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 I_f, 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.