The hyperpolarization‐activated cyclic nucleotide‐gated 4 channel as a potential anti‐seizure drug target

Abstract

Background and Purpose Hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels are encoded by four genes (HCN1–4) with distinct biophysical properties and functions within the brain. HCN4 channels activate slowly at robust hyperpolarizing potentials, making them more likely to be engaged during hyperexcitable neuronal network activity seen during seizures. HCN4 channels are also highly expressed in thalamic nuclei, a brain region implicated in seizure generalization. Here, we assessed the utility of targeting the HCN4 channel as an anti‐seizure strategy using pharmacological and genetic approaches. Experimental Approach The impact of reducing HCN4 channel function on seizure susceptibility and neuronal network excitability was studied using an HCN4 channel preferring blocker (EC18) and a conditional brain specific HCN4 knockout mouse model. Key Results EC18 (10 mg·kg−1) and brain‐specific HCN4 channel knockout reduced seizure susceptibility and proconvulsant‐mediated cortical spiking recorded using electrocorticography, with minimal effects on other mouse behaviours. EC18 (10 μM) decreased neuronal network bursting in mouse cortical cultures. Importantly, EC18 was not protective against proconvulsant‐mediated seizures in the conditional HCN4 channel knockout mouse and did not reduce bursting behaviour in AAV‐HCN4 shRNA infected mouse cortical cultures. Conclusions and Implications These data suggest the HCN4 channel as a potential pharmacologically relevant target for anti‐seizure drugs that is likely to have a low side‐effect liability in the CNS.

Expression of the pacemaker channel HCN4 in excitatory interneurons in the dorsal horn of the murine spinal cord

“…Two weeks after starting DOX administration, we could not observe obvious alteration in general behavior of HCN4-knockdown mice. This finding was in good agreement with the previous report on brain-specific HCN4 conditional knockout mice [ 31 ]. We then examined the nocifensive behavior in HCN4-knockdown mice using von Frey assay.…”

Testing broad-spectrum and isoform-preferring HCN channel blockers for anticonvulsant properties in mice

“…EC18 is a compound that is preferentially selective for HCN4 channels, with reduced selectivity for HCN1 channels and HCN2 channels (Del Lungo et al, 2012;Postea and Biel, 2011). EC18 at 5 mg/kg had a protective effect against s.c.PTZ-induced seizures in J o u r n a l P r e -p r o o f male wildtype mice, producing a right shift in latency to hindlimb extension seizures consistent with previously published data at 10 mg/kg (Kharouf et al, 2020) (Fig. 3A, Table S4).…”

“…Additionally, we demonstrate that ivabradine is effective at reducing thermogenic seizure susceptibility in wildtype mice and in the Scn1a R1407X mouse model of Dravet syndrome (Cao et al, 2012). The anticonvulsant properties of EC18, a HCN4 isoformpreferring drug, highlights the HCN4 channel as a potential anticonvulsant target (Kharouf et al, 2020). In contrast, the HCN2/1 isoform-preferring drug, MEL55A, caused an increase in seizure susceptibility.…”

“…EC18, for example, is known to also block K + channels (Romanelli et al, 2019). This said, we have confidence that EC18 is acting through HCN4 channels given that its antiseizure activity is blunted in a conditional HCN4 channel knockout mouse (Kharouf et al, 2020). However, we cannot rule out the fact that the impact of MEL55A on seizure susceptibility may be due to off-target effects.…”

“…Firstly, whilst pharmacokinetic data in mice is available for EC18 (Kharouf et al, 2020), pharmacokinetic studies have not yet been carried out for MEL55A and MEL57A. Whilst we know that EC18 is brain penetrant, it is possible that the lack of anticonvulsant efficacy of MEL57A is due to poor brain penetration given its physico-chemical properties (MW: 737.9 Da; hydrophilicity (ClogP): 5.92) are not ideal for CNS bioavailability.…”