Rattlesnake Phospholipase A2 Increases CFTR-Chloride Channel Current and Corrects ∆F508CFTR Dysfunction: Impact in Cystic Fibrosis.

Abstract

Deletion of Phe508 in the nucleotide binding domain (∆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 ∆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 ΔF508CFTR 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 ABCβ and F1-like ATP-binding subdomains of ΔF508-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 ∆F508CFTR-cytokeratin 8 complex, allowing for the escape of ∆F508CFTR from degradation. Therefore CB, as a dual modulator of ΔF508CFTR, constitutes a template for the development of new anti-CF agents.

• Authors: Grazyna Faure, Naziha Bakouh, Stéphane Lourdel, Norbert Odolczyk, Aiswarya Premchandar, Nathalie Servel, Aurélie Hatton, Maciej K Ostrowski, Haijin Xu, Frederick A Saul, Christelle Moquereau, Sara Bitam, Iwona Pranke, Gabrielle Planelles, Jacques Teulon, Harald Herrmann, Ariel Roldan, Piotr Zielenkiewicz, Michal Dadlez, Gergely L Lukacs, Isabelle Sermet-Gaudelus, Mario Ollero, Pierre-Jean Corringer, Aleksander Edelman
• Journal: Journal of molecular biology (J Mol Biol) Vol. 428 Issue 14 Pg. 2898-915 (Jul 17 2016) ISSN: 1089-8638 [Electronic] Netherlands
• PMID: 27241308 (Publication Type: Journal Article)
• Copyright: Copyright © 2016 Elsevier Ltd. All rights reserved.

Chemical References

• Chloride Channels
• Snake Venoms
• cystic fibrosis transmembrane conductance regulator delta F508
• Cystic Fibrosis Transmembrane Conductance Regulator
• Cyclic AMP
• Phospholipases A2

Topics

• Animals
• Cell Line, Tumor
• Chloride Channels (genetics)
• Crotalus (genetics)
• Cyclic AMP (genetics)
• Cystic Fibrosis (genetics)
• Cystic Fibrosis Transmembrane Conductance Regulator (genetics)
• Female
• HeLa Cells
• Humans
• Ion Channel Gating (genetics)
• Kinetics
• Male
• Mice
• Molecular Docking Simulation (methods)
• Mutation (genetics)
• Oocytes (metabolism)
• Phospholipases A2 (genetics)
• Protein Binding (genetics)
• Sequence Deletion (genetics)
• Snake Venoms (genetics)
• Xenopus laevis (genetics)