Comprehensive classification of HCN1 variants linked to neurodevelopmental disorders with and without epilepsy

Hyperpolarization-activated cyclic nucleotide-gated 1 (HCN1) channels mediate the cationic I_h current, a fundamental regulator of neuronal excitability and synaptic integration, and are predominantly expressed in the cortex and hippocampus. Although HCN1 variants have been increasingly identified in patients with epilepsy, from milder forms to developmental and epileptic encephalopathies (DEE), genotype-phenotype correlations remain poorly defined. Here, we used patch-clamp electrophysiology and confocal imaging in HEK293 cells to functionally characterize 43 HCN1 variants identified in patients with neurodevelopmental disorders, with or without epilepsy. Based on biophysical properties, we established a classification system comprising four distinct functional classes: I) low or no current; II) hyperpolarizing shift in voltage dependence; III) depolarizing shift in voltage dependence; and IV) generation of an instantaneous current. The molecular basis for this classification was further interpreted using available cryo-EM structures of HCN1. Integration with clinical data from 50 patients revealed a striking correlation between functional class and clinical presentation. Loss-of-function variants prevailed among individuals with milder phenotypes or without epilepsy, with the latter distinctively clustering within the channel selectivity filter. In contrast, gain-of-function and mixed variants were predominantly associated with DEE. Finally, we demonstrate that allosteric modulators, including peptides NB6 and TRIP8b_nano and the small molecule J&J12e, effectively normalize channel function across three of the four classes in vitro. These findings establish a biophysical framework for interpreting HCN1 variants, demonstrating that the direction of channel dysfunction is a major determinant of epilepsy risk and severity, and providing a conceptual foundation for future class-based precision medicine approaches.