ATP-independent CFTR channel gating and allosteric modulation by phosphorylation

Area of Science:

• Molecular Biology
• Biophysics
• Ion Channel Physiology

Background:

• Cystic fibrosis (CF) results from mutations in the CFTR channel, an ABC transporter.
• CFTR gating depends on ATP binding, NBD dimerization, and R domain phosphorylation.
• The precise allosteric mechanisms governing CFTR gating remain incompletely understood.

Purpose of the Study:

• To investigate the allosteric gating mechanisms of the CFTR channel.
• To identify mutations that confer ATP-independent CFTR channel activity.
• To elucidate the role of R domain phosphorylation in CFTR function.

Main Methods:

• Site-directed mutagenesis of CFTR cytosolic loops.
• Electrophysiological recordings to assess channel activity.
• Analysis of ATP sensitivity and R domain phosphorylation requirements.

Main Results:

• Specific point mutations in CFTR cytosolic loops induced significant ATP-independent channel activity.
• These constitutive mutations localized to the putative symmetry axis of CFTR.
• Mutations enhanced ATP sensitivity and rescued activity in non-functional CFTR variants (G551D, NBD2-deletion).
• Constitutive CFTR mutants still required R domain phosphorylation for optimal activity.

Conclusions:

• CFTR gating exhibits allosteric properties shared with conventional ligand-gated channels.
• ATP is not essential for CFTR opening but modulates its equilibrium between active and inactive states.
• The R domain regulates CFTR activity independently of ATP-induced NBD dimerization.