Cystic fibrosis transmembrane conductance regulator Cl- channels with R domain deletions and translocations show phosphorylation-dependent and -independent activity

Phosphorylation of the R domain regulates cystic fibrosis transmembrane con ductance regulator Cl- channel activity. Earlier studies suggested that the R domain controls activity via more than one mechanism; a phosphorylated R domain may stimulate activity, and an unphosphorylated R domain may preven t constitutive activity, i.e, opening with ATP alone. However, the mechanis ms responsible for these two regulatory properties are not understood. In t his study we asked whether the two effects are dependent on its position in the protein and whether smaller regions from the R domain mediate the effe cts. We found that several portions of the R domain conferred phosphorylati on-stimulated activity. This was true whether the R domain sequences were p resent in their normal location or were translocated to the C terminus. We also found that some parts of the R domain could be deleted without inducin g constitutive activity. However, when residues 760-783 were deleted, chann els opened without phosphorylation, Translocation of the R domain to the C terminus did not prevent constitutive activity, These results suggest that different parts of the phosphorylated R domain can stimulate activity and t hat their location within the protein is not critical. In contrast, prevent ion of constitutive activity required a short specific sequence that could not be moved to the C terminus. These results are consistent with a recent model of an R domain composed primarily of random coil in which more than o ne phosphorylation site is capable of stimulating channel activity, and net activity reflects interactions between multiple sites in the R domain and the rest of the channel.