Chronic exposure to EGF affects trafficking and function of ENaC channel in cystic fibrosis cells

Using the whole-cell patch-clamp technique, we identified an amiloride (AMI)-sensitive Na+ current in cystic fibrosis cells, JME/ CF15, growing in standard medium. The reversal potential of this current depended on Na+ concentrations and the cation selectivity was much higher for Na+ than for K+, indicating that the current is through ENaC channels. In contrast, cells front EGF-containing medium lacked AMI-sensitive Na+ currents. In permeabilized cells growing in EGF-containing medium, alpha ENaC was mainly detected in a perinuclear region, while in cells from standard medium it was distributed over the cell body. Western-blot analysis showed that in standard medium cells expressed fast-migrating EndoH-insensitive and slow-migrating EndoH-sensitive alpha ENaC fractions, while in cells growing in the presence of EGF, alpha ENaC was only detected as the fast-migrating EndoH-insensitive fraction. Long-term incubation of cells with EGF resulted in an increased basal Ca2+ level, [Ca2+](i). A similar increase of [Ca2+], was also observed in the presence of 2 mu M thapsigargin, resulting in inhibition of ENaC function. Thus, in JME/CF15 cells inhibition of the ENaC function by chronic incubation with EGF is a Ca2+-mcdiated process that affects trafficking and surface expression of ENaC channels. (c) 2005 Elsevier Inc. All rights reserved.