A cluster of inhibitory residues in the regulatory domain prevents activation of the cystic fibrosis transmembrane conductance regulator

Activation of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^‒ channel requires PKA phosphorylation at the regulatory (R) domain to relieve inhibition of ATP-dependent channel activity. This study aimed to identify the primary inhibitory site that prevents channel activation. CFTR mutants with deletion of residues 760 to 783 (ΔR_760-783) elicited constitutive macroscopic and single-channel Cl^‒ currents in the presence of ATP before PKA phosphorylation, suggesting that protein segment R_760-783 in the R domain blocks CFTR activation. With the background of ΔR_760-835, further deletion of R_708-759 led to fully active channels in the presence of ATP, but the absence of PKA, suggesting that R_708-759 prevents the activation of ΔR_760-835-CFTR. R_760-783 peptides were unstructured in buffered solutions in CD spectroscopy and the N771P mutation that interrupts the α-helix formation induced no apparent constitutive current before PKA phosphorylation. These data suggest that interpeptide interactions by α-helices likely contribute trivially to the blocking effect of R_760-783. CFTR mutants with small deletions or alanine replacements containing any one of residues R⁷⁶⁶ and S⁷⁶⁸ in a PKA consensus sequence and M⁷⁷³ and T⁷⁷⁴ generated PKA-independent CFTR Cl^‒ currents. Similarly, introducing the mutations Q767C or T774C into a control CFTR construct produced constitutive CFTR Cl^‒ currents by positively charged 2-(trimethylammonium)ethylmethanethiosulfonate modification of target cysteines. Moreover, PKA-independent single-channel activity was evidently observed in R766K-, S768K-, and T774K-CFTR mutants. Therefore, the four residues, R⁷⁶⁶, S⁷⁶⁸, M⁷⁷³, and T⁷⁷⁴, may form an inhibitory module that precludes CFTR activation through side-chain interactions. This inhibitory mechanism might be emulated by other PKA-dependent proteins.