State-dependent modulation of CFTR gating by pyrophosphate

Area of Science:

• Biophysics
• Molecular Biology
• Ion Channel Physiology

Background:

• The cystic fibrosis transmembrane conductance regulator (CFTR) functions as an ATP-gated chloride channel.
• ATP binding and hydrolysis regulate CFTR channel gating, with dimerization of nucleotide-binding domains (NBDs) linked to the open state.

Purpose of the Study:

• To investigate the effects of pyrophosphate (PPi) and related analogues on CFTR gating.
• To elucidate the distinct closed states of CFTR and their modulation by nucleotides.

Main Methods:

• Utilized excised membrane patch electrophysiology to record CFTR channel activity.
• Applied ATP, pyrophosphate (PPi), and non-hydrolyzable ATP analogues (MgPPi, MgAMP-PNP) to study CFTR gating kinetics.
• Investigated the influence of specific CFTR mutations (W401G, Y1219G) and ATP analogues on channel states.

Main Results:

• MgPPi alone can induce CFTR channel opening, but typically results in short-lived events, suggesting it doesn't stabilize NBD dimers.
• MgPPi elicits prolonged CFTR opening when applied after ATP washout, indicating stabilization of a specific closed state (C2).
• The C2 state's stability is influenced by prior ATP analogue binding and CFTR mutations, suggesting a role for bound ATP at NBD1.

Conclusions:

• CFTR exhibits at least two distinct closed states (C1 and C2) with differential responses to MgPPi.
• The C2 closed state is stabilized by MgPPi and potentially involves a bound ATP molecule at NBD1.
• MgAMP-PNP acts similarly to MgPPi, suggesting a conserved mechanism for nucleotide-mediated stabilization of CFTR closed states.