The Role of ROS in Tethering CFTR within Ceramide Platforms at the Plasma Membrane

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel which is expressed at the apical surface of many secretory epithelia. CFTR and reactive oxygen species (ROS) are both involved in innate immune responses against bacterial pathogens, however the link between them during host defense is not well understood. Here we study the surface expression and dynamics of CFTR in epithelial cells during protein kinase C (PKC) stimulation using fluorescence microscopy and k-space image correlation spectroscopy (kICS). kICS analysis was used to measure the confined dynamics of CFTR under control conditions and during PKC stimulation while simultaneously monitoring CFTR expression at the cell surface as total fluorescence intensity. During PKC stimulation, CFTR became aggregated into large (1 −2 µm diameter) structures on the plasma membrane called platforms. PKC induced a significant (1.5-fold) increase in CFTR surface expression and increased the CFTR confinement within platforms, as indicated by a decline in its confined diffusion coefficient from 0.011±0.001 µm2/s to 0.0033±0.0002 µm2/s. Pharmacological inhibition of ROS or acid sphingomyelinase (ASM) prevented the PKC-induced aggregation and tethering of CFTR. This suggests a scheme in which PKC stimulates the production of ROS, which activates ASM and ceramide synthesis, leading to the formation of platforms in the outer leaflet of the plasma membrane. Ceramide platforms may help stabilize CFTR in the membrane so that its surface expression and channel function are increased during secretagogue stimulation.