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Potentiation of the cystic fibrosis transmembrane conductance regulator by VX‐770 involves stabilization of the pre‐hydrolytic, O 1 state
Author(s) -
Langron Emily,
Prins Stella,
Vergani Paola
Publication year - 2018
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/bph.14475
Subject(s) - cystic fibrosis transmembrane conductance regulator , long term potentiation , regulator , cystic fibrosis , conductance , chemistry , biophysics , membrane potential , transmembrane protein , neuroscience , medicine , biochemistry , biology , physics , gene , condensed matter physics , receptor
Background and Purpose 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 (O 1 ). ATP hydrolysis leads it to a short‐lived post‐hydrolytic open state (O 2 ), 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↔O 1 ); K464A‐CFTR has a destabilized O 1 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 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 O 1 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.