Acute downregulation of ENaC by EGF involves the PY motif and putative ERK phosphorylation site

The epithelial sodium channel (ENaC) is expressed in a variety of tissues, including the renal collecting duct, where it constitutes the rate-limiting step for sodium reabsorption. Liddle's syndrome is caused by gain-of-function mutations in the beta and gamma subunits of ENaC, resulting in enhanced Na reabsorption and hypertension. Epidermal growth factor ( EGF) causes acute inhibition of Na absorption in collecting duct principal cells via an extracellular signal-regulated kinase (ERK)-dependent mechanism. In experiments with primary cultures of collecting duct cells derived from a mouse model of Liddle's disease (beta-ENaC truncation), it was found that EGF inhibited short-circuit current (Isc) by 24 +/- 5% in wild-type cells but only by 6 +/- 3% in homozygous mutant cells. In order to elucidate the role of specific regions of the beta-ENaC C terminus, Madin-Darby canine kidney (MDCK) cell lines that express beta-ENaC with mutation of the PY motif ( P616L), the ERK phosphorylation site (T613A), and C terminus truncation (R564stop) were created using the Phoenix retroviral system. All three mutants exhibited significant attenuation of the EGF-induced inhibition of sodium current. In MDCK cells with wild-type beta-ENaC, EGF-induced inhibition of Isc (< 30 min) was fully reversed by exposure to an ERK kinase inhibitor and occurred with no change in ENaC surface expression, indicative of an effect on channel open probability (P-o). At later times (> 30 min), EGF-induced inhibition of Isc was not reversed by an ERK kinase inhibitor and was accompanied by a decrease in ENaC surface expression. Our results are consistent with an ERK-mediated decrease in ENaC open probability and enhanced retrieval of sodium channels from the apical membrane.