Introduction

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are activated by membrane hyperpolarization and conduct mixed Na+/K+ currents, thus contributing to pacemaker depolarization, which generates rhythmic activity in sinoatrial node cells and spontaneously active neurons [1,2,3,4,5]. One notable characteristic of HCN channels is that they are dually activated by voltage hyperpolarization and intracellular cyclic adenosine monophosphate (cAMP) [6,7,8,9,10]. To date, four mammalian HCN isoforms (HCN 1–4) have been identified, each with distinct biophysical, modulatory properties, and diverse tissue distributions [4, 5, 11, 12]. HCN4 is preferentially expressed in the sinoatrial node cells of mammals [13, 14]. Loss-of-function mutations of the HCN4 gene have been identified as responsible for several heart channelopathies [15,16,17,18]. All HCN isoforms are widely expressed in the central and peripheral nervous system. Dysfunctional HCN channels in the nervous system are involved in numerous pathologic processes such as pain, epilepsy, autism spectrum disorder, schizophrenia, and mood disorders [5, 19]. HCN channels have been proposed as a novel target for pharmacological intervention for angina, heart failure, pain, and epilepsy [20].

Two classes of HCN channel inhibitors have been identified and broadly studied, including “bradine” compounds (zatebradine, cilobradine, and ivabradine) and ZD7288 (chemical structures in Fig. 1). Among all “bradine” compounds, ivabradine is the only one that is clinically approved for the treatment of angina pectoris and heart failure [20,21,22]. ZD7288 is another HCN channel blocker, which is structurally unrelated to “bradine” compounds and is broadly used as a tool to study the biophysical and physiological properties of HCN channels [23, 24]. Both ivabradine and ZD7288 block all HCN isoforms to a similar extent, and they are thought to be pore blockers with similar action sites on HCN channels [24,25,26,27]. Recently, structural modifications of the nonselective inhibitor zatebradine generated several new inhibitors (EC18, MEL55A and MEL57A), which show subtype selectivity towards different HCN subtypes [28, 29]. The discovery of inhibitors with novel structural backbones and subtype selectivities could be beneficial for both the biophysical and pharmacological study of HCN channels.

Fig. 1
figure 1

Structures of known HCN channel inhibitors

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During our initial efforts to identify novel HCN channel inhibitors, we serendipitously discovered that compound 4a inhibited the HCN2 channel significantly. Furthermore, we designed and synthesized a series of novel alkanol amine compounds based on compound 4a and evaluated their ability to inhibit the HCN channels. The most potent compound, 4e, blocked the HCN2 current with an IC50 of 2.9 ± 1.2 µM at − 120 mV. In addition to blocking channel conductance, 4e caused a slowing of activation and a hyperpolarizing shift of the voltage dependence of HCN2 channel activation. Moreover, we found that 4e blocked Ih in small dorsal root ganglion (DRG) neurons and inhibited its action potential firing. Our study may provide a new structural backbone for the development of future HCN channel inhibitors.

Materials and methods

Chemistry

All of the compounds were synthesized according to Scheme 1; 4-bromobut-1-ene and mCPBA were used to synthesize 2-(2-bromoethyl)oxirane. The 2-(2-phenoxyethyl)oxirane was synthesized by substituted phenol derivatives and 2-(2-bromoethyl)oxirane. Connection with 3,5-dimethylpyrazole and substituted benzyl chloride in weak base resulted in the 1-benzyl-3,5-dimethyl-1H-pyrazole derivatives. The corresponding secondary amine compounds were prepared with paraformaldehyde and methylamine hydrochloride in ethanol through the Mannish reaction. 2-(2-phenoxyethyl)oxirane and a secondary amine were refluxed in ethanol to give the targeted products (4a–h).

Scheme 1
scheme 1

Synthesis of alkanol amine derivatives. Reagents and conditions. (i) mCPBA, CH2Cl2, rt, 24 h. (ii) K2CO3, acetone, Rf, 16 h. (iii) 3,5-dimethyl-1H-pyrazole, KOH, DMSO, 80 °C, 1.5 h; then benzyl cholide, rt, 2 h. (iv) CH3NH2, (CHO)n, EtOH, Rf, 16 h. (v) EtOH, Rf, 24 h

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Cell culture and transfection

COS7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, USA) containing 10% fetal bovine serum (FBS) and maintained at 37 °C with a 5% CO2 atmosphere.

