FMRFamide (Phe-Met-Arg-Phe-NH 2 )-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 specificinsertion motifs among the ENaC/DEG superfamily, residing at the putative ␣4-␣5 linker of the upper thumb domain and the ␣6-␣7 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.The epithelial sodium channel/Degenerin (ENaC/DEG) 4 superfamily, cloned in the early 1990s, is nonvoltage-gated, amiloride-sensitive, and sodium-selective ion channels, including ASIC, ENaC, DEG, FaNaC, bile acid-sensitive ion channel (BASIC), and pickpocket (PPK) channels, etc. (1, 2). ENaC/DEG ion channels are implicated in many physiological and pathological functions such as synaptic plasticity, learning and memory, emotion regulation, neurodegenerative diseases, epileptic seizures, pain sensation, mechano-sensation, blood pressure regulation, and cystic fibrosis (1-3), which makes them potential drug targets for those disorders. The members of the ENaC/ DEG superfamily differ markedly by means of activation. For example, DEG channels are mechano-sensitive; ENaC channels open spontaneously; ASIC channels are capable of sensing tissue acidosis, while FaNaC is activated by RFamide peptides, etc. (4). Recently, great advances have been made in the exploration of the activation mechanism of this superfamily. For example, it is now known that ENaC, ASIC3, BASIC, and ASIC1a channels can be activated by the small molecules S3969 (5), GMQ (6), bile acid (7), and peptide toxin (8, 9) respectively. These new findings sugge...

The degenerin/epithelial sodium channel (DEG/ENaC) superfamily of ion channels contains subfamilies with diverse functions that are fundamental to many physiological and pathological processes, ranging from synaptic transmission to epileptogenesis. The absence in mammals of some DEG/ENaCs subfamily orthologues such as FMRFamide peptide-activated sodium channels (FaNaCs), which have been identified only in mollusks, indicates that the various subfamilies diverged early in evolution. We recently reported that the nonproton agonist 2-guanidine-4-methylquinazoline (GMQ) activates acid-sensing ion channels (ASICs), a DEG/ENaC subfamily mainly in mammals, in the absence of acidosis. Here, we show that GMQ also could directly activate the mollusk-specific FaNaCs. Differences in ion selectivity and unitary conductance and effects of substitutions at key residues revealed that GMQ and FMRFamide activate FaNaCs via distinct mechanisms. The presence of two activation mechanisms in the FaNaC subfamily diverging early in the evolution of DEG/ENaCs suggested that dual gating is an ancient feature in this superfamily. Notably, the GMQ-gating mode is still preserved in the mammalian ASIC subfamily, whereas FMRFamide-mediated channel gating was lost during evolution. This implied that GMQ activation may be essential for the functions of mammalian DEG/ENaCs. Our findings provide new insights into the evolution of DEG/ENaCs and may facilitate the discovery and characterization of their endogenous agonists.