Increased Diffusional Mobility of CFTR at the Plasma Membrane after Deletion of Its C-terminal PDZ Binding Motif

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a cAMP-regulated Cl^- channel expressed at the apical plasma membrane. It has been proposed that the C-terminal PDZ binding motif of CFTR is required for its apical membrane targeting and that PDZ-domain interactions may tether CFTR to the actin cytoskeleton via soluble proteins including EBP50/NHERF1 and ezrin. We measured the diffusional mobility of human CFTR in the plasma membrane of Madin-Darby canine kidney cells by photobleaching of green fluorescent protein (GFP)-CFTR chimeras. After bleaching by a focused laser beam, GFP-CFTR fluorescence in the bleached membrane region recovered to ∼90% of its initial level, indicating that nearly all of the CFTR was mobile. The GFP-CFTR diffusion coefficient (D) was 0.99 ± 0.09 × 10^-10 cm²/s at 37 °C, similar to that of other membrane proteins. GFP-CFTR diffusion was not altered by protein kinase A or C activators but was blocked by paraformaldehyde and filipin. CFTR mutants lacking functional PDZ-binding domains (GFPCFTR-ΔTRL and GFP-CFTR-ΔTRA) were also mobile with D significantly increased by ∼60% compared with GFP-CFTR. However, GFP-CFTR, GFP-CFTR-Δ TRL, and GFP-CFTR-ΔTRA had similar mobilities (D ∼12 × 10^-10 cm²/s) at the endoplasmic reticulum in brefeldin A-treated cells. Agents that modulate the actin cytoskeleton (cytochalasin D and jasplakinolide) altered the plasma membrane mobility of CFTR but not CFTR- ΔTRL. EBP50 (NHERF1), a PDZ domain-containing protein that interacts with the C terminus of CFTR, diffused freely in the cytoplasm with a diffusion coefficient of 0.9 ± 0.1 × 10^-7 cm²/s. EBP50 diffusion increased by ∼2-fold after deletion of its ezrin-binding domain. These results indicate that wild-type CFTR is not tethered statically at the plasma membrane but that its diffusion is dependent on PDZ-domain interactions and an intact actin skeleton. PDZ-domain interactions of CFTR are thus dynamic and occur on a time scale of seconds or faster.