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.

Structural basis for modulation and agonist specificity of HCN pacemaker channels


The family of hyperpolarization-activated, cyclic nucleotide-modulated (HCN) channels are crucial for a range of electrical signalling, including cardiac and neuronal pacemaker activity, setting resting membrane electrical properties and dendritic integration1. These nonselective cation channels, underlying the If, Ih and Iq currents of heart and nerve cells, are activated by membrane hyperpolarization and modulated by the binding of cyclic nucleotides such as cAMP and cGMP2. The cAMP-mediated enhancement of channel activity is largely responsible for the increase in heart rate caused by β-adrenergic agonists3. Here we have investigated the mechanism underlying this modulation by studying a carboxy-terminal fragment of HCN2 containing the cyclic nucleotide-binding domain (CNBD) and the C-linker region that connects the CNBD to the pore. X-ray crystallographic structures of this C-terminal fragment bound to cAMP or cGMP, together with equilibrium sedimentation analysis, identify a tetramerization domain and the mechanism for cyclic nucleotide specificity, and suggest a model for ligand-dependent channel modulation. On the basis of amino acid sequence similarity to HCN channels, the cyclic nucleotide-gated, and eag- and KAT1-related families of channels are probably related to HCN channels in structure and mechanism.

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: HCN2 channel topology and alignment with related channels.
Figure 2: Structure of the mouse HCN2 C-linker and CNBD construct bound to cAMP.
Figure 3: cAMP and cGMP bind to the same site with different stereochemistry.
Figure 4: C-linker interactions and cyclic nucleotide-dependent tetramer formation.


  1. Robinson, R. B. & Siegelbaum, S. A. Hyperpolarization-activated cation currents: from molecules to physiological function. Annu. Rev. Physiol. 65, 453–480 (2003)

    CAS  Article  Google Scholar 

  2. Biel, M., Schneider, A. & Wahl, C. Cardiac HCN. channels: structure, function, and modulation. Trends Cardiovasc. Med. 12, 206–212 (2002)

    CAS  Article  Google Scholar 

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

    ADS  CAS  Article  Google Scholar 

  4. Zagotta, W. N. & Siegelbaum, S. A. Structure and function of cyclic nucleotide-gated channels. Annu. Rev. Neurosci. 19, 235–263 (1996)

    CAS  Article  Google Scholar 

  5. Bruggemann, A., Pardo, L. A., Stuhmer, W. & Pongs, O. Ether-a-go-go encodes a voltage-gated channel permeable to K+ and Ca2+ and modulated by cAMP. Nature 365, 445–448 (1993)

    ADS  CAS  Article  Google Scholar 

  6. Hoshi, T. Regulation of voltage dependence of the KAT1 channel by intracellular factors. J. Gen. Physiol. 105, 309–328 (1995)

    CAS  Article  Google Scholar 

  7. Doyle, D. A. et al. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science 280, 69–77 (1998)

    ADS  CAS  Article  Google Scholar 

  8. Wainger, B. J., DeGennaro, M., Santoro, B., Siegelbaum, S. A. & Tibbs, G. R. Molecular mechanism of cAMP modulation of HCN. pacemaker channels. Nature 411, 805–810 (2001)

    ADS  CAS  Article  Google Scholar 

  9. Weber, I. T. & Steitz, T. A. Structure of a complex of catabolite gene activator protein and cyclic AMP refined at 2.5 Å resolution. J. Mol. Biol. 198, 311–326 (1987)

    CAS  Article  Google Scholar 

  10. Su, Y. et al. Regulatory subunit of protein kinase A: structure of deletion mutant with cAMP binding domains. Science 269, 807–813 (1995)

    ADS  CAS  Article  Google Scholar 

  11. Chen, S., Wang, J. & Siegelbaum, S. A. Properties of hyperpolarization-activated pacemaker current defined by coassembly of HCN1 and HCN2 subunits and basal modulation by cyclic nucleotide. J. Gen. Physiol. 117, 491–504 (2001)

    CAS  Article  Google Scholar 

  12. Varnum, M. D., Black, K. D. & Zagotta, W. N. Molecular mechanism for ligand discrimination of cyclic nucleotide-gated channels. Neuron 15, 619–625 (1995)

    CAS  Article  Google Scholar 

  13. Matulef, K., Flynn, G. E. & Zagotta, W. N. Molecular rearrangements in the ligand-binding domain of cyclic nucleotide-gated channels. Neuron 24, 443–452 (1999)

