Fig. 3: Two amino acids on the 4–5 intracellular linker are essential for heat sensing in HCN4.
From: HCN4 channels sense temperature and determine heart rate responses to heat
a Schematic of HCN4 subunit, highlighting the pore, voltage-sensing, intracellular linker, disc, and cyclic nucleotide binding (CNBD) domains. b Ribbon diagram showing superimposed HCN4 in apo/closed (6GYN, purple) and holo/activated (cAMP bound, 6GYO, orange) conformations. c Total solvent accessible surface area (SASA) exposed or buried in the residues of the S4-5 intracellular linker (top), transmembrane domain (TM), disk, and CNBD in response to channel opening. Apolar (green), polar (blue), and amphipathic (orange) residues are shown. d Left, representative HCN4 currents at lower (LT: 24–30 °C) and higher temperatures (HT: 34–43 °C) from cultured HEK 293 cells expressing heat-insensitive HCN4 (M407Q/Y409F). Scale bars: 500 ms (horizontal), 5 pA/pF (vertical). Middle, summary Q10 data for wild type (n = 31) and M407Q/Y409F (n = 13) HCN4. Data are mean values +/- SEM. Unpaired Student’s t-test(two-tailed), ****p < 0.0001. Right, mean activation curves (from absolute tail current values) from HEK 293 cells expressing M407Q/Y409F HCN4 channels, before (LT) and after temperature increase (HT). Data are mean values +/- SEM. Solid lines show Boltzmann fitting (Eq. 1, see Methods). V1/2 of LT or HT curves: -107 ± 0.7 mV or -113 ± 1.5 mV. The Gmax values of LT or HT curves: 41.5 or 45.9. n = 13 for LT or HT groups. e Sequence alignment of the 4-5 intracellular linker of human and mouse HCN1-4. f Mean activation curves from normalized tail currents of WT (left, n = 20 LT, n = 21 HT) and mutant 407/409 (center, n = 27 LT, n = 13 HT) HCN4 in HEK 293 cells, at low (LT) and high temperature (HT) (WT data are the same as in Fig. 2c). Data are presented as mean values +/- SEM. Solid lines show Boltzmann fitting (values in Supplementary Table 2). Right panel, data at LT for WT and HCN4M407Q/Y409F mutant are replotted for comparison. g Summary data (mean values +/- SEM) for midpoint voltage of activation (V1/2). One-way ANOVA with Tukey’s multiple comparisons test, ****p < 0.0001, p-value for NS is 0.49. n = 20, HCN4-WT-LT, n = 21, HCN4-WT-HT, n = 27, HCN4−407/409-LT, n = 13, HCN4-407/409-HT. See Supplementary Table 2 for values. Created in BioRender. Aguilar, E. (2025) https://BioRender.com/o33f198.
