Conformational changes in the pore of CLC-0

Area of Science:

• Molecular Biology
• Ion Channel Physiology
• Pharmacology

Background:

• The Torpedo CLC-0 channel is a chloride channel.
• Clofibric acid derivatives inhibit CLC-0 from the intracellular side.
• Understanding the mechanism of CLC-0 inhibition is crucial for ion channel research.

Purpose of the Study:

• To investigate the mechanism of block of the CLC-0 channel by p-chlorophenoxy-acetic acid (CPA).
• To determine if channel opening involves more than a local conformational rearrangement.
• To test the hypothesis that mutations affecting CPA binding should impact open and closed channel affinities similarly.

Main Methods:

• Utilized a slow gate-deficient mutant CLC-0C212S.
• Performed electrophysiological recordings to assess CPA block kinetics and affinity.
• Introduced point mutations in key residues near the pore mouth and selectivity filter.

Main Results:

• CPA blocks open CLC-0 channels with low affinity and fast dissociation.
• CPA binds to closed channels with higher affinity and slower kinetics, indicating state-dependent block.
• Mutations S123T, Y512A, and K519Q significantly affected open channel block, while T471S exclusively altered closed channel binding.
• Mutation of Tyr 512 had minimal impact on conductance and selectivity.

Conclusions:

• The CPA binding site structure differs between open and closed CLC-0 channel states.
• Channel opening likely involves conformational changes beyond a single side chain movement.
• Other channel regions and the selectivity filter are probably involved in CLC-0 gating.