Increased diffusional mobility of CFTR at the plasma membrane after deletion of its C-terminal PDZ binding motif

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 similar 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 x 10(-10) cm(2)/ s at 37degreesC, 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 ( GFP-CFTRDelta TRL and GFP- CFTR- Delta TRA) were also mobile with D significantly increased by similar to 60% compared with GFP- CFTR. However, GFP- CFTR, GFP- CFTR- Delta TRL, and GFP- CFTR- Delta TRA had similar mobilities ( D similar to 12 x 10(-10) cm(2)/ 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-Delta TRL. 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 x 10(-7) cm(2)/ s. EBP50 diffusion increased by similar to 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.