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 conductance 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 prevent constitutive activity, i.e. opening with ATP alone. However, the mechanisms responsible for these two regulatory properties are not understood. In this 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 effects. We found that several portions of the R domain conferred phosphorylation-stimulated activity. This was true whether the R domain sequences were present 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 inducing constitutive activity. However, when residues 760-783 were deleted, channels 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 that their location within the protein is not critical. In contrast, prevention 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 one 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.
Baldursson
O
O
Howard Hughes Medical Institute, Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
Ostedgaard
L S
LS
Rokhlina
T
T
Cotten
J F
JF
Welsh
M J
MJ
D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D010766:Phosphorylation; D021381:Protein Transport; D017384:Sequence Deletion
D019005:Cystic Fibrosis Transmembrane Conductance Regulator