    CAS  Article  Google Scholar 

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

    ADS  CAS  Article  Google Scholar 

  15. Wang, J., Chen, S. & Siegelbaum, S. A. Regulation of hyperpolarization-activated HCN. channel gating and cAMP modulation due to interactions of COOH terminus and core transmembrane regions. J. Gen. Physiol. 118, 237–250 (2001)

    CAS  Article  Google Scholar 

  16. Weber, I. T., Shabb, J. B. & Corbin, J. D. Predicted structures of the cGMP binding domains of the cGMP-dependent protein kinase: a key alanine/threonine difference in evolutionary divergence of cAMP and cGMP binding sites. Biochemistry 28, 6122–6127 (1989)

    CAS  Article  Google Scholar 

  17. Kumar, V. D. & Weber, I. T. Molecular model of the cyclic GMP-binding domain of the cyclic GMP-gated ion channel. Biochemistry 31, 4643–4649 (1992)

    CAS  Article  Google Scholar 

  18. Altenhofen, W. et al. Control of ligand specificity in cyclic nucleotide-gated channels from rod photoreceptors and olfactory epithelium. Proc. Natl Acad. Sci. USA 88, 9868–9872 (1991)

    ADS  CAS  Article  Google Scholar 

  19. Gauss, R., Seifert, R. & Kaupp, U. B. Molecular identification of a hyperpolarization-activated channel in sea urchin sperm. Nature 393, 583–587 (1998)

    ADS  CAS  Article  Google Scholar 

  20. Liu, D. T., Tibbs, G. R. & Siegelbaum, S. A. Subunit stoichiometry of cyclic nucleotide-gated channels and effects of subunit order on channel function. Neuron 16, 983–990 (1996)

    CAS  Article  Google Scholar 

  21. Ulens, C. & Siegelbaum, S. A. Tetramer constructs reveal stoichiometry of cAMP modulation of hyperpolarization-activated HCN2 channels. Biophys. J. 84, 137a (2003)

    Google Scholar 

  22. Rothberg, B. S., Shin, K. S., Phale, P. S. & Yellen, G. Voltage-controlled gating at the intracellular entrance to a hyperpolarization-activated cation channel. J. Gen. Physiol. 119, 83–91 (2002)

    CAS  Article  Google Scholar 

  23. Shin, K. S., Rothberg, B. S. & Yellen, G. Blocker state dependence and trapping in hyperpolarization-activated cation channels: evidence for an intracellular activation gate. J. Gen. Physiol. 117, 91–101 (2001)

    CAS  Article  Google Scholar 

  24. Jiang, Y. et al. Crystal structure and mechanism of a calcium-gated potassium channel. Nature 417, 515–522 (2002)

    ADS  CAS  Article  Google Scholar 

  25. Johnson, J. P. Jr & Zagotta, W. N. Rotational movement during cyclic nucleotide-gated channel opening. Nature 412, 917–921 (2001)

    ADS  CAS  Article  Google Scholar 

  26. Guerrero, S. A., Hecht, H. J., Hofmann, B., Biebl, H. & Singh, M. Production of selenomethionine-labelled proteins using simplified culture conditions and generally applicable host/vector systems. Appl. Microbiol. Biotechnol. 56, 718–723 (2001)

    CAS  Article  Google Scholar 

  27. Collaborative Computational Project, N, The CCP4 suite: programs for X-ray crystallography. Acta Crystallogr. D 50, 760–763 (1994)

    Article  Google Scholar 

  28. Jones, T. & Kjeldgaard, M. Electron-density map interpretation. Methods Enzymol. 277, 173–208 (1997)

    CAS  Article  Google Scholar 

  29. Brunger, A. T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

    CAS  Article  Google Scholar 

  30. Nicholls, A., Sharp, K. A. & Honig, B. Protein folding and association: Insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11, 281–296 (1991)

    CAS  Article  Google Scholar 

Download references


We thank J. Lidestri for support of the X-ray facility at Columbia University; M. A. Gawinowicz for assistance with protein analysis by mass spectrometry; and S. Siegelbaum for comments and the HCN2 channel cDNA. X-ray diffraction data sets were collected at the MacCHESS synchrotron facility and at NSLS, and we thank the beamline personnel for their assistance. N.O. and R.O. were supported in part by a NIH training grant in Molecular Biophysics. Funding for the analytical ultracentrifuge was provided by the NIH. W.N.Z. and E.G. are investigators with the Howard Hughes Medical Institute.

Author information

Authors and Affiliations


Corresponding authors

Correspondence to William N. Zagotta or Eric Gouaux.

Ethics declarations

Competing interests

The authors declare that they have no competing financial interests.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


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