Fig. 1: Hydrophobic and hydrophilic interactions of the D and E helices with the C-helix.

a Ribbon representation of human HCN1 tetrameric structure bound to cAMP (PDB: 6UQF). Helices D and E of each monomer are shown in yellow and labeled. Boxed: Blow-up showing residues L601, L602 of D-helix and D629 of E-helix engaging hydrophobic and hydrophilic interactions with I588 and R593 on the C-helix of their own subunit. Numbering refers to hHCN1 sequence. b Multiple sequence alignment and secondary structure elements in the region of the D and E helices of human and mouse HCN1 (hHCN1 and mHCN1, Gene ID: 348980 and 15165, respectively), human and mouse HCN2 (hHCN2 and mHCN2, Gene ID: 610 and 15166, respectively) and rabbit HCN4 (rbHCN4, Gene ID: 100009452). Residues shown in the blow-up in panel a, are highlighted in gray and in yellow. Symbols below denote residue identity (*) and conservative (:) or semi-conservative (.) substitutions. Arrowheads indicate the last residue of the truncated constructs, color-coded as follow: ΔC-term gray, ΔE green, ΔDE’ blue and ΔDE red. c Representative whole cell currents of HCN4 full length (HCN4 FL) and ΔC-term recorded at −30, −90 and −150 mV in control solution and with 1 µM cAMP in the patch pipette. Scale bars: 200 pA and 500 ms. Right: mean activation curves in control solution (full symbols) and with cAMP (empty symbols) from HCN4 FL (black) and ΔC-term (gray). Data are presented as mean ± SEM. Data fit with a Boltzmann function are plotted as solid (control) and dashed line (+1 µM cAMP). Calculated half-activation voltages (V_1/2) and inverse slope factors (k) are reported in Supplementary Table 2 together with the details on statistical analysis.