Skip to main content

Thank you for visiting nature.com. 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.

How subunits cooperate in cAMP-induced activation of homotetrameric HCN2 channels

Nature Chemical Biology volume 8, pages 162–169 (2012)Cite this article

Subjects

Abstract

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are tetrameric membrane proteins that generate electrical rhythmicity in specialized neurons and cardiomyocytes. The channels are primarily activated by voltage but are receptors as well, binding the intracellular ligand cyclic AMP. The molecular mechanism of channel activation is still unknown. Here we analyze the complex activation mechanism of homotetrameric HCN2 channels by confocal patch-clamp fluorometry and kinetically quantify all ligand binding steps and closed-open isomerizations of the intermediate states. For the binding affinity of the second, third and fourth ligand, our results suggest pronounced cooperativity in the sequence positive, negative and positive, respectively. This complex interaction of the subunits leads to a preferential stabilization of states with zero, two or four ligands and suggests a dimeric organization of the activation process: within the dimers the cooperativity is positive, whereas it is negative between the dimers.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Enhancement of activation in HCN2 channels by cAMP and fcAMP.
Figure 2: Binding of fcAMP to HCN2 channels on concentration jumps.
Figure 3: Fit of ligand binding and activation by a Markovian state model.
Figure 4: Time courses of the subunit action.
Figure 5: Equilibrium constants and type of cooperativity.
Figure 6: Gibbs free energy profiles.
Figure 7: Replacement of saturating cAMP by fcAMP in activated HCN2 channels.

References

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

    Article  CAS  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. Moosmang, S., Biel, M., Hofmann, F. & Ludwig, A. Differential distribution of four hyperpolarization-activated cation channels in mouse brain. Biol. Chem. 380, 975–980 (1999).

    Article  CAS  Google Scholar 

  5. Santoro, B. et al. Molecular and functional heterogeneity of hyperpolarization-activated pacemaker channels in the mouse CNS. J. Neurosci. 20, 5264–5275 (2000).

    Article  CAS  Google Scholar 

  6. Chan, C.S., Shigemoto, R., Mercer, J.N. & Surmeier, D.J. HCN2 and HCN1 channels govern the regularity auf autonomous pacemaking and synaptic resetting in globus pallidus neurons. J. Neurosci. 24, 9921–9932 (2004).

    Article  CAS  Google Scholar 

  7. Banks, M.I., Pearce, R.A. & Smith, P.H. Hyperpolarization-activated cation current (Ih) in neurons of the medical nucleus of the trapezoid body: voltage-clamp analysis and enhancement by norepinephrine and cAMP suggest a modulatory mechanism in the auditory brain system. J. Neurophysiol. 70, 1420–1432 (1993).

    Article  CAS  Google Scholar 

  8. Cuttle, M.F., Rusznak, Z., Wong, A.Y., Owens, S. & Forsythe, I.D. Modulation of a presynaptic hyperpolarization-activated cationic current (Ih) at an excitory synaptic terminal in the rat auditory brain stem. J. Physiol. (Lond.) 534, 733–744 (2001).

    Article  CAS  Google Scholar 

  9. Ingram, S.L. & Williams, J.T. Modulation of the hyperpolarization-activated current (Ih) by cyclic nucleotides in guinea-pig primary afferent neurons. J. Physiol. (Lond.) 492, 97–106 (1996).

    Article  CAS  Google Scholar 

  10. Saitow, F. & Konishi, S. Excitability increase induced by beta-adrenergic receptor-mediated activation of hyperpolarization-activated cation channels in rat cerebellar basket cells. J. Neurophysiol. 84, 2026–2034 (2000).

    Article  CAS  Google Scholar 

  11. Notomi, T. & Shigemoto, R. Immunohistochemical localization of Ih channel subunits, HCN1–4, in the rat brain. J. Comp. Neurol. 471, 241–276 (2004).

    Article  CAS  Google Scholar 

  12. Brown, H.F., DiFrancesco, D. & Noble, S.J. How does adrenaline accelerate the heart? Nature 280, 235–236 (1979).

    Article  CAS  Google Scholar 

  13. Ludwig, A. et al. Two pacemaker channels from human heart with profoundly different activation kinetics. EMBO J. 18, 2323–2329 (1999).

    Article  CAS  PubMed Central  Google Scholar 

  14. Gauss, R. & Seifert, R. Pacemaker oscillations in heart and brain: a key role for hyperpolarization-activated cation channels. Chronobiol. Int. 17, 453–469 (2000).

    Article  CAS  Google Scholar 

  15. Biel, M., Wahl-Schott, C., Michalakis, S. & Zong, X. Hyperpolarization-activated cation channels: from genes to function. Physiol. Rev. 89, 847–885 (2009).

    Article  CAS  Google Scholar 

  16. DiFrancesco, D. Characterization of single pacemaker channels in cardiac sino-atrial node cells. Nature 324, 470–473 (1986).

    Article  CAS  Google Scholar 

  17. Santoro, B. & Tibbs, G.R. The HCN gene family: molecular basis of the hyperpolarization-activated pacemaker channels. Ann. NY Acad. Sci. 868, 741–764 (1999).

    Article  CAS  Google Scholar 

  18. DiFrancesco, D. Dual allosteric modulation of pacemaker (f) channels by cAMP and voltage in rabbit SA node. J. Physiol. (Lond.) 515, 367–376 (1999).

    Article  CAS  Google Scholar 

  19. 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