The open reading frames of human HCN1 (NM_021072.3), mouse HCN2 (NM_008226.2), and mouse HCN4 (NM_001081192.1) were synthesized, sequenced, and further cloned into pEGFP-N1 vectors.

To express the HCN channels, COS7 cells were cultured in a six-well plate, maintained overnight, and then transiently transfected with 1.5 μg plasmid DNA (HCN1, HCN2, or HCN4) using Lipofectamine 2000 reagent (Life Technologies) according to the manufacturer’s instructions. After 24–48 h, cells were split and redistributed onto coverslips coated with 0.1 mg/mL poly-l-lysine (Sigma-Aldrich, USA) for the following electrophysiological experiments.

Acute dissociation of DRG neurons

DRG neurons were isolated from 4–6-week-old wild-type C57BL/6 male mice. In brief, all mice were killed by cervical dislocation followed by decapitation, and DRG neurons were dissected from the entire length of the spinal column and placed directly into cooled phosphate-buffered saline as a cleaning step. The DRG neurons were digested with DMEM containing 2 mg/mL collagenase type IA (Sigma-Aldrich, USA) and 0.5 mg/mL trypsin (Solarbio, USA) for 40 min at 37 °C. After terminating the enzymatic treatment by FBS, DRG neurons were centrifuged at 1000 r/min for 5 min, and the supernatant was discarded and resuspended in 1 mL DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin. Cells were plated onto coverslips coated with 0.1 mg/mL poly-l-lysine and kept at 37 °C in an incubator with 5% CO2. All electrophysiological recordings were made within 4−72 h after dissociation.

Whole-cell patch clamp recording

Whole-cell HCN channel currents were recorded using an Axopatch-700B amplifier interfaced to a Digidata 1550B data acquisition system using the pClamp 10.6 software (Molecular Devices, USA). Recording pipettes were pulled from borosilicate glass capillaries using a P-97 horizontal micropipette puller (Sutter Instrument, USA) and had an average resistance of 2–5 MΩ. The data were filtered at 2 kHz and digitized at 20 kHz. The pipette electrode for the COS7 cells contained (in mM): 140 KCl, 1 MgCl2, 5 EGTA, and 10 HEPES (pH 7.4 with KOH). The extracellular solution contained (in mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES (pH 7.4 with NaOH). Patch pipettes for the DRG neurons were filled with an internal solution containing (in mM): 140 K-aspartate, 5 EGTA, 2 MgCl2, 10 HEPES, 0.5 NaGTP and 2 MgATP (pH 7.4 with KOH). All whole-cell recordings were completed at room temperature (25 ± 2 °C).

Electrophysiological data were processed using Clampfit 10.6 (Molecular Devices, USA) and then analyzed in GraphPad Prism 5 (GraphPad Software, USA). Voltage-dependent activation curves were fitted with the Boltzmann equation G = Gmin + (Gmax−Gmin)/(1 + exp(V−V1⁄2)/S), where Gmax is the maximum conductance, Gmin is the minimum conductance, V1⁄2 is the voltage for half-activation potential, and S is the slope factor. The time constants for activation (τact) or deactivation (τdeact) were obtained by fitting the HCN current tracings at − 120 mV with a monoexponential function using Clampfit 10.6. Dose–response curves were obtained by fitting to the Hill equation, E = Emin + (Emax−Emin)/(1 + 10^((LogIC50−C)n)), where E = Idrug/Icontrol, Emax, and Emin are the maximally and minimally inhibited responses, respectively, IC50 corresponds to the concentration that provokes a response halfway between Emax and Emin, C corresponds to the drug concentration, and n is the Hill coefficient. Data are expressed as the mean ± SEM. Statistical analysis was conducted using Student’s t test or one-way analysis of variance where appropriate, and results were considered significant when P < 0.05.

Application of compounds

All compounds were dissolved in dimethyl sulfoxide to prepare a stock solution with a concentration of 100 mM, from which the appropriate volumes were added to the extracellular solution to obtain the desired concentrations and applied to the cells through perfusion.

