Langron, E;
Prins, S;
Vergani, P;
(2018)
Potentiation of the cystic fibrosis transmembrane conductance regulator by VX‐770 involves stabilization of the pre‐hydrolytic, O₁ state.
British Journal of Pharmacology
, 175
(20)
pp. 3990-4002.
10.1111/bph.14475.
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Abstract
BACKGROUND AND PURPOSE: Cystic fibrosis (CF) is a debilitating hereditary disease caused by mutations in the CFTR gene, which encodes an anion channel. WT-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 greatly 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 RESULTS: VX-770 strongly potentiates 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 stabilising the pre-hydrolytic O1 state with respect to the closed state. Potentiation of K464A-CFTR channels suggests action of the drug does 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.
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