Transmembrane helical interactions in the CFTR channel pore

Area of Science:

• Structural Biology
• Biophysics
• Molecular Medicine

Background:

• Cystic Fibrosis (CF) is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, impacting epithelial fluid transport.
• Understanding CFTR channel gating mechanisms is essential for developing effective pharmaceutical strategies for CF.
• Existing CFTR homology models lack accuracy due to low sequence similarity with bacterial ABC transporters.

Purpose of the Study:

• To refine existing and develop new 3D structural models of CFTR in inward (IWF) and outward (OWF) facing conformations.
• To elucidate the structural determinants of CFTR channel gating and identify mechanisms responsible for ion permeation.
• To investigate the role of pore blockage versus open-close state switching in CFTR gating.

Main Methods:

• Integrated experimental and molecular dynamics (MD) simulations (200 ns) to generate IWF and OWF CFTR models.
• Cysteine cross-linking with bifunctional and monofunctional reagents to probe residue positions and orientations within transmembrane helices (TMHs).
• Comparison of pore profiles between OWF and IWF conformations to assess ion permeability.

Main Results:

• Developed accurate IWF and OWF CFTR models, with the IWF model showing good agreement with cryo-EM structures.
• Cysteine cross-linking verified TMH structures and revealed a physiologically relevant pore in the OWF conformation.
• Pore radius analysis indicated hydrated Cl- ion passage in OWF but not IWF states.
• Cross-linking experiments demonstrated that pore blockage, rather than simple open-close gating, is the primary mechanism affecting ion currents.

Conclusions:

• The study provides refined 3D structural models of CFTR, advancing the understanding of its gating mechanism.
• Pore blockage is identified as the primary mechanism responsible for regulating ion flow in CFTR.
• These findings offer critical insights for the rational design of CFTR-targeting therapeutics.