cAMP binding to closed pacemaker ion channels is non-cooperative

Area of Science:

• Molecular Biology
• Neuroscience
• Cardiology

Background:

• Electrical activity in the brain and heart relies on pacemaker ion channels (HCN) regulated by cyclic AMP (cAMP) binding.
• Previous studies on cAMP binding to HCN channels showed conflicting results due to limitations of bulk measurements.
• Direct observation of individual ligand binding to membrane receptors at physiological concentrations remained a challenge.

Purpose of the Study:

• To directly resolve the binding dynamics of individual cAMP ligands to multimeric HCN1 and HCN2 ion channels.
• To investigate the cooperativity and stepwise binding of cAMP to HCN channel subunits at physiological concentrations.
• To validate the ligand-induced flip-state model for HCN channel regulation.

Main Methods:

• Utilized nanophotonic zero-mode waveguides to monitor single-molecule binding events.
• Directly observed the binding dynamics of individual cAMP ligands to HCN1 and HCN2 ion channels.
• Analyzed conformational changes and isomerization states following ligand binding.

Main Results:

• Demonstrated that cAMP binds independently to all four subunits of HCN channels when the pore is closed.
• Observed a subsequent conformational isomerization to a flip state at each binding site.
• Identified distinct dynamics in cAMP binding and isomerization for different HCN isoforms.

Conclusions:

• The independent binding and distinct dynamics of cAMP with HCN channel subunits explain isoform-specific physiological responses.
• Provides direct validation for the ligand-induced flip-state model in HCN channel regulation.
• Establishes a novel single-molecule approach to study allosteric binding in intact membrane proteins at physiological concentrations.