Fig. 3

Molecular dynamics simulations reveal a stable Ca²⁺-binding site in the ASIC channels. a cASIC1 homotrimer embedded in the POPE lipid bilayer and close-up view of the channel pore. The lipid phosphorus atoms are showed as magenta spheres and the three protein subunits are represented as cyan, orange and ice blue ribbons, respectively, with the pore gate highlighted in red. The extracellular and intracellular vestibules are indicated by dotted lines. The Cα atoms of the residues at positions 429 (target mutation site), 433, and 436 are displayed as spheres. b Probability distribution of the area of the triangle formed by the Cα atoms of symmetry-related Gly429 (or Glu429 in the mutant systems) (upper), Asp433 (middle), and Gly436 (lower). The red and magenta curves correspond to the cASIC1 G429E systems with 1 Ca²⁺ (mtASIC1_1Ca²⁺) and without Ca²⁺ (mtASIC1_0Ca²⁺) bound, respectively, and the green and cyan curves denote the wild-type systems with 1 Ca²⁺ (wtASIC1_1Ca²⁺) and without Ca²⁺ (wtASIC1_0Ca²⁺), respectively. The dashed and dashed-dotted lines denote the area values calculated from the crystal structures in desensitized and wide open states (PDB codes: 4NYK & 4NTW), respectively. c, d The Ca²⁺ block site identified at the pore entrance with the Ca²⁺ point charge (c) and dummy atom models (d). The Ca²⁺ in the mutant channel (mtASIC1_1Ca²⁺) forms both pentagonal bi-pyramidal and octahedral coordination (first and second panels), whereas the Ca²⁺ binding in the wild channel (wtASIC1_1Ca²⁺) assumes an exclusive octahedral geometry (third panel). The residues Glu429 and Asp33 and water molecules coordinating Ca²⁺ are represented in stick and ball-and-stick models, respectively, and the Cα atom of Gly429 in the wild-type system is shown as a sphere. The coordination number (CN) is indicated at the upper-right corner of each panel. For clarity, the dummy complex in d has been shown as a Lennard–Jones sphere as in c. Statistical analysis of the coordination bonds and angles is reported in Supplementary Table 2. e Inter-subunit ionic interactions of Glu426 in TM2 and Arg65 in TM1 stabilizing a putative intermediate state of the G429E mutant