Evidence for direct CFTR inhibition by CFTR_inh-172 based on Arg³⁴⁷ mutagenesis

CFTR (cystic fibrosis transmembrane conductance regulator) is an epithelial Cl⁻ channel inhibited with high affinity and selectivity by the thiazolidinone compound CFTR_inh-172. In the present study, we provide evidence that CFTR_inh-172 acts directly on the CFTR. We introduced mutations in amino acid residues of the sixth transmembrane helix of the CFTR protein, a domain that has an important role in the formation of the channel pore. Basic and hydrophilic amino acids at positions 334–352 were replaced with alanine residues and the sensitivity to CFTR_inh-172 was assessed using functional assays. We found that an arginine-to-alanine change at position 347 reduced the inhibitory potency of CFTR_inh-172 by 20–30-fold. Mutagenesis of Arg³⁴⁷ to other amino acids also decreased the inhibitory potency, with aspartate producing near total loss of CFTR_inh-172 activity. The results of the present study provide evidence that CFTR_inh-172 interacts directly with CFTR, and that Arg³⁴⁷ is important for the interaction.