensitive distal nephron) to increase Na reabsorption. This occurs at least in part through stimulation of the uptake of Na from the urine across the apical membrane through epithelial Na channels (ENaC). How this occurs, however, is incompletely understood. In the most prevalent model of this process, aldosterone stimulates the synthesis of a key enzyme, the serum and glucocorticoid induced kinase (SGK1). SGK1 then phosphorylates the ubiquitin ligase Nedd4-2, inhibiting its interaction with ENaC and diminishing the rate of channel internalization. Na reabsorption is then enhanced due to increased residence times of the channels at the apical surface. However, several lines of evidence suggest that this is not the main mechanism through which the hormone operates. First, the effects of inhibiting the binding of Nedd4-2 to ENaC by truncating the C-terminal of the ENaC surface (mimicking Liddle’s syndrome in humans) are synergistic with those of elevated aldosterone levels (5,10). This is not expected if the two manipulations affect the same cellular processes. Second, analysis of ENaC distribution in the cell and the excretion of ENaC protein in urinary exosomes suggests that the major effect of aldosterone is to increase forward trafficking to the apical membrane (18). Finally, measurement of the ubiquitination state of ENaC indicates that when transport is stimulated by aldosterone the number of ubiquitinated subunits increases, whereas the standard model predicts a decrease. In the work described in this application, we will examine a revised model of ENaC trafficking that is more consistent with these data. Here subunit protein is expressed in excess even under basal conditions when the need for transport is minimal. The main hormone-stimulated event is transport of ENaC protein from the ER to the plasma membrane. Arrival at the apical membrane exposes the channels to urinary proteases, which mediate the final proteolytic processing and activation of the channels. This increases the susceptibility of the subunits to ubiquitination, limiting their lifetime at the surface and/or their active states. This model will be tested using a variety of experimental techniques including quantitative Western blots to estimate subunit numbers, immunocytochemistry, in situ biotinylation and Western blotting to assess the cleavage states of ENaC subunits, electrophysiology to measure overall channel function, and ubiquitin assays to assess the modification of channels is subcellular compartments.