Extracellular activation of HCN4 by a subtype-specific nanobody

Hyperpolarization-activated cyclic nucleotide-gated channels (HCN1-4) control cardiac and neuronal firing and their dysfunction leads to cardiac arrythmias (HCN4), epilepsy (HCN1) and chronic pain (HCN2). Prompted by the urgent need for HCN subtype-specific treatments, we screened a recombinant nanobody library in search of HCN4-specific binders. Here we show that nanobody 5 (NB5) binds to the extracellular side of HCN4 with high specificity and nanomolar affinity and activates the channel by a non-canonical electromechanical coupling path. In ex vivo and in vitro experiments, NB5 acts as an agonist of the pacemaker current If, increasing the firing rate of rabbit cardiac pacemaker myocytes and of human derived cardiomyocytes. Notably in vitro, NB5 rescued the loss-of-function effects on HCN4 current caused by a mutation found in a patient with sinus node dysfunction. Our work illustrates that animal-free recombinant nanobodies have strong potential as next generation modulators for clinical application in symptomatic bradycardia.