Gating of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl-channel is activated by PKA-dependent phosphorylation on its regulatory domain (R domain).Phosphorylation causes the relief of channel inhibition and also stimulation on the gating activity of the CFTR Cl-channel.However, it is unclear whether there are specific regions in the R domain generating these activation mechanisms.To test this hypothesis, we constructed a variety of deletion mutants within the R domain region from residue 708 to 835 and studied CFTR activation by the patch-clamp method.Our data indicate that deleting region 760-783, 760-769 or 770-776 all produced large constitutive Cl-currents.These data suggest that region 760-776 may form an inhibitory motif that prevents CFTR activation.Using the alanine residue to replace 4 or 2 residues within this region each time identified an inhibitory motif, whose inhibition could be relieved by an introduced cysteine residue with a positive modifier MTSET.However, deleting region 784-835 generated little constitutive Cl-currents in the presence of 1 mM ATP alone, but after PKA phosphorylation, greatly reduced the activity of the CFTR Cl-channel.Studying channel gating of the CFTR mutants with small deletions in this region demonstrates that region 784-835 regulates the channel closing rate, whereas region 829-835 also modulates the channel opening rate.In addition, deletions of region 708-759 and region 760-783 had small effects on channel activity.Therefore, these data suggest that the C-terminal part of the R domain is required for normal CFTR gating.We speculate that phosphorylation on these specific R domain regions may relief the channel inhibition by region 760-776 and endow the stimulatory function of region 784-835 on channel gating for CFTR activation.