Rattlesnake Phospholipase A2 Increases CFTR-Chloride Channel Current and Corrects ΔF508CFTR Dysfunction: Impact in Cystic Fibrosis

Deletion of Phe508 in the nucleotide binding domain (Delta F508-NBD1) of the cystic fibrosis transmembrane regulator (CFTR; a cyclic AMP-regulated chloride channel) is the most frequent mutation associated with cystic fibrosis. This mutation affects the maturation and gating of CFTR protein. The search for new high-affinity ligands of CFTR acting as dual modulators (correctors/activators) presents a major challenge in the pharmacology of cystic fibrosis. Snake venoms are a rich source of natural multifunctional proteins, potential binders of ion channels. In this study, we identified the CB subunit of crotoxin from Crotalus durissus terrificus as a new ligand and allosteric modulator of CFTR. We showed that CB interacts with NBD1 of both wild type and Delta F508CFTR and increases their chloride channel currents. The potentiating effect of CB on CFTR activity was demonstrated using electrophysiological techniques in Xenopus laevis oocytes, in CFTR HeLa cells, and ex vivo in mouse colon tissue. The correcting effect of CB was shown by functional rescue of CFTR activity after 24-h Delta F508CFTR treatments with CB. Moreover, the presence of fully glycosylated CFTR was observed. Molecular docking allowed us to propose a model of the complex involving of the ABC beta and F1-like ATP-binding subdomains of Delta F508-NBD1. Hydrogen deuterium exchange analysis confirmed stabilization in these regions, also showing allosteric stabilization in two other distal regions. Surface plasmon resonance competition studies showed that CB disrupts the Delta F508CFTR-cytokeratin 8 complex, allowing for the escape of Delta F508CFTR from degradation. Therefore CB, as a dual modulator of Delta F508CFTR, constitutes a template for the development of new anti-CF agents. (C) 2016 Elsevier Ltd. All rights reserved.