Exploration of the Peptide Recognition of an Amiloride-sensitive FMRFamide Peptide-gated Sodium Channel

FMRFamide (Phe-Met-Arg-Phe-NH2)-activated sodium channel (FaNaC) is an amiloride-sensitive sodium channel activated by endogenous tetrapeptide in invertebrates, and belongs to the epithelial sodium channel/degenerin (ENaC/DEG) superfamily. The ENaC/DEG superfamily differs markedly in its means of activation, such as spontaneously opening or gating by mechanical stimuli or tissue acidosis. Recently, it has been observed that a number of ENaC/DEG channels can be activated by small molecules or peptides, indicating that the ligand-gating may be an important feature of this superfamily. The peptide ligand control of the channel gating might be an ancient ligand-gating feature in this superfamily. Therefore, studying the peptide recognition of FaNaC channels would advance our understanding of the ligand-gating properties of this superfamily of ion channels. Here we demonstrate that Tyr-131, Asn-134, Asp-154, and Ile-160, located in the putative upper finger domain of Helix aspersa FaNaC (HaFaNaC) channels, are key residues for peptide recognition of this ion channel. Two HaFaNaC specific-insertion motifs among the ENaC/DEG superfamily, residing at the putative α4-α5 linker of the upper thumb domain and the α6-α7 linker of the upper knuckle domain, are also essential for the peptide recognition of HaFaNaC channels. Chemical modifications and double mutant cycle analysis further indicated that those two specific inserts and key residues in the upper finger domain together participate in peptide recognition of HaFaNaC channels. This ligand recognition site is distinct from that of acid-sensing ion channels (ASICs) by a longer distance between the recognition site and the channel gate, carrying useful information about the ligand gating and the evolution of the trimeric ENaC/DEG superfamily of ion channels.