Deletion of phenylalanine 508 causes attenuated phosphorylation-dependent activation of CFTR chloride channels

1. In cell-attached patches stimulated with cAMP agonists, the single-chann el open probability (P-o) of the phenylalanine 508-deleted cystic fibrosis transmembrane conductance regulator (Delta F508-CFTR) channel, the most com mon disease-associated mutation in cystic fibrosis, was abnormally low (a f unctional defect). To investigate the mechanism for the poor response of De lta F508-CFTR to cAMP stimulation, we examined, in excised inside-out patch es, protein kinase A (PKA)-dependent phosphorylation activation and ATP-dep endent gating of wild-type (WT) and Delta F508-CFTR channels expressed in N IH3T3 mouse fibroblasts. 2. For WT-CFTR, the activation time course of CFTR channel current upon add ition of PKA and ATP followed a sigmoidal function with time constants that decreased as [PKA] was increased. The curvilinear relationship between [PK A] and the apparent activation rate suggests an incremental phosphorylation -dependent activation of CFTR at multiple phosphorylation sites. 3. The time course of PKA-dependent activation of Delta F508-CFTR channel c urrent also followed a sigmoidal function, but the rate of activation was a t least 7-fold slower than that with WT channels. This result suggests that deletion of phenylalanine 508 causes attenuated PKA-dependent phosphorylat ion of the CFTR chloride channel. 4. Once Delta F508-CFTR channels were maximally activated with PKA, the mut ant channel and WT channel and indistinguishable steady-state P-o values, A TP dose-response relationships and single-channel kinetics, indicating that Delta F508-CFTR is not defective in ATP-dependent gating. 5. By measuring whole-cell current density we compared the number of functi onal channels in WT- and Delta F508-CFTR cell membrane. Our data showed tha t the estimated channel density for Delta F508-CFTR was similar to 10-fold lower than that fbr WT-CFTR, but the cAMP-dependent whole-cell current dens ity differed by similar to 200-fold. We thus conclude that the functional d efect (a decrease in P-o) of Delta F508-CFTR is as important as the traffic king defect (a decrease in the number of functional channels in the plasma membrane) in cystic fibrosis pathogenesis.