Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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


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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

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.


  1. DiFrancesco, D. Pacemaker mechanisms in cardiac tissue. Annu. Rev. Physiol. 55, 455–472 (1993).

    CAS  Article  Google Scholar 

  2. Ludwig, A., Zong, X., Jeglitsch, M., Hofmann, F. & Biel, M. A family of hyperpolarization-activated mammalian cation channels. Nature 393, 587–591 (1998).

    CAS  Article  Google Scholar 

  3. Santoro, B. et al. Identification of a gene encoding a hyperpolarization-activated pacemaker channel of brain. Cell 93, 717–729 (1998).

    CAS  Article  Google Scholar 

  4. Zagotta, W.N. et al. Structural basis for modulation and agonist specificity of HCN pacemaker channels. Nature 425, 200–205 (2003).

    CAS  Article  Google Scholar 

  5. Craven, K.B., Olivier, N.B. & Zagotta, W.N. C-terminal movement during gating in cyclic nucleotide-modulated channels. J. Biol. Chem. 283, 14728–14738 (2008).

    CAS  Article  Google Scholar 

  6. Clayton, G.M., Silverman, W.R., Heginbotham, L. & Morais-Cabral, J.H. Structural basis of ligand activation in a cyclic nucleotide regulated potassium channel. Cell 119, 615–627 (2004).

    CAS  Article  Google Scholar 

  7. Schunke, S., Stoldt, M., Novak, K., Kaupp, U.B. & Willbold, D. Solution structure of the Mesorhizobium loti K1 channel cyclic nucleotide-binding domain in complex with cAMP. EMBO Rep. 10, 729–735 (2009).

    Article  Google Scholar 

  8. Romling, U., Galperin, M.Y. & Gomelsky, M. Cyclic di-GMP: the first 25 years of a universal bacterial second messenger. Microbiol. Mol. Biol. Rev. 77, 1–52 (2013).

    Article  Google Scholar 

  9. Chen, Z.H. & Schaap, P. The prokaryote messenger c-di-GMP triggers stalk cell differentiation in Dictyostelium. Nature 488, 680–683 (2012).

    CAS  Article  Google Scholar 

  10. Wu, J. et al. Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA. Science 339, 826–830 (2013).

    CAS  Google Scholar 

  11. Burdette, D.L. et al. STING is a direct innate immune sensor of cyclic di-GMP. Nature 478, 515–518 (2011).

    CAS  Article  Google Scholar 

  12. DiFrancesco, D. & Tortora, P. Direct activation of cardiac pacemaker channels by intracellular cyclic AMP. Nature 351, 145–147 (1991).

    CAS  Article  Google Scholar 

  13. Chandler, N.J. et al. Molecular architecture of the human sinus node: insights into the function of the cardiac pacemaker. Circulation 119, 1562–1575 (2009).

    Article  Google Scholar 

  14. Lolicato, M. et al. Tetramerization dynamics of C-terminal domain underlies isoform-specific cAMP gating in hyperpolarization-activated cyclic nucleotide-gated channels. J. Biol. Chem. 286, 44811–44820 (2011).

    CAS  Article  Google Scholar 

  15. Davies, B.W., Bogard, R.W., Young, T.S. & Mekalanos, J.J. Coordinated regulation of accessory genetic elements produces cyclic di-nucleotides for V. cholerae virulence. Cell 149, 358–370 (2012).

    CAS  Article  Google Scholar 

  16. Gao, P. et al. Cyclic [G(2′,5′)pA(3′,5′)p] is the metazoan second messenger produced by DNA-activated cyclic GMP-AMP synthase. Cell 153, 1094–1107 (2013).

    CAS  Article  Google Scholar 

  17. Diner, E.J. et al. The innate immune DNA sensor cGAS produces a noncanonical cyclic dinucleotide that activates human STING. Cell Rep. 3, 1355–1361 (2013).

    CAS  Article  Google Scholar 

  18. Ablasser, A. et al. cGAS produces a 2′-5′-linked cyclic dinucleotide second messenger that activates STING. Nature 498, 380–384 (2013).

    CAS  Article  Google Scholar 

  19. Zhang, X. et al. Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING. Mol. Cell 51, 226–235 (2013).

    CAS  Article  Google Scholar 

  20. Yin, Q. et al. Cyclic di-GMP sensing via the innate immune signaling protein STING. Mol. Cell 46, 735–745 (2012).

    CAS  Article  Google Scholar 

  21. DiFrancesco, D., Ducouret, P. & Robinson, R.B. Muscarinic modulation of cardiac rate at low acetylcholine concentrations. Science 243, 669–671 (1989).

    CAS  Article  Google Scholar 

  22. Santoro, B. et al. TRIP8b splice variants form a family of auxiliary subunits that regulate gating and trafficking of HCN channels in the brain. Neuron 62, 802–813 (2009).

    CAS  Article  Google Scholar 

  23. Zolles, G. et al. Association with the auxiliary subunit PEX5R/Trip8b controls responsiveness of HCN channels to cAMP and adrenergic stimulation. Neuron 62, 814–825 (2009).

    CAS  Article  Google Scholar 

  24. DiFrancesco, J.C. et al. Recessive loss-of-function mutation in the pacemaker HCN2 channel causing increased neuronal excitability in a patient with idiopathic generalized epilepsy. J. Neurosci. 31, 17327–17337 (2011).

    CAS  Article  Google Scholar 

  25. Bucchi, A., Tognati, A., Milanesi, R., Baruscotti, M. & DiFrancesco, D. Properties of ivabradine-induced block of HCN1 and HCN4 pacemaker channels. J. Physiol. (Lond.) 572, 335–346 (2006).

    CAS  Article  Google Scholar 

  26. Li, X.D. et al. Pivotal roles of cGAS-cGAMP signaling in antiviral defense and immune adjuvant effects. Science 341, 1390–1394 (2013).

    CAS  Article  Google Scholar 

  27. Sun, L., Wu, J., Du, F., Chen, X. & Chen, Z.J. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science 339, 786–791 (2013).

    CAS  Article  Google Scholar 

  28. Uhlen, M. et al. Towards a knowledge-based Human Protein Atlas. Nat. Biotechnol. 28, 1248–1250 (2010).

    CAS  Article  Google Scholar 

  29. Zorn-Pauly, K. et al. Endotoxin impairs the human pacemaker current If . Shock 28, 655–661 (2007).

    CAS  PubMed  Google Scholar 

  30. Werdan, K. et al. Impaired regulation of cardiac function in sepsis, SIRS, and MODS. Can. J. Physiol. Pharmacol. 87, 266–274 (2009).

    CAS  Article  Google Scholar 

  31. Klöckner, U. et al. Differential reduction of HCN channel activity by various types of lipopolysaccharide. J. Mol. Cell. Cardiol. 51, 226–235 (2011).

    Article  Google Scholar 

  32. Ablasser, A. et al. Cell intrinsic immunity spreads to bystander cells via the intercellular transfer of cGAMP. Nature 503, 530–534 (2013).

    CAS  Article  Google Scholar 

  33. Leslie, A.G.M. MOSFLM User Guide, Mosflm Version 6.2.3 (MRC laboratory of Molecular Biology, Cambridge, UK, 2003).

  34. Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D Biol. Crystallogr. 62, 72–82 (2006).

    Article  Google Scholar 

  35. Vagin, A. & Teplyakov, A. MOLREP: an automated program for molecular replacement. J. Appl. Crystallogr. 30, 1022–1025 (1997).

    CAS  Article  Google Scholar 

  36. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  37. Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).

    CAS  Article  Google Scholar 

  38. Laskowski, R.A., Macarthur, M.W., Moss, D.S. & Thornton, J.M. Procheck—a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291 (1993).

    CAS  Article  Google Scholar 

  39. Krissinel, E. & Henrik, K. in Computational Life Sciences, Vol. 3695 (eds. Berthold, M.R., Glen, R.C., Diederichs, K., Kohlbacher, O. & Fischer, I.) 163–174 (2005).

  40. Accili, E.A. & DiFrancesco, D. Inhibition of the hyperpolarization-activated current (If) of rabbit SA node myocytes by niflumic acid. Pflugers Arch. 431, 757–762 (1996).

    CAS  Article  Google Scholar 

  41. DiFrancesco, D. & Mangoni, M. Modulation of single hyperpolarization-activated channels (If) by cAMP in the rabbit sino-atrial node. J. Physiol. (Lond.) 474, 473–482 (1994).

    CAS  Article  Google Scholar 

  42. Baruscotti, M. et al. Deep bradycardia and heart block caused by inducible cardiac-specific knockout of the pacemaker channel gene Hcn4. Proc. Natl. Acad. Sci. USA 108, 1705–1710 (2011).

    CAS  Article  Google Scholar 

  43. Bucchi, A., Baruscotti, M., Robinson, R.B. & DiFrancesco, D.I. If-dependent modulation of pacemaker rate mediated by cAMP in the presence of ryanodine in rabbit sino-atrial node cells. J. Mol. Cell. Cardiol. 35, 905–913 (2003).

    CAS  Article  Google Scholar 

  44. Kranzusch, P.J., Lee, A.S., Berger, J.M. & Doudna, J. A. Structure of human cGAS reveals a conserved family of second-messenger enzymes in innate immunity. Cell Rep. 3, 1362–1368 (2013).

    CAS  Article  Google Scholar 

  45. Zsoldos, Z., Szabo, I., Szabo, Z. & Johnson, A.P. Software tools for structure based rational drug design. J. Mol. Struct. THEOCHEM 666, 659–665 (2003).

    Article  Google Scholar 

  46. Zsoldos, Z., Reid, D., Simon, A., Sadjad, B.S. & Johnson, A.P. eHiTS: an innovative approach to the docking and scoring function problems. Curr. Protein Pept. Sci. 7, 421–435 (2006).

    CAS  Article  Google Scholar 

  47. Zsoldos, Z., Reid, D., Simon, A., Sadjad, S.B. & Johnson, A.P. eHiTS: a new fast, exhaustive flexible ligand docking system. J. Mol. Graph. Model. 26, 198–212 (2007).

    CAS  Article  Google Scholar 

Download references


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.

Author information

Authors and Affiliations



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.

Corresponding author

Correspondence to Anna Moroni.

Ethics declarations

Competing interests

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.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–8 and Supplementary Tables 1–3. (PDF 1265 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing