Structural basis for CFTR inhibition by CFTR _inh -172

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that regulates electrolyte and fluid balance in epithelial tissues. Whereas activation of CFTR is vital to treating cystic fibrosis, selective inhibition of CFTR is a potential therapeutic strategy for secretory diarrhea and autosomal dominant polycystic kidney disease (ADPKD). Although several CFTR inhibitors have been developed by high-throughput screening, their modes of action remain elusive. In this study, we determined the structure of CFTR in complex with the inhibitor CFTR _inh - 172 to 2.7 Å resolution by cryogenic electron microscopy (cryo-EM). We observe that CFTR _inh - 172 binds inside the pore near transmembrane helix 8 (TM8), a critical structural element that links ATP hydrolysis with channel gating. Binding of CFTR _inh -172 stabilizes a conformation in which the chloride selectivity filter is collapsed and the pore is blocked from the extracellular side of the membrane. Single molecule fluorescence resonance energy transfer (smFRET) experiments indicate that CFTR _inh -172 inhibits channel gating without compromising nucleotide-binding domain (NBD) dimerization. Together, these data show that CFTR _inh -172 acts as both a pore blocker and a gating modulator, setting it apart from typical ion channel inhibitors. The dual functionality of CFTR _inh -172 reconciles previous biophysical observations and provides a molecular basis for its activity.