Results

Synthesis of alkanol amine derivatives

4a: Yield 84%; 1H NMR (400 MHz, CDCl3) δ 7.37 (d, J = 8.0 Hz, 1 H), 7.23–7.11 (m, 2 H), 6.84 (d, J = 1.6 Hz, 4 H), 6.52–6.48 (m, 1 H), 5.32 (s, 2 H), 4.13–3.91 (m, 5 H), 3.53–3.25 (m, 2 H), 2.48–2.31 (m, 2 H), 2.27 (s, 3 H), 2.23 (s, 3 H), 2.12 (s, 3 H), 1.93–1.78 (m, 2 H), 1.42–1.37 (m, 3 H). 13C NMR (101 MHz, CDCl3) δ 153.08, 152.88, 147.83, 134.98, 131.72, 129.21, 128.57, 127.38, 127.24, 115.42, 115.40, 115.35, 77.27, 66.62, 65.28, 64.19, 63.95, 62.81, 51.35, 50.08, 41.49, 34.61, 32.62, 14.88, 12.13, 9.65. ESI-MS (Electrospray ionization-mass spectrometry): m/z calcd for C26H34ClN3O3 (M + H+) 471.2, found 471.4.

4b: Yield 75%; 1H NMR(400 MHz, CDCl3) δ 7.38 (d, J = 8.0 Hz, 1 H), 7.25–7.13 (m, 2 H), 7.09 (d, J = 7.6 H Hz, 2 H), 6.83 (d, J = 8.4 Hz, 2 H), 6.52 (d, J = 7.6 Hz, 1 H), 5.33 (s, 2 H), 4.15–4.11 (m, 2 H), 3.99 (s, 1 H), 3.56–3.50 (m, 1 H), 3.36–3.31 (m, 1 H), 2.53–2.48 (m, 1 H), 2.42–2.37 (m, 1 H), 2.32–2.24 (m, 9 H), 2.14 (s, 3 H), 1.98–1.79 (m, 2 H). 13C NMR (101 MHz, CDCl3) δ 156.64, 147.82, 139.60, 138.59, 138.32, 137.91, 129.80, 129.80, 129.20, 128.57, 127.36, 127.23, 114.33, 77.16, 64.70, 64.27, 62.82, 51.35, 50.71, 50.04, 41.52, 34.53, 20.38, 12.06, 9.59. ESI-MS: m/z calcd for C25H32ClN3O2 (M + H+) 441.2, found 441.4.

4c: Yield 79%; 1H NMR (400 MHz, CDCl3) δ 7.38 (d, J = 7.6 Hz,1 H), 7.22–7.16 (m, 2 H), 7.10 (d, J = 8.0 Hz, 2 H), 6.85 (d, J = 8.4 Hz, 2 H), 6.53 (d, J = 7.6 Hz, 1 H), 5.33 (s, 2 H), 4.14 (t, J = 7.6 Hz, 2 H), 4.03 (s, 1 H), 3.58 (s, 1 H), 3.39 (s, 1 H), 2.72–2.54 (m, 3 H), 2.45–2.41 (m, 1 H), 2.28 (s, 6 H), 2.16 (s, 3 H), 1.97–1.83 (m, 2 H), 1.23 (t, J = 7.6 Hz, 3 H). 13C NMR (101 MHz, CDCl3) δ 156.79, 147.83, 141.44, 136.41, 134.96, 131.72, 129.21, 128.63, 128.63, 127.38, 127.23, 114.34, 114.34, 98.22, 64.67, 64.25, 62.82, 51.35, 50.76, 50.06, 41.50, 34.55, 27.90, 15.81, 12.10, 9.63. ESI-MS: m/z calcd for C26H34ClN3O2 (M + H+) 455.3, found 455.4.

