Nanobody-mediated modulation of HCN1 channels in epileptic disorders

Hyperpolarization-activated cyclic-nucleotide gated 1 (HCN1) channels contribute to the cationic Ih current in neurons and are involved in the control of neuronal excitability. Several mutations in the HCN1 gene have been correlated with the insurgence of Early Infantile Epileptic Encephalopathy (EIEE), a severe form of epilepsy characterized by recurrent seizures with impaired cognitive and motor development with onset in the first year of life, that doesn’t have a targeted treatment yet. In search of modulators that can reduce, and possibly abolish, HCN1-related phenotypes, nanobodies (NBs) represents a future therapeutic tool with great potential. By screening a synthetic library, we have isolated several nanobodies that specifically target HCN channel subtypes. We show here that nanobody #5 is able to revert the phenotype, 8 mV right shift in voltage-dependency, caused by the pathogenic R548H mutation of the hHCN1 channel. Notably, the nanobody acts from the extracellular side of the channel presumably binding to the voltage sensor, while the mutation, previously characterized, affects cAMP binding to the intracellular Cyclic Nucleotide Binding Domain (CNBD) of the channel. This result opens valuable therapeutical perspectives for EIEE patients and further highlights that in HCN channels voltage sensing at the membrane and ligand binding at the cytosolic domain are mutually interconnected.