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Proc Natl Acad Sci U S A
2024 Oct 08;12141:e2409097121. doi: 10.1073/pnas.2409097121.
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A conserved peptide-binding pocket in HyNaC/ASIC ion channels.
Ortega-Ramírez AM
,
Albani S
,
Bachmann M
,
Schmidt A
,
Pinoé-Schmidt M
,
Assmann M
,
Augustinowski K
,
Rossetti G
,
Gründer S
.
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The only known peptide-gated ion channels-FaNaCs/WaNaCs and HyNaCs-belong to different clades of the DEG/ENaC family. FaNaCs are activated by the short neuropeptide FMRFamide, and HyNaCs by Hydra RFamides, which are not evolutionarily related to FMRFamide. The FMRFamide-binding site in FaNaCs was recently identified in a cleft atop the large extracellular domain. However, this cleft is not conserved in HyNaCs. Here, we combined molecular modeling and site-directed mutagenesis and identified a putative binding pocket for Hydra-RFamides in the extracellular domain of the heterotrimeric HyNaC2/3/5. This pocket localizes to only one of the three subunit interfaces, indicating that this trimeric ion channel binds a single peptide ligand. We engineered an unnatural amino acid at the putative binding pocket entrance, which allowed covalent tethering of Hydra RFamide to the channel, thereby trapping the channel in an open conformation. The identified pocket localizes to the same region as the acidic pocket of acid-sensing ion channels (ASICs), which binds peptide ligands. The pocket in HyNaCs is less acidic, and both electrostatic and hydrophobic interactions contribute to peptide binding. Collectively, our results reveal a conserved ligand-binding pocket in HyNaCs and ASICs and indicate independent evolution of peptide-binding cavities in the two subgroups of peptide-gated ion channels.
Fig. 1. Photo-cross-linking reveals the subunit arrangement of HyNaC subunits. (A) Homology models of the two possible orders of the HyNaC subunits, based on the closed conformation of cASIC1. Left, the clockwise 2-3-5 order, and Right, the clockwise 2-5-3 order, viewed from the top. HyNaC2 is depicted in green, HyNaC3 in blue, and HyNaC5 in yellow. Residues 3-N213 and 3-F330 are highlighted with purple sticks. (B) Western blot analysis illustrating cross-linking of two subunits after UV irradiation. HyNaC2, HyNaC3, and HyNaC5 were coexpressed in HEK293 cells; subunits carried HA or Flag epitopes as indicated. The red arrow indicates putative cross-linked dimeric complexes with an apparent molecular weight of ~150 kDa. On the Left, HyNaC3-containing complexes were detected with an anti-HA antibody, and on the Right, HyNaC2- or HyNaC5-containing complexes were detected on the same blot with an anti-FLAG antibody.
Fig. 2. Site-directed mutagenesis identifies residues at the 5-3 interface important for activation by RFamide I. Top, sideview on the three interfaces in a homology model of HyNaC2/3/5, based on the open conformation of cASIC1. The position of substituted amino acids is indicated. HyNaC2 is shown in green, HyNaC3 in blue, and HyNaC5 in yellow. Bottom, EC50 values (mean ± SD) for activation by RFamide I for wild-type HyNaC2/3/5 and mutant channels. One subunit carrying the indicated mutations was coexpressed with the other two wild-type subunits. Mutants are grouped by interfaces. The dashed black line indicates the mean EC50 value of the wt. *P < 0.05; **P < 0.01; ***P < 0.001 (one-way ANOVA).
Fig. 3. Alanine scanning of RFamide II reveals residues important for activation of HyNaCs. (A) Representative current trace for activation with the F7A mutant. Wild-type RFamide II (1 µM) was used as control. (B) As in (A), but for G5A. (C) Concentration–response relationships (mean ± SD) for wild-type RFamide II (black circles; n = 15), G5A (blue circles; n = 5), pQ1A (n = 11), W2A (n = 10), F3A (n = 5), N4A (n = 8), R6A (n = 5), and F7A (n = 6) (open circles).
Fig. 4. Molecular docking results and pose selection. (A) Protein–ligand interaction fingerprint of the selected poses (see Methods for details) for RFamide I (Left panel) and RFamide II (Right panel). For each residue, the average number of interactions with the bound poses is reported. The bar color represents whether an interaction is formed between the ligand and the backbone (blue) or the side chain (gold) of the residue. Only bars with a value larger than 0.3 are displayed. (B) Top and (C) side view of the bound pose within the homology model. Green, yellow, and light blue ribbons and sticks represent HyNaC2, HyNaC5, and HyNaC3, respectively. The ligand is colored in reddish purple. (D) Detailed stick representation of RFamide I in the binding pocket. All residues within 3.0 Å of the neuropeptide are shown. Point of view as indicated in (C). For RFamide II, see SI Appendix, Fig. S4. (E) Schematic representation of the hydrogen bonds (blue arrows) and aromatic interactions (green dashed lines) between the top-scoring pose of the neuropeptide and the protein.
Fig. 5. Covalent cross-linking of RFamide II constitutively activates HyNaC2/3/5. (A) Scheme illustrating how cross-linking RFamide peptides (RFa) to HyNaC might constitutively open the ion pore. (B) Left, representative current traces for 3-F330azF coexpressed with HyNaC2/5 wild-type. Top, for cells that had been treated with UV light. Bottom, for cells that had been treated with UV light in the presence of 5 µM RFamide II (RF II). Right, normalized summary data (mean ± SD). Absolute current amplitudes (mean ± SD) were 445 ± 40 pA for cells treated with UV light and 438 ± 86 pA for cells treated with UV light in the presence of RFamide II. (C) As in (B) but for 3-E338azF. Absolute current amplitudes were 403 ± 61 pA for cells treated with UV light, and 387 ± 58 pA for cells treated with UV light in the presence of RFamide II. *P < 0.05; **P < 0.01; ***P < 0.001 (one-way ANOVA).
Fig. 6. Key amino acids are conserved between the peptide-binding pocket of HyNaCs and the acidic pocket of ASICs. (A) Alignment of sequences contributing to the peptide-binding site in HyNaCs with the corresponding sequence of cASIC1. The linker following β2 is in the lower finger, β4-β7 are in the β-ball, and α5 is in the thumb domain. Conserved residues on the principal side of the HyNaC binding site are in bold red, those on the complementary side are in bold green, and amino acids contributing to the acidic pocket of cASIC1 are in bold blue. Note that the β6-β7 linker is shorter in HyNaCs than in cASIC1 and the alignment in this region is ambiguous. The sequences of the β11- β12 linker, which is not involved in peptide binding, are also shown. Numbers above sequences indicate positions in HyNaC3, HyNaC5, and cASIC1, respectively; positions of α-helices and β-sheets above sequences are based on cASIC1 (12). (B) View under a comparable angle into the peptide binding pocket at the 5-3 interface (Left) and into the acidic pocket of cASIC1 (Right). Amino acids highlighted in (A) are indicated. Green, yellow, and light blue subunits correspond to HyNaC2, HyNaC5, and HyNaC3, respectively; the three cASIC1a subunits are distinguished by similar colors. (C) Scheme illustrating how a single peptide binding site may be sufficient to open the HyNaC ion pore. Binding of a single RFamide could induce movement of a single thumb domain, closing the binding pocket which is transmitted to the lower ECD to open the ion pore. However, the incomplete movement of all three thumb domains might prohibit gating movements that are associated with desensitization in ASICs. In ASICs all three thumb domains move upon binding of protons, opening the ion pore and allowing concomitant desensitization.