4d: Yield 82%; 1H NMR (400 MHz, CDCl3) δ 7.38 (d, J = 7.6 Hz, 1 H), 7.23–7.13 (m, 2 H), 7.10 (d, J = 8.4 Hz, 2 H), 6.84 (d, J = 8.4 Hz, 2 H), 6.52 (d, J = 7.6 Hz, 1 H), 5.33 (s, 2 H), 4.16–4.11 (m, 2 H), 4.02–3.94 (m, 1 H), 3.56–3.50 (m, 1 H), 3.36–3.30 (m, 1 H), 2.54 (t, J = 7.6 Hz, 2 H), 2.49–2.45 (m, 1 H), 2.41–2.35 (m, 1 H), 2.28–2.25 (m, 5 H), 2.14 (s, 3 H), 1.97–1.77 (m, 2 H), 1.66–1.59 (m, 2 H), 1.29–1.24 (m, 1 H), 0.95 (t, J = 7.3 Hz, 3 H). 13C NMR (101 MHz, CDCl3) δ 156.84, 147.81, 138.51, 135.04, 134.79, 131.71, 129.23, 129.23, 129.23, 129.20, 128.55, 127.37, 127.23, 114.24, 114.17, 64.67, 64.25, 62.85, 51.37, 50.06, 41.52, 37.09, 34.58, 24.71, 13.72, 12.12, 9.60. ESI-MS: m/z calcd for C27H36ClN3O2 (M + H+) 469.3, found 469.3.

4e: Yield 77%; 1H NMR (400 MHz, CDCl3) δ 7.39 (d, J = 6.8 Hz, 1 H), 7.31 (d, J = 8.4 Hz, 2 H), 7.22–7.15 (m, 2 H), 6.86 (d, J = 8.8 Hz, 2 H), 6.52 (d, J = 7.6 Hz, 1 H), 5.33 (s, 2 H), 4.15 (t, J = 5.2 Hz, 2 H), 4.01 (s, 1 H), 3.58–3.52 (m, 1 H), 3.40–3.34 (m, 1 H), 2.54 (s, 1 H), 2.42 (s, 1 H), 2.28 (s, 6 H), 2.15 (s, 3 H), 1.95–1.83 (m, 2 H), 1.32 (s, 9 H). 13C NMR (101 MHz, CDCl3) δ 156.53, 147.80, 143.28, 138.46, 135.07, 131.70, 129.19, 128.54, 127.37, 127.23, 126.13, 126.13, 113.91, 113.91, 64.63, 64.26, 62.88, 51.39, 50.06, 41.54, 34.58, 33.99, 31.47, 31.47, 31.47, 31.47, 12.12, 9.59. ESI-MS: m/z calcd for C28H38ClN3O2 (M + H+) 483.2, found 483.3.

4f: Yield 82%; 1H NMR (400 MHz, CDCl3) δ 7.27–7.22 (m, 1 H), 7.11–7.01 (m, 2 H), 6.85 (s, 5 H), 5.29 (s, 2 H), 4.10 (t, J = 6.4 Hz, 2 H), 4.00 (q, J = 7.2 Hz, 3 H), 3.56–3.50 (m, 1 H), 3.36–3.30 (m, 1 H), 2.52 (s, 1 H), 2.42–2.36 (m, 1 H), 2.26 (s, 6 H), 2.18 (s, 3 H), 1.91–1.80 (m, 2 H), 1.41 (t, J = 7.0 Hz, 3 H). 13C NMR (101 MHz, CDCl3) δ 153.04, 152.93, 147.57, 138.10, 129.07, 128.49, 124.48, 124.34, 115.39, 115.33, 115.33, 115.19, 114.98, 113.36, 65.33, 64.25, 63.92, 62.93, 51.40, 46.11, 46.06, 41.53, 34.61, 14.89, 12.09, 9.51. ESI-MS: m/z calcd for C26H34FN3O3 (M + H+) 455.3, found 455.5.

4g: Yield 69%; 1H NMR (400 MHz, CDCl3) δ 7.17 (d, J = 8.4 Hz, 2 H), 7.12–7.08 (m, 1 H), 6.85 (s, 4 H), 6.45 (d, J = 7.2 Hz, 1 H), 5.21 (s, 2 H), 4.14–4.08 (m, 2 H), 4.00 (q, J = 7.2 Hz, 3 H), 3.56–3.50 (m, 1 H), 3.36–3.30 (m, 1 H), 2.55–2.47 (m, 1 H), 2.40 (s, 1 H), 2.35 (s, 3 H), 2.27 (s, 6 H), 2.12 (s, 3 H), 1.97–1.80 (m, 2 H), 1.41 (t, J = 7.2 Hz, 3 H). 13C NMR (101 MHz, CDCl3) δ 153.05, 152.97, 147.34, 138.35, 135.41, 134.61, 130.08, 127.19, 126.34, 125.67, 115.41, 115.41, 115.35, 115.35, 65.34, 64.23, 63.95, 62.87, 51.41, 50.71, 50.68, 41.50, 34.62, 19.00, 14.89, 12.09, 9.70. ESI-MS: m/z calcd for C27H37N3O3 (M + H+) 451.3, found 451.6.

4h: Yield 68%; 1H NMR (400 MHz, CDCl3) δ 7.30 (s, 1 H), 7.28 (s, 1 H), 7.00 (d, J = 8.0 Hz, 2 H), 6.85 (s, 4 H), 5.20 (s, 2 H), 4.12–4.08 (m, 2 H), 4.00 (q, J = 6.8 Hz, 3 H), 3.56–3.50 (m, 1 H), 3.36–3.30 (m, 1 H), 2.55–2.46 (m, 1 H), 2.41–2.35 (m, 1 H), 2.26 (s, 6 H), 2.14 (s, 3 H), 1.97–1.75 (m, 2 H), 1.41 (t, J = 6.8 Hz, 3 H). 13C NMR (101 MHz, CDCl3) δ 153.07, 152.90, 147.51, 135.73, 133.29, 128.84, 128.84, 127.84, 127.84, 115.39, 115.39, 115.35, 115.35, 77.15, 65.28, 64.20, 63.95, 62.89, 52.06, 51.34, 41.49, 34.60, 14.89, 12.07, 9.77. ESI-MS: m/z calcd for C26H34ClN3O3 (M + H+) 471.2, found 471.3.

Identification of a series of HCN channel blockers

We first examined the effects of the synthesized compounds on the HCN2 channel, which was heterologously expressed in COS7 cells. The HCN2 current was elicited by a hyperpolarizing potential of − 120 mV, and the effects on the amplitude of the inward current (I/I0) were analyzed. The lead compound 4a (10 μM) blocked the HCN2 current at − 120 mV to 56% (n = 14) (Table 1), which was a stronger inhibitory effect on the HCN2 channel compared with ZD7288 (n = 4; P < 0.01) (Supplementary Fig. 1).

Table 1 Effects of alkanol amine compounds on HCN2 currents in COS7 cells
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Comparing the compounds from 4b to 4e showed a substitution of the para- position on the R group with a series of extended carbon chains, which markedly enhanced the inhibitory efficacy. The inhibitory effect significantly increased in particular with the tert-butyl group in 4e, which indicated the large hindrance substitution was helpful for the activity (Table 1).

Replacement of the oCl on the R’ group with an electron-withdrawing group (4f) also strongly augmented the compound’s inhibitory effect on the HCN2 channel, whereas adding a weak electron-donating group (4g) had no significant effect on its inhibitory action. Moreover, substitution of the oCl with pCl (4h) also had no significant influence on the compound’s inhibitory action (Table 1).

Electrophysiological properties of 4e blockage on HCN2 channel

We selected the most potent inhibitor, 4e, for further characterization of its inhibitory properties on HCN channels. We first tested the inhibitory effect of 4e on the HCN2 channel under a testing voltage of − 120 mV and used different concentrations to evaluate the IC50. Fig. 2a showed the typical HCN2 current traces in the absence or presence of 10 μM 4e. Dose–response curves showed that the IC50 of 4e on the HCN2 channel was 2.9 ± 1.2 μM (Fig. 2b), which was similar to the reported IC50 of ivabradine on heterologously expressed HCN channels [30]. Then, we elicited HCN2 current at different voltages from − 50 to − 140 mV. The data showed that 10 µM 4e markedly decreased the HCN2 current in all tested voltages, (Fig. 2c, d) and the normalized I–V relationship that was determined for multiple COS7 cells (n = 5) was plotted in Fig. 2e. The voltage dependence of the current activation normalized to Gmax for each cell was plotted in Fig. 2f, demonstrating that the voltage-dependent activation was drastically shifted in a more hyperpolarized direction after the treatment with 10 µM 4e (ΔV1/2 = −30.2 ± 2.9 mV, n = 5) (Table 2). In addition, 4e slowed down the activation speed dramatically at − 120 mV, but it had no effect on the deactivation process that was measured at + 50 mV after a test pulse to − 130 mV (Table 2).

Fig. 2
figure 2

Electrophysiological properties of 4e blocking HCN2 current. a Typical HCN2 current traces in the absence or presence of 4e (10 μM). HCN2 current was elicited by a 2 s hyperpolarizing step at − 120 mV from a holding potential of − 40 mV, tail current was measured at + 50 mV and the protocol was applied every 15 s until achieving stable inhibition. b Dose–response relationship of 4e on the HCN2 channel recorded at − 120 mV at different concentrations (0.1, 0.5, 3, 5, 10, or 30 μM, as indicated). c, d Representative traces of HCN2 current in the control condition c or with the perfusion of 10 μM 4e d. HCN2 current was recorded by a series of hyperpolarizing steps ranging from − 50 mV to − 140 mV in − 10 mV decrements from a holding potential of − 40 mV. Voltage was then returned to + 50 mV to measure the tail current. € Normalized I–V relationships recorded before and after the application of 10 μM 4e on HCN2 channel. f Normalized voltage-dependent activation curves of the HCN2 channel before and after treatment with 10 μM 4e. The dotted curve represented data normalized to its own peak amplitude. Data were fitted with a Boltzmann equation to estimate the V1/2 for channel activation. Error bars indicate S.E.M

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Table 2 Effects of 10 μM 4e on voltage dependence and kinetics of activation and deactivation on HCN1, HCN2, and HCN4 channels
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Subtype selectivity of 4e on HCN channels

To investigate the selectivity of 4e toward other HCN subtypes, compound 4e was further tested on the HCN1 and HCN4 channels. Representative current tracings from COS7 cells expressing HCN1 and HCN4 before and after the application of 10 µM 4e are shown in Fig. 3a, b. In total, 10 µM 4e blocked both HCN1 and HCN4 but to a less potent extent compared with its effect on HCN2, whose I/I0 = 0.65 ± 0.01 (n = 15) for HCN1 and I/I0 =  0.56 ± 0.04 (n = 9) for HCN4 (Fig. 3c). Moreover, dose–response curves determined that the IC50 of 4e was 17.2 ± 1.3 μM for HCN1 and 7.3 ± 1.2 μM for HCN4, respectively (Fig. 3d, e). The effects of 4e on HCN1 and HCN4 under different voltages were further investigated. Similarly, to its effect of 4e on the HCN2 channel, it also reduced the HCN1 and HCN4 currents in all tested voltages (Fig. 4c, d). Moreover, 10 μM 4e induced hyperpolarizing shifts on the voltage-dependent activation curves of the HCN1 and HCN4 channels (ΔV1/2 = − 30.4 ± 3.7 mV, n = 4 for HCN1; ΔV1/2 = − 20.3 ± 3.4 mV, n = 4 for HCN4) (Fig. 4e, f). In addition, we noted that the rate constants for activation, which were obtained from each isoform at − 120 mV, were significantly increased in the presence of 10 μM 4e, whereas there were no significant differences in deactivation rate, similar to what was described for the HCN2 channel (Table 2).

Fig. 3
figure 3

Inhibitory effect of 4e on HCN1 and HCN4 channel. a, b Typical current traces in the absence or presence of 4e (10 μM) on the HCN1 a and HCN4 b channel are as indicated. To elicit the HCN1 current, the holding potential was at − 30 mV followed by a 2 s hyperpolarizing step at − 120 mV, and a depolarization step to + 50 mV was used to record the tail current, whereas the HCN4 current was recorded as previously described for HCN2 in Fig. 2a. c Summary of the inhibitory effect of 10 μM 4e on HCN channels with a testing voltage at − 120 mV. d, e Dose–response curves of 4e on the HCN1 d and HCN4 e channel recorded at − 120 mV with different concentrations (0.1, 0.5, 3, 5, 10, 30, 50, 100, and 200 μM, as indicated). Error bars indicate S.E.M

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Fig. 4
figure 4

Effect of 4e on voltage-dependent activation of HCN1 and HCN4 channel. a, b Representative traces of the HCN1 (a) and HCN4 (b) current in the absence (upper) or presence (bottom) of 10 μM 4e; the HCN1 current was evoked by a series of hyperpolarizing steps ranging from 0 mV to − 130 mV in − 10 mV decrements from a holding potential of − 30 mV, whereas the HCN4 current was recorded as previously described for HCN2 in Fig. 2c. c, d Normalized I–V relationships recorded before and after the application of 10 μM 4e on the HCN1 c and HCN4 d channel. e, f Normalized voltage-dependent activation curves of the HCN1 e and HCN4 f channel before and after exposure to 10 μM 4e. The dotted curve represents data normalized to its own peak amplitude with the treatment with 10 μM 4e. Error bars indicate S.E.M

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4e inhibited HCN channels in a non-use-dependent manner

Consistent with previous research, ZD7288 was an apparent non-use-dependent inhibitor of the HCN1 channel [31] (Supplementary Fig. 2). Then, we examined whether 4e was a use-dependent inhibitor of HCN channels with the same protocol. In all tested isoforms of the HCN channels, application of 10 µM 4e led to a reduction of HCN-mediated current when cells were held at − 30 mV, indicating that 4e could inhibit HCN channels regardless of channel conformation (Fig. 5). Applying 10 µM 4e without activation of the HCN1 or HCN4 channel initially resulted in a similar extent of inhibition compared to the condition of when HCN1 and HCN4 were activated every 15 s (Fig. 5a, c). For the HCN2 channel, 4e initially resulted in an inhibition by 39% ± 5% (n = 4) of the HCN2 current, and the inhibition gradually reached the stable state after the channel activation was resumed, resulting in a final inhibition of 69% ± 2% (n = 4) (Fig. 5b). The results indicated that 4e was a non-use-dependent blocker of HCN channels.

Fig. 5
figure 5

The blockade of HCN channels by 4e is non-use-dependent. a–c Upper: time course of the inhibition of HCN channels by 10 μM 4e using the protocol shown in Fig. 3a (HCN1: n = 7, HCN2: n = 8, HCN4: n = 5). Below: Time course of the inhibition of HCN channels by 10 μM 4e using a standard activation/deactivation protocol and HCN current was measured as shown before. At the beginning of the perfusion with 10 μM 4e, the protocol was interrupted for several minutes (HCN1 was interrupted for 5 min, HCN2 for 8 min and HCN4 for 4 min), whereas the cell was held at the membrane voltage at − 30 mV, then channel activation was resumed to test the use-dependence of 4e (HCN1: n = 4, HCN2: n = 4, HCN4: n = 4)

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Influence of 4e on I h and action potential firing in small DRG neurons

As reported previously, HCN2 is the predominant HCN subunit that is expressed in small DRG neurons [32]. Cs+ (5 mM) and ZD7288 (10 μM) were used as positive controls to confirm native Ih in the small DRG neurons in our study. The results show that I/I0 is 0.04 ± 0.02 (n = 4) for Cs+ and 0.51 ± 0.04 (n = 7) for ZD7288, respectively (Fig. 6a, b and d). The inhibitory properties of Cs+ and ZD7288 were consistent with earlier studies [33,34,35]. Next, we examined whether 10 μM 4e could block native Ih in small DRG neurons. The results show that its I/I0 is 0.28 ± 0.04 (n = 13) (Fig. 6c, d). Furthermore, we examined the influence of 4e on the firing properties of small DRG neurons. As shown in Fig. 6e–g, the application of 10 μM 4e caused remarkably decreased action potential firing, which was induced by a 30 pA current injection.

Fig. 6
figure 6

The effect of 4e in small DRG neurons. a–c Representative current traces of Ih measured with response to 5 mM CsCl a, 10 μM ZD7288 b and 10 μM 4e c. The Ih current was elicited by a 2 s hyperpolarizing step at − 120 mV from a holding potential of − 60 mV. d Summary of inhibitory effect of 5 mM CsCl, 10 μM ZD7288, and 10 μM 4e in small DRG neurons with a testing voltage of − 120 mV. e–f Induced action potential firing of small DRG neurons was recorded under conditions without e, with f and wash g of 10 μM 4e. A 1 s depolarization 30 pA current was injected into small DRG neurons to elicit the action potential through the recording electrode

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Discussion

HCN channels play an important role in the heart and nervous system, in particular; HCN4 is expressed mostly in the heart and especially in the sinoatrial node, whereas the HCN1 and HCN2 channels are the predominant isoforms in neurons, and both contribute to spontaneous rhythm and firing frequency. Interestingly, it has been recently established that HCN channels are involved in autism spectrum disorder by interacting with Shank3 [36]. Therefore, HCN channels are an ideal new pharmacological targets for the treatment of disorders linked to abnormal excitability, such as inflammatory and neuropathic pain, epilepsy, arrhythmias, and tachycardia.

In the present study, we designed and synthesized novel alkanol amine compounds based on the compound 4a and evaluated their ability to inhibit HCN channels (HCN1, 2, and 4). The most potent inhibitor in our study, 4e, which was chemically distinct from previously reported HCN channel inhibitors, not only blocked HCN channel conductance but also dramatically left-shifted their voltage-dependent activation, which indicates that it is a negative gating modifier other than a pure pore blocker. Compound 4e blocked the HCN2 channel with an IC50 of 2.9 ± 1.2 µM at − 120 mV and showed an inhibitory preference toward HCN2 compared with HCN1 or HCN4.

Previous studies have shown that ivabradine acts as a use-dependent blocker of HCN4 whose suppression effect only worked when the channel was in the “open state”, and it acts as a non-open-dependent blocker of the HCN1 channel whose inhibition could only occur when channel was closed [26]. On the other hand, ZD7288 is an obvious non-use-dependent blocker of the HCN1 channel [31]. In our study, we found that 4e blocked HCN channels (HCN1, 2, and 4) both in the “closed state” and in the “open state” of channels, suggesting that 4e was a non-use-dependent blocker of HCN channels.

DRG neurons are the first level of sensory afferent neurons, which are located in the peripheral nervous system, playing an indispensable role in the transmission of pain signals that transmit the information after painful injuries to the higher central nervous system. Therefore, new therapeutic strategies that target DRG neurons in the peripheral nervous system to alleviate chronic pain have the advantage of minimizing side effects. Several ion channels (TRPA1, Nav1.7, and HCN channels) that are expressed in DRG neurons have been reported to play important roles in pain perception and transmission pathways [37]. Mouse DRG neurons were classified based on diameter of somata as follows: small ( < 20 μm), medium (20–30 μm), and large ( > 30 μm). HCN1 and HCN2 are predominantly expressed in DRG neurons and play a critical role in modulating firing frequency [38]. In particular, large DRG neurons mainly express the HCN1 channel, which has faster activation kinetics and is less cAMP sensitive, and HCN1−/− mice exhibited substantially less cold allodynia than wild-type littermates in a neuropathic pain model, suggesting an important role for HCN1 in neuropathic pain [38]. In the majority of small neurons, Ih has slower kinetics and is sensitive to intracellular cAMP, suggesting that it is mediated mainly by the HCN2 channel. Studies have shown that after knocking out the HCN2 channel in DRG neurons, thermal stimulation no longer caused hyperalgesia in inflammatory pain. Mechanical hyperalgesia, thermal hyperalgesia, and cold hyperalgesia were also significantly reduced in a neuropathic pain model. This study indicates that HCN2 plays an important role in both inflammatory pain and neuropathic pain in DRG neurons [39]. Furthermore, it was reported that ivabradine could suppress both the inflammatory and neuropathic pain by peripheral blockage of HCN channels [35]. Therefore, we tested the effect of 4e on small DRG neurons to estimate the possibility of 4e for the treatment of pain. The results showed that 4e significantly reduced Ih in small DRG neurons and inhibited its action potential firing property.

Future animal behavior experiments are needed to investigate the possibility of the compound series for pain treatment identified in our study. In addition, 4e provides a structural backbone for further modification, which would be valuable to identify more efficient and subtype-selective HCN channel inhibitors.