HCN4 channels sense temperature and determine heart rate responses to heat

Abstract

The hyperpolarization-activated cyclic nucleotide-gated ion channel 4 (HCN4) current increases due to cAMP binding and is well-recognized to contribute to adrenergically driven heart rate acceleration. HCN4 current also increases with heat by an unknown mechanism(s). We use thermodynamical and homology computational modeling, site-directed mutagenesis, and mouse models to identify a concise motif on the S4-S5 linker of HCN4 channels (M407/Y409) that determines HCN4 current (I_f) responses to heat. This motif is required for heat-triggered rate acceleration in cardiac pacemaker cells, isolated hearts and in vivo. Surprisingly, a loss of function M407/Y409 motif mutation prevented not only normal heat but also cAMP responses, suggesting that the heat-sensing machinery within the S4-S5 linker is essential for operating the cAMP allosteric pathway and is central to HCN4 gating modulation. The M407/Y409 motif is conserved across all HCN family members suggesting that HCN channels participate broadly in coupling heat to changes in cell membrane excitability.

Fig. 1. HCN4 antagonists or Hcn4 knock out prevent action potential rate increases by heat in SAN cells.

a Isolated SAN cell in whole cell mode current clamp configuration, scale bar = 50 µm. b Representative SAN cell action potential (AP) tracings recorded under lower (left) and increased temperature (right) from the same SAN cell. c Representative SAN cell AP tracings recorded in the presence of HCN4 antagonist agents Ivabradine (4 μM) or (d) ZD7288 (4 μM) under lower (left) and increased temperature (right). The AP was recorded at different temperatures but from the same SAN cell in each case. e upper panel, Example of adenovirus-infected SAN cells from floxed mice expressing mCherry and Cre (red, right) (scale bar = 50 µm). lower panel, Representative AP tracings recorded under lower basal (left) and increased temperature (right) conditions from the same cultured single SAN cells isolated from Hcn4^flox/flox mice cultured in the presence of adenovirus expressing Cre recombinase (see Methods). b–e the horizontal lines mark 0 mV. Scale bars are 200 ms horizontal and 20 mV vertical. f Summary data for Q₁₀ from SAN cell AP rate responses to temperature increases. The presence of isoproterenol (ISO, 1 μM), Ivabradine (Iva, 4 μM), or ZD7288 (ZD, 4 μM) in the bath solution is indicated. Data are presented as mean values +/- SEM. One-way ANOVA, ***p < 0.001, ****p < 0.0001, p (baseline vs. ISO) 0.8, p-values for comparisons of right 5 groups are from 0.52- > 1. (n = 20 Baseline, n = 17 ISO, n = 5 Iva, n = 9 ZD, n = 7 ZD-ISO, n = 8 Iva-ISO, n = 6 HCN4^-/-).

Fig. 2. HCN4 current (I_f) has a Q₁₀ of ~2.0 that is independent of cAMP signaling.

Representative I_f (HCN4 current) tracings recorded under lower and increased temperatures (left panels) from (a), the same isolated SAN cell or (b) in the same human embryonic kidney cell (HEK) transfected with HCN4 (see Methods). Current-voltage relationship from the same cell (right panels, filled circles lower temperature and open circles higher temperature). The right-most panels expanded to highlight the physiological cell membrane potential range for SAN cells (-40 – -80 mV). The horizontal scale bar is 500 ms and the vertical scale bar is 5 pA/pF in both (a) and (b). c Activation curves from absolute (left) or normalized tail current values (right) from HEK 293 cells expressing HCN4, before and after temperature increase (empty circle: lower temperature, filled circle: higher temperature). Data are presented as mean values +/- SEM. Solid lines show Boltzmann fitting to the data (Eq.1, see Methods) yielding the following values of V_1/2: -107 ± 1 mV (LT) and -97 ± 2 mV (HT) in left panel and -107 ± 0.7 (LT) and -96 ± 1.2 (HT) in right panel. The Gmax values: 35.9 (LT) and 57.3 (HT), and A values (offset): 2.11 (LT) and 6.42 (HT) in left panel. Student’s t-test or Mann-Whitney Rank Sum Test applied when appropriate, (two-tailed) *p < 0.05, **p < 0.01, ***p < 0.001. n = 15 for both LT or HT groups. d Time constant of activation at -130 mV from HCN4 channel current (n = 17, empty circle) at lower temperature and higher temperature (n = 5, filled circle). Data are presented as mean values +/- SEM. ****p < 0.0001 by Student’s t-test (two-tailed). e Summary data for Q₁₀ from HCN4 channel currents at −60 mV from HEK cells expressing HCN4 in the presence (n = 12) or absence of cAMP (n = 34). Data are presented as mean values +/- SEM. f Summary Q₁₀ data for I_f at −60 mV from isolated SAN cells treated with ISO (1 µM, n = 13), and/or Ivabradine (Iva, 4 μM, n = 6, Iva+ISO, n = 6) or ZD7288 (ZD, 4 μM, n = 5, ZD + ISO n = 4), n = 10 Baseline, n = 26, HEK. Data are presented as mean values +/- SEM. One-way ANOVA *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 3. Two amino acids on the 4–5 intracellular linker are essential for heat sensing in HCN4.

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). V_1/2 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 HCN4^M407Q/Y409F mutant are replotted for comparison. g Summary data (mean values +/- SEM) for midpoint voltage of activation (V_1/2). 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.

Fig. 4. Mice with Hcn4^M407Q/Y409F mutant channels exhibit reduced rate responses to heat.

a I_f currents under LT (left) and HT (middle) from SAN cells of Hcn4^+/QF mice. Bar graph: Q₁₀ at −60 mV from Hcn4^+/QF (n = 7) and WT SAN cells (n = 10). Data are mean +/- SEM, unpaired Student’s t-test (two-tailed), ****p < 0.0001. Scale bar: 5 pA/pF (vertical) and 500 ms (horizontal) (same for e and f). b AP under LT (left) and HT (middle) from Hcn4^+/QF SAN cells. Bar graph: Q₁₀ on AP rates from Hcn4^+/QF (n = 4) and WT SAN cells (n = 20), mean +/- SEM, unpaired Student’s t-test (two-tailed), ****p < 0.0001. Horizontal lines mark 0 mV. Scale bars: 200 ms (horizontal) and 20 mV (vertical) (same for e and f). c ECG under LT (left) and HT (middle) from isolated hearts of WT (upper) or Hcn4^+/QF (lower) mice. Bar graph: Q₁₀ on ECG rates (beats/min, BPM) for Hcn4^+/QF mice (n = 11) and WT mice (n = 8). Data are mean +/- SEM, unpaired Student’s t-test (two-tailed), ***p = 0.0002. d ECG under lower or increased body temperatures from unrestrained WT (upper) or Hcn4^+/QF (lower) mice. Bar graph: in vivo heart rates (upper, n = 5 Hcn4^+/QF, n = 8 WT) and body temperature (lower, n = 5 Hcn4^+/QF, n = 8 WT) used to define HT and LT. Data are mean +/- SEM. *p = 0.048, by one-way ANOVA with Tukey’s test. e Representative I_f (left) and AP (middle) under LT or HT from cultured Hcn4^flox/flox SAN cells infected with Ad-mCherry-Cre and Ad-HCN4-WT. Right panels: I_f current density-voltage relationship calculated from the same cell. The right-most panel expanded to highlight the physiological cell membrane potential range for SAN cells. f Similar to (e), I_f and AP recordings and I_f current density-voltage relationship from cultured Hcn4^flox/flox SAN cells infected with Ad-mCherry-Cre and Ad-HCN4^QF/QF. g Q₁₀ data for I_f at −60 mV from Hcn4^flox/flox SAN cells expressing HCN4 WT (n = 11), or HCN4^QF/QF (n = 5). Data are mean +/- SEM; unpaired Student’s t-test (two-tailed) ***p = 0.0003. h Q₁₀ data for AP rates from cultured Hcn4^flox/flox SAN cells expressing HCN4 WT (n = 8), or HCN4^QF/QF (n = 7). Data are mean +/- SEM; unpaired Student’s t-test (two-tailed), ***p = 0.0006.

Fig. 5. Heat sensing is required for cAMP-dependent activation of HCN4.

a Representative I_f currents recorded from HCN4 transfected HEK 293 cells with or without intracellular dialysis of cAMP (1 mM) (left panel). Red traces indicated current amplitude at -90 mV for comparison. Boltzmann distribution for HCN4 transfected HEK 293 cells (middle panel) and summary data for midpoint voltage of activation (right panel, V_1/2). Data are presented as mean values +/- SEM. Unpaired Student’s t-test (two-tailed), ****p < 0.0001, n = 20, HCN4-WT, n = 14 HCN4-WT+cAMP. b Representative I_f currents recorded from mutant HCN4 (EA) transfected HEK 293 cells with or without intracellular dialysis of cAMP (1 mM) (left panel). Red traces indicated current amplitude at -100 mV for comparison. Data are analyzed and displayed in (b) middle and right panels as in (a) panel (above). Data are presented as mean values +/- SEM. n = 22 HCN4-EA, n = 11 +cAMP. c Representative I_f currents recorded under lower (left) and increased (middle) temperature conditions from cultured HEK 293 cells expressing HCN4 EA mutants. Summary Q₁₀ data for I_f currents at −60 mV from HEK 293 cells (right panel). Data are presented as mean values +/- SEM, n = 31 HCN4-WT, n = 22 HCN4-EA. Example tracings and summary data are calculated using data from the same cell. d Representative I_f currents in the presence or absence of intracellular dialysis with cAMP (1 mM) (left panels) in HCN4^407/409 mutant expressing HEK 293 cells. Red traces indicated current amplitude at -90 mV for comparison. Right two panels, HCN4^407/409 current activation curves without (empty symbols) and with cAMP (1 mM) (filled symbols) dialysis. Summary data for midpoint voltage of activation (V1/2) with(n = 13) or without cAMP(n = 27) (right). Data are presented as mean values +/- SEM. Solid lines show Boltzmann fitting to the data (middle, see Methods). Values are reported in Supplementary Table 2. a–d Scale bars are 500 ms horizontal and 5 pA/pF vertical.