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

## TL;DR

The study identifies a cluster of residues in the CFTR protein that prevents its activation until phosphorylated, offering insights into cystic fibrosis.

## Contribution

The discovery of an inhibitory module formed by residues R766, S768, M773, and T774 that blocks CFTR activation.

## Key findings

- Deleting residues 760–783 in CFTR leads to constitutive channel activity without PKA phosphorylation.
- Residues R766, S768, M773, and T774 form an inhibitory module preventing CFTR activation.
- Modifying these residues with cysteine mutations results in PKA-independent channel activity.

## 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 (ΔR760–783) elicited constitutive macroscopic and single-channel Cl‒ currents in the presence of ATP before PKA phosphorylation, suggesting that protein segment R760–783 in the R domain blocks CFTR activation. With the background of ΔR760–835, further deletion of R708–759 led to fully active channels in the presence of ATP, but the absence of PKA, suggesting that R708–759 prevents the activation of ΔR760–835-CFTR. R760–783 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 R760–783. 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.