Fig. 6: Structural features of the TadNaC2 channel.

a Ribbon diagrams of the chick ASIC1 homotrimeric channel crystal structure (left, PDB number 6VTK), and the AlphaFold-predicted tertiary structure of the mouse ASIC1a subunit (right). The three separate subunits of the homotrimeric channel are colored in red, white, and gray, and the colored circles denote the β carbon atoms of critical residues corresponding to the back-colored residues of the mouse ASIC1a channel in the protein alignment shown in (b) (i.e., atoms in blue are within the acid pocket, pink are within the wrist, green are within the palm, and purple are within the finger). The dashed boxes denote structural regions of the single mASIC1a subunit structure bearing these same critical residues. b Protein sequence alignment of the acid pocket (enclosed by blue dashed boxes), wrist (pink), palm (green), and finger (purple) regions of select proton-activated Deg/ENaC channels from cnidarians and bilaterians with TadNaC2, TadNaC6, HhoNaC2, and HhoNaC4/6/7 channels from the placozoans Trichoplax adhaerens and Hoilungia hongkongensis. Residues that are back-colored in black represent conserved residues for proton activation of ASIC channels, while those back-colored red denote key residues that render the ASIC2b splice variant insensitive to external protons. Residues that are back-colored in gray denote protonatable amino acids in TadNaC2 within these key structural regions, some of which are conserved in cnidarian and bilaterian homologs, while those back-colored in brown denote cationic residues in TadNaC2 that flank the critical K211 residue of ASIC channels, also found in several other channels. Notable is the complete conservation of the critical residues H73 and K211 in all included ASIC channels, and their absence in most non-ASIC proton-activated channels including TadNaC2. c Homology model of the homotrimeric TadNaC2 channel structure (left), and AlphaFold-predicted structure of the single subunit, with a similar annotation as described for (a). d Left panels: Close-up view of the acid pocket region of mASIC1a (top) and TadNaC2 (bottom) within corresponding AlphaFold-predicted structures. The six rendered residues in the TadNaC2 channel correspond to residues that align with the six acid pocket residues in mASIC1a as depicted in (a). Right panels: Surface rendering of the acid pocket region of mASIC1a (top) and TadNaC2 (bottom) reveals a stark difference in the electrostatic potential between the two channel subunits. e Close-up view of the wrist and palm regions of mASIC1a and TadNaC2. Apparent in the wrist region is the absence of a critical H73 proton-sensing residue in TadNaC2, but conservation of the aromatic amino acids F70 and W276, which in mASIC1a (i.e., Y71 and W278) form an aromatic bridge critical for channel gating. Instead, TadNaC2 bears a putative proton-sensing amino acid (H80) at the opposite end of a β strand that projects from the first transmembrane helix in wrist region (TMH1) to the palm domain, placing it near the residues R201 and K203 that flank the critical K211 residue of mASIC1a. f Close-up view of the finger and acid pocket regions, with rendered amino acids corresponding to the positions in the ASIC2b splice variant that make the channel insensitive to protons. Also labeled are the equivalent acid pocket residues, and the predicted α1 to α3 helices in the finger region.