Comparative analysis defines a broader FMRFamide-gated sodium channel family and determinants of neuropeptide sensitivity

Abstract

FMRFamide (Phe-Met-Arg-Phe-amide, FMRFa) and similar neuropeptides are important physiological modulators in most invertebrates, but the molecular basis of FMRFa activity at its receptors is unknown. We therefore sought to identify the molecular determinants of FMRFa potency against one of its native targets, the excitatory FMRFa-gated sodium channel (FaNaC) from gastropod mollusks. Using molecular phylogenetics and electrophysiological measurement of neuropeptide activity, we identified a broad FaNaC family that includes mollusk and annelid channels gated by FMRFa, FVRIamides, and/or Wamides (or myoinhibitory peptides). A comparative analysis of this broader FaNaC family and other channels from the overarching degenerin (DEG)/epithelial sodium channel (ENaC) superfamily, incorporating mutagenesis and experimental dissection of channel 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 but enhanced by FMRFa, 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 FMRFa neuropeptide activity at FaNaC receptor channels and illuminates the evolution of ligand recognition in one branch of the DEG/ENaC superfamily of ion channels.

Keywords: acid-sensing ion channel; electrophysiology; evolution; invertebrate; ion channel; neuropeptide; phylogenetics.

Figure 1

Phylogenetic and experimental characterization of the FaNaC family.A, FaNaC branch from DEG/ENaC phylogeny (full phylogeny in Fig. S1). ∗Genes tested here. ∞Genes tested elsewhere. Genes in bold encode peptide-gated channels. aLRT support for key branches is shown. Blue background, broader FaNaC family; green background, annelid-specific branch of FaNaCs with diverse ligand sensitivity; pink background, Wamide-activated channels (WaNaCs). B, example peptide-gated currents at Xenopus oocytes expressing indicated genes. All peptide applications 100 μM, except for some at Capitella FaNaC: FMRFa, FLRPa, FLRFa, and FMKFa (3 μM); LFRYa (30 μM). The scale bars represent x, 10 s; y, 5 μA. C, mean (± SEM) normalized current response to different peptides (concentrations as in (B)) at oocytes expressing indicated channels. Lighter symbols are individual data points, n = 4 to 7. FaNaC, FMRFa-gated sodium channel; FMRFa, Phe-Met-Arg-Phe-amide.

Figure 2

Neuropeptide sensitivity of different members of the FaNaC family.A, example currents in response to increasing concentrations of FMRFa, ARSGFVRIa, or KWGGNSRMWa at oocytes expressing indicated annelid (Malacoceros fuliginosus) channels. The scale bars represent x, 10 s; y, 1 μA. B, mean (± SEM, n = 4–5) normalized current responses to increasing concentrations of neuropeptides (as indicated) at FaNaCs and WaNaCs. FaNaC, FMRFa-gated sodium channel; FMRFa, Phe-Met-Arg-Phe-amide; WaNaC, Wamide-gated sodium channel.

Figure 3

Comparative analysis of amino acid residues determining FMRFa sensitivity.A, part of amino acid sequence alignment comparing verified FMRFa-gated channels with other channels (complete alignment in Fig. S3). Examples of FaNaC-specific amino acid residues, that is, those that are conserved—either completely or in terms of physicochemical properties—in FaNaCs but different in other channels, are indicated by arrowheads. B, left, Aplysia kurodai FaNaC homology model. Different subunits in different shades of blue, approximate position of cell membrane in gray. Right, 43 FaNaC-specific amino acid residues (dark blue spheres) in a single subunit of model. C, example current responses to FMRFa at oocytes expressing WT or mutant Aplysia kurodai FaNaC channels. The scale bars represent x, 5 s; y, 1 μA. D, mean (± SEM) FMRFa potency (3 μM FMRFa-gated current amplitude/30 μM FMRFa-gated current amplitude: “I_{3 μM}/I_{30 μM}”) was compared by one-way ANOVA with Dunnett's multiple comparisons test: dark blue, not significantly different to WT or significantly greater than WT; light blue, significantly less than WT (p < 0.05); white, not significantly different from zero. Individual data points shown as faint symbols, n = 3 to 14. FaNaC, FMRFa-gated sodium channel; FMRFa, Phe-Met-Arg-Phe-amide.

Figure 4

Isolation of individual determinants of FMRFa sensitivity in Aplysia FaNaC.A, example current responses to FMRFa at oocytes expressing mutant Aplysia kurodai FaNaC channels. The scale bars represent x, 5 s; y, 200 nA. B, mean (± SEM) FMRFa potency (3 μM FMRFa-gated current amplitude/30 μM FMRFa-gated current amplitude: “I_{3 μM}/I_{30 μM}”), WT repeated from Figure 3. Dark blue, not significantly different to WT; light blue, significantly less than WT (p < 0.05); white, not significantly different from zero. Individual data points as faint symbols, n = 4 to 14. C, example responses to increasing FMRFa concentrations at indicated Aplysia FaNaC mutants. The scale bars represent x, 10 s; y, 10 μA (WT and S184A) or 500 nA (F231G). D, mean (±SEM, n =3–6) normalized current amplitude in response to increasing FMRFa concentrations. Each individual data point was normalized to mean maximum current amplitude at WT (n = 3) on the same day. E, extracellular domain of Aplysia FaNaC homology model showing selected residues (sticks) at the interface of two adjacent subunits (one cyan, one blue). FaNaC, FMRFa-gated sodium channel; FMRFa, Phe-Met-Arg-Phe-amide.

Figure 5

Determinants of neuropeptide activity in different FaNaCs and WaNaCs.A, example current responses to indicated neuropeptide at oocytes expressing WT or mutant Aplysia FaNaC (AkFaNaC), Capitella FaNaC⁵²⁸³³ (CtFaNaC), or Capitella WaNaC²¹²⁹¹² (CtWaNaC). The scale bars represent x, 10 s; y, 5 μA. B, average (± SEM, n = 4–5) FMRFa potency (I_{3 μM}/I_{30 μM} at Aplysia FaNaC, I_{0.3 μM}/I_{3 μM} at Capitella FaNaC) and AWVGDKSLSWa potency (I_{3 μM}/I_{30 μM} at Capitella WaNaC), grouped according to the equivalent amino acid residue. FaNaC, FMRFa-gated sodium channel; WaNaC, Wamide-gated sodium channel.

Figure 6

Determinants of FRRFa modulation of rat ASIC1a.A, pH 5.3 (filled black bars)-gated currents after preincubation in desensitizing pH (unfilled bars) alone or in 50 μM FRRFa (blue bars) at oocytes expressing WT or mutant rat ASIC1a (rASIC1a). The scale bars represent x, 10 s; y, 200 nA. Desensitizing pH described in Figure S4. B, mean (± SEM, n = 4–7) enhancement (current_b/current_a from (A)) of desensitized current amplitude by 50 μM FRRFa at indicated rat ASIC1a mutants. ASIC, acid-sensing ion channel.