A conserved peptide-binding pocket in HyNaC/ASIC ion channels

Area of Science:

• Ion channel research
• Molecular biology
• Neuroscience

Background:

• Peptide-gated ion channels, including FaNaCs/WaNaCs and HyNaCs, are crucial in signaling and belong to the DEG/ENaC family.
• FaNaCs bind FMRFamide, while HyNaCs bind Hydra RFamides, with distinct evolutionary origins.
• The ligand-binding site in FaNaCs is known, but the HyNaC binding site remained elusive.

Purpose of the Study:

• To identify the Hydra RFamide binding pocket in the heterotrimeric HyNaC2/3/5 channel.
• To understand the molecular basis of peptide binding in HyNaCs.
• To compare the ligand-binding mechanisms between different peptide-gated ion channel subgroups.

Main Methods:

• Molecular modeling to predict the binding pocket.
• Site-directed mutagenesis to confirm the pocket's role.
• Engineering an unnatural amino acid for covalent tethering of the ligand.
• Comparative analysis with acid-sensing ion channels (ASICs).

Main Results:

• A putative Hydra RFamide binding pocket was identified in the extracellular domain of HyNaC2/3/5, located at a subunit interface.
• This pocket binds a single peptide ligand, unlike some other channels.
• Covalent tethering of Hydra RFamide to the engineered channel trapped it in an open state.
• The HyNaC binding pocket shares regional homology with the ASIC acidic pocket but differs in acidity and interaction types.

Conclusions:

• A conserved ligand-binding pocket in HyNaCs and ASICs was revealed.
• Peptide binding in HyNaCs involves both electrostatic and hydrophobic interactions.
• This study suggests independent evolution of peptide-binding cavities within DEG/ENaC family subgroups.