Potentiation of the cystic fibrosis transmembrane conductance regulator by VX-770 involves stabilization of the pre-hydrolytic, O1 state

Cystic fibrosis (CF) is a debilitating hereditary disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes an anion channel. Wild type-CFTR gating is a non-equilibrium process. After ATP binding, CFTR enters a stable open state (O1 ). ATP hydrolysis leads it to a short-lived post-hydrolytic open state (O2 ), from which channels close. Here, we use mutations to probe the mechanism of VX-770, the first compound directly targeting the CFTR protein approved for treatment of CF. D1370N and K1250R mutations reduce or abolish catalytic activity, simplifying the gating scheme to an equilibrium (C↔O1 ); K464A-CFTR has a destabilized O1 state and rarely closes via hydrolysis. Experimental approach: Potentiation by VX-770 was measured using microscopic imaging of HEK293 cells expressing an anion-sensitive YFP-CFTR. A simple mathematical model was used to predict fluorescence quenching following extracellular iodide addition and estimate CFTR conductance. Membrane density of CFTR channels was measured in a parallel assay, using CFTR-pHTomato. Key

Cystic fibrosis (CF) is a debilitating hereditary disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes an anion channel. Wild type-CFTR gating is a non-equilibrium process. After ATP binding, CFTR enters a stable open state (O1 ). ATP hydrolysis leads it to a short-lived post-hydrolytic open state (O2 ), from which channels close. Here, we use mutations to probe the mechanism of VX-770, the first compound directly targeting the CFTR protein approved for treatment of CF. D1370N and K1250R mutations reduce or abolish catalytic activity, simplifying the gating scheme to an equilibrium (C↔O1 ); K464A-CFTR has a destabilized O1 state and rarely closes via hydrolysis. Experimental approach: Potentiation by VX-770 was measured using microscopic imaging of HEK293 cells expressing an anion-sensitive YFP-CFTR. A simple mathematical model was used to predict fluorescence quenching following extracellular iodide addition and estimate CFTR conductance. Membrane density of CFTR channels was measured in a parallel assay, using CFTR-pHTomato. Key

VX-770 strongly potentiated WT-CFTR, D1370N-CFTR and K1250R-CFTR. K464A-CFTR was also strongly potentiated, regardless of whether it retained catalytic activity or not. Conclusions and implications: Similar potentiation of hydrolytic and non-hydrolytic mutants suggests that VX-770 increases CFTR open probability mainly by stabilizing pre-hydrolytic O1 states with respect to closed states. Potentiation of K464A-CFTR channels suggests action of VX-770 did not strongly alter conformational dynamics at site 1. Understanding potentiator mechanism could help develop improved treatment for CF patients. The fluorescence assay presented here is a robust tool for such investigations.