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 from EGF-containing medium lacked AMI-sensitive Na(+) currents. In permeabilized cells growing in EGF-containing medium, alphaENaC 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 alphaENaC fractions, while in cells growing in the presence of EGF, alphaENaC 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 2muM 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.
Cao
Lishuang
L
Department of Physiology, KU Leuven, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium.
Owsianik
Grzegorz
G
Becq
Frédéric
F
Nilius
Bernd
B
D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D003550:Cystic Fibrosis / Q000378:metabolism* / Q000473:pathology*; D004553:Electric Conductivity; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D053503:Epithelial Sodium Channels; D006031:Glycosylation; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D017136:Ion Transport / Q000187:drug effects; D018408:Patch-Clamp Techniques; D021381:Protein Transport / Q000187:drug effects; D012964:Sodium / Q000378:metabolism; D015222:Sodium Channels / Q000737:chemistry / Q000378:metabolism*; D019284:Thapsigargin / Q000494:pharmacology; D013997:Time Factors
D053503:Epithelial Sodium Channels; D015222:Sodium Channels; D004815:Epidermal Growth Factor; D019284:Thapsigargin; D012964:Sodium; D002118:Calcium