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Cyclic dinucleotides bind the C-linker of HCN4 to control channel cAMP responsiveness

An Erratum to this article was published on 18 July 2014

This article has been updated

Abstract

cAMP mediates autonomic regulation of heart rate by means of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which underlie the pacemaker current If. cAMP binding to the C-terminal cyclic nucleotide binding domain enhances HCN open probability through a conformational change that reaches the pore via the C-linker. Using structural and functional analysis, we identified a binding pocket in the C-linker of HCN4. Cyclic dinucleotides, an emerging class of second messengers in mammals, bind the C-linker pocket (CLP) and antagonize cAMP regulation of the channel. Accordingly, cyclic dinucleotides prevent cAMP regulation of If in sinoatrial node myocytes, reducing heart rate by 30%. Occupancy of the CLP hence constitutes an efficient mechanism to hinder β-adrenergic stimulation on If. Our results highlight the regulative role of the C-linker and identify a potential drug target in HCN4. Furthermore, these data extend the signaling scope of cyclic dinucleotides in mammals beyond their first reported role in innate immune system.

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Figure 1: Crystal structure of the soluble portion of the CL-CNBD of HCN4 in complex with cGMP.
Figure 2: Chemical structures of cyclic dinucleotides.
Figure 3: Effect of c-di-GMP on voltage dependence of activation of wild-type and mutant HCN4 channels.
Figure 4: Effects of c-di-GMP and 2′3′-cGAMP on voltage dependence of If activation and spontaneous rate in SAN.
Figure 5: Effect of BPU on HCN4, If channels and spontaneous rate in SAN.

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Change history

  • 16 June 2014

    In the version of this article initially published, a data point and one of its two error bars were missing from Figure 3e in the row depicting mutant F564T. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank Xention Ltd. (Cambridge, UK) for the generous gift of HCNs cDNA, J. Berger and J.A. Doudna for hSTING cDNA and C. Deutscher (BioLog, Bremen) for technical help. This work was supported by SAL-49 Progetto di Cooperazione Scientifica e Tecnologica Regione Lombardia, Programmi di Ricerca di Rilevante Interesse Nazionale 2010CSJX4F and Ministero Affari Esteri 01467532013-06-27 to A.M.; by European Drug Initiative on Channels and Transporters to K.S., A.P.J., C.W.G.F. and A.M; and by Bundesministerium für Bildung und Forschung (GREVIS) project to G.T.

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Contributions

M.L. purified, crystallized, collected, processed and refined X-ray data. M.N. processed and refined X-ray data, prepared some of the figures and, with M.B., revised the manuscript. C.A. prepared the mutants and, with M.A., performed the whole-cell experiments. S.Z. and I.S. performed the inside-out experiments. A.B. made measurements and, with D.D., designed and analyzed the experiments on mouse SAN cells. K.S. performed the docking studies with guidance from A.P.J. and C.W.G.F. F.S. and D.K. synthesized cyclic dinucleotides and contributed information on their signaling properties. G.T. and A.M. designed the study, analyzed data and wrote the paper.

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Correspondence to Anna Moroni.

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F.S. and D.K. are Head of Research and Development and staff member, respectively, at the BIOLOG Life Science Institute, which sells cyclic nucleotide analogs for research purposes.

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Lolicato, M., Bucchi, A., Arrigoni, C. et al. Cyclic dinucleotides bind the C-linker of HCN4 to control channel cAMP responsiveness. Nat Chem Biol 10, 457–462 (2014). https://doi.org/10.1038/nchembio.1521

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