Work

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

Year: 2025

Type: article

Abstract: 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 R 766 and S 768 in a PKA consensus sequence and M 773 and T 774 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, R 766 , S 768 , M 773 , and T 774 , may form an inhibitory module that precludes CFTR activation through side-chain interactions. This inhibitory mechanism might be emulated by other PKA-dependent proteins.

Source: Journal of Biological Chemistry

DOI: 10.1016/j.jbc.2025.108460