NOVEL THERAPEUTIC APPROACHES EMERGING FROM HCN CHANNELS STRUCTURAL AND FUNCTIONAL STUDIES

Hyperpolarization-activated cyclic-nucleotide gated 1 (HCN1) channels are the molecular determinants of the cationic Ih current that regulates spontaneous electrical activity and synaptic integration in neurons. Mutations in the HCN1 gene have been linked to Early Infantile Epileptic Encephalopathy (EIEE) and a spectrum of neurodevelopmental disorders (ND). Despite the growing number of identified HCN1 variants, the mechanisms by which they alter channel function and contribute to pathogenesis remain poorly understood. Here, we combine molecular, cellular, and system-level neuroscience approaches to functionally characterize HCN1 mutations and explore targeted strategies for therapeutic rescue. Using in vitro electrophysiology in HEK293 cells, we analyze a panel of patient-derived variants, and we propose a systematic classification of HCN1 mutations into four distinct classes (I-IV) according to their functional properties. This framework reveals correlations between mutation location, channel dysfunction, and clinical phenotype. Interestingly, we identify a novel correlation between loss-of-function mutations in the pore-helix / selectivity filter domain of HCN1 that are associated with ND and no history of seizures. We further explore the use of pore blockers and allosteric modulators to restore wild-type-like function in mutant HCN1 channels in vitro, highlighting the potential of precision medicine approaches tailored to mutation-specific phenotypes. Extending these studies ex vivo, we demonstrate that the allosteric modulator J&J12e effectively rescues altered intrinsic properties and excitability in hippocampal CA1 pyramidal neurons from a knock-in mouse model carrying the HCN1 M153I mutation. Additionally, we show that NB6 can modulate native Ih currents in native brain tissue, underscoring its promise as a peptide-based therapeutic tool. At the system level, in vivo electrophysiological recordings reveal increased interictal spike activity in knock-in mice, which is further amplified following lamotrigine administration. Sleep architecture is also disrupted in these animals, suggesting that HCN1 dysfunction impacts broader aspects of brain function beyond seizure susceptibility. Together, these findings provide a comprehensive understanding of the functional impact of HCN1 dysfunction and lay the basis for the development of targeted, mutation-specific therapies aimed at restoring normal channel function and improving clinical outcomes in patients with HCN1-related disorders.