Medical Faculty, Institute of Physiology, Rheinisch-Westfälische Technische Hochschule Aachen University, 52074 Aachen, Germany.
Computational Biomedicine-Institute for Advanced Simulation/Institute of Neuroscience and Medicine, Forschungszentrum Jülich, 52425 Jülich, Germany.
Proc Natl Acad Sci U S A. 2024 Oct 8;121(41):e2409097121. doi: 10.1073/pnas.2409097121. Epub 2024 Oct 4.
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.
已知的唯一肽门控离子通道-FaNaCs/WaNaCs 和 HyNaCs-属于 DEG/ENaC 家族的不同进化枝。FaNaCs 被短神经肽 FMRFamide 激活，而 HyNaCs 被 Hydra RFamides 激活，后者与 FMRFamide 没有进化关系。FaNaCs 中的 FMRFamide 结合位点最近在大细胞外结构域的裂隙顶端被鉴定出来。然而，该裂隙在 HyNaCs 中并不保守。在这里，我们结合了分子建模和定点突变，并在三聚体 HyNaC2/3/5 的细胞外结构域中鉴定出了一个用于 Hydra-RFamides 的假定结合口袋。该口袋仅位于三个亚基界面之一上，表明这种三聚体离子通道结合单个肽配体。我们在假定的结合口袋入口处设计了一个非天然氨基酸，这使得 Hydra RFamide 可以与通道共价连接，从而将通道固定在开放构象中。鉴定出的口袋位于与酸性感应离子通道 (ASIC) 结合肽配体的酸性口袋相同的区域。HyNaCs 中的口袋酸性较弱，静电和疏水相互作用都有助于肽结合。总的来说，我们的结果揭示了 HyNaCs 和 ASICs 中保守的配体结合口袋，并表明在肽门控离子通道的这两个亚群中，肽结合腔的独立进化。
