cAMP binding to closed pacemaker ion channels is cooperative.

Area of Science:

• Molecular and Cellular Biology
• Biophysics
• Neuroscience

Background:

• Oligomeric receptors fine-tune activation through subunit cooperation.
• Voltage-activated HCN pacemaker ion channels regulate electric rhythmicity in neurons and cardiomyocytes.
• Conflicting evidence exists regarding cooperativity in HCN channel ligand binding, particularly its independence from channel activation.

Purpose of the Study:

• To investigate positive cooperativity in ligand binding to closed HCN2 channels in native cell membranes.
• To determine if cooperativity is causally linked to voltage-activated channel opening.
• To assess the role of subunit cooperation in HCN channel regulation.

Main Methods:

• Single-molecule fluorescence imaging of individual labeled cAMP binding events.
• Utilizing native cell membranes for studying HCN2 channels.
• Applying kinetic modeling to analyze binding data and receptor states.

Main Results:

• Demonstrated positive cooperativity in cAMP binding to closed HCN2 channels.
• Showed that binding affinity increases with site occupation in closed channels.
• Revealed that cooperativity is not dependent on prior voltage-activated channel opening.

Conclusions:

• Ligand binding to HCN2 channel subunits is cooperative even in the absence of voltage activation.
• Cooperativity in HCN2 channels is an intrinsic property, not solely a consequence of voltage-induced activation.
• Single-molecule binding assays at equilibrium can effectively quantify receptor cooperativity in native membranes.