Functional and structural characterization of interactions between opposite subunits in HCN pacemaker channels

Area of Science:

• Molecular biology
• Biophysics
• Ion channel function

Background:

• Hyperpolarization-activated and cyclic nucleotide (HCN) modulated channels are tetrameric cation channels crucial for neuronal excitability.
• The cyclic nucleotide-binding domain (CNBD) is coupled to the transmembrane domain via the C-linker, mediating functional interactions between subunits.
• The C-linker's role in coupling CNBD conformational changes to the channel pore is critical but not fully understood.

Purpose of the Study:

• To investigate the role of the C-linker in mHCN2 channel gating.
• To elucidate the contribution of specific C-linker residues to channel stabilization and activation.
• To understand how C-linker mutations affect the coupling between the intracellular domain and the ion pore.

Main Methods:

• Site-directed mutagenesis of the mHCN2 channel.
• Electrophysiological recordings using the patch-clamp technique.
• Confocal patch-clamp fluorometry for real-time measurements.
• All-atom molecular dynamics (MD) simulations to model channel behavior.

Main Results:

• Identified residue K464 in the C-linker as essential for stabilizing the closed state of the mHCN2 channel via inter-subunit interactions.
• MD simulations revealed that the K464E mutation induces a rotation of the intracellular domain relative to the pore, mimicking cAMP-induced conformational changes.
• This rotation weakens the autoinhibitory effect of the CL-CNBD region, suggesting its indirect involvement in gate modulation but direct role in activation-induced affinity increase.

Conclusions:

• The C-linker, specifically residue K464, plays a key role in maintaining mHCN2 channel closure through inter-subunit contacts.
• Conformational changes in the C-linker, such as rotation, are critical for modulating channel activity and affinity.
• These findings provide insights into the molecular mechanisms underlying HCN channel gating and regulation.