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FMRFamide and similar neuropeptides are important physiological modulators in most invertebrates, but the molecular basis of FMRFamide activity at its receptors is unknown. We therefore sought to identify the molecular determinants of FMRFamide potency in one of its native targets, the excitatory FMRFamide-gated sodium channel (FaNaC) from gastropod mollusks. Using molecular phylogenetics and electrophysiological measurement of function, we identified a broad FaNaC family that includes mollusk and annelid channels gated by FMRFamide, FVRIamides, and/or Wamides (or myoinhibitory peptides). A comparative analysis of this broader FaNaC family and other channels from the overarching DEG/ENaC superfamily, incorporating mutagenesis and experimental dissection of function, identified a pocket of amino acid residues that determines activation of FaNaCs by neuropeptides. Although this pocket has diverged in distantly related DEG/ENaC channels that are activated by other ligands, such as mammalian acid-sensing ion channels, we show that it nonetheless contains residues that determine enhancement of those channels by similar peptides. This study thus identifies amino acid residues that determine FMRFamide activity at FaNaCs and illuminates evolution of ligand recognition in one branch of the DEG/ENaC superfamily of ion channels.

Rapid chemo-electric signaling between neurons is mediated by ligand-gated ion channels, cell-surface proteins with an extracellular ligand-binding domain and a membrane-spanning ion channel domain. The degenerin/epithelial sodium channel (DEG/ENaC) superfamily, which occurs throughout the animal kingdom, is unique in its diversity of gating stimuli, with some DEG/ENaCs gated by conventional ligands such as neuropeptides, and others gated by e.g. pH, mechanical force, or enzymatic activity. The mechanism by which ligands bind to and activate DEG/ENaCs is poorly understood. We have therefore dissected the structural basis for neuropeptide binding and gating in a neuropeptide-gated DEG/ENaC, FMRFamide-gated sodium channel 1 (FaNaC1) from the annelid worm Malacoceros fuliginosus, using cryo-electron microscopy. High-resolution structures of FaNaC1 in the ligand-free resting state and in several ligand-bound states reveal the ligand-binding site and capture the ligand-induced conformational changes that mediate channel gating. Complementary mutagenesis experiments confirm the functional roles of particular amino acid residues implicated by the structures. Our results illuminate ligand-induced channel gating in DEG/ENaCs and offer a structural template for the experimental dissection of channel pharmacology and ion conduction in a characteristically metazoan ion channel superfamily.

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