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 Ca 2+ level, [Ca 2+] i. A similar increase of [Ca 2+] i was also observed in the presence of 2 μM thapsigargin, resulting in inhibition of ENaC function. Thus, in JME/CF15 cells inhibition of the ENaC function by chronic incubation with EGF is a Ca 2+-mediated process that affects trafficking and surface expression of ENaC channels.