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<sub>f</sub>, 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.
Authors
- Alessandro Porro (ORCID: https://orcid.org/0000-0003-4845-6165)
- Andrea Saponaro (ORCID: https://orcid.org/0000-0001-5035-5174)
- Roberta Castelli (ORCID: https://orcid.org/0009-0000-6773-7486)
- Gerhard Thiel (ORCID: https://orcid.org/0000-0002-2335-1351)
- Luca Palloni (ORCID: https://orcid.org/0000-0002-9004-4415)
- Matteo E. Mangoni (ORCID: https://orcid.org/0000-0002-8892-3373)
- Ana M. Gómez (ORCID: https://orcid.org/0000-0003-0009-2884)
- Dario DiFrancesco (ORCID: https://orcid.org/0000-0002-7322-1790)
- Albano C. Méli (ORCID: https://orcid.org/0000-0002-1727-3027)
- Atiyeh Sadat Sharifzadeh
- Anna Moroni (ORCID: https://orcid.org/0000-0002-1860-406X)
- Romain Perrier (ORCID: https://orcid.org/0000-0003-2229-4700)
- Pietro Mesirca (ORCID: https://orcid.org/0000-0002-9538-3096)
- Nadia Mekrane
- Hugo Benoit
Institutions
- Centre National de la Recherche Scientifique (FR)
- Inserm (FR)
- University of Milan (IT)
- Université de Montpellier (FR)
- Université Paris-Saclay (FR)
- Physiologie et Médecine Expérimentale du Coeur et des Muscles (FR)
- Laboratory Signaling and Cardiovascular Pathophysiology (FR)
- Institut de Génomique Fonctionnelle (FR)
- Bocconi University (IT)
Publication Details
- Journal
- Nature Communications
- Published
- 2025-12-01
- DOI
- https://doi.org/10.1038/s41467-025-65852-3
- Primary Topic
- Monoclonal and Polyclonal Antibodies Research
- Type
- article
- Field-Weighted Citation Impact
- 0.00
Funders
- European Commission
- Agence Nationale de la Recherche
- Fondation Leducq
- Fondazione Telethon