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

Abstract: 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 from EGF-containing medium lacked AMI-sensitive Na+ currents. In permeabilized cells growing in EGF-containing medium, αENaC 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 αENaC fractions, while in cells growing in the presence of EGF, αENaC 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+]i was also observed in the presence of 2μM 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+-mediated process that affects trafficking and surface expression of ENaC channels. [Copyright &y& Elsevier]