Differential effects of new allosteric modulators of HCN channel function on intrinsic excitability in CA1 pyramidal neurons

This is the authors' abstract. We don't add key points for this paper.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are key regulators of neuronal excitability, with the HCN1 isoform deemed an especially promising therapeutic target for neurological and neuropsychiatric disorders. However, currently available HCN modulators are limited by poor brain penetration, insufficient isoform selectivity and off-target effects. Here, we performed a comparative characterization of recently developed HCN-targeting small molecule drugs and biologic tools in native hippocampal CA1 pyramidal neurons ex vivo and heterologously expressed HCN1 channels in vitro , to determine how distinct mechanisms of HCN modulation influence neuronal excitability. Two new high-potency drug inhibitors, Org-2D and J&J12e, reduced the HCN-mediated current, I h , in neurons at nanomolar concentrations, causing changes in resting membrane potential, input resistance, rheobase and firing while preserving action potential waveform. Despite these similar effects, the two compounds differentially altered voltage sag and rebound depolarization, consistent with distinct allosteric mechanisms revealed by testing in vitro . In contrast, the HCN1-selective RO-275 compound showed limited efficacy in neurons, an effect potentially explained by its strong preference for binding to the closed state of HCN1 channels. Finally, the nanobody activator NB6 induced opposite effects on HCN-dependent membrane properties, showing that extracellular targeting can effectively enhance channel activity ex vivo . These findings show that the mechanism of HCN modulation critically determines the physiological effects of HCN-targeting compounds while highlighting both the promise and current limitations of small-molecule and biologic approaches for selectively targeting HCN channels in the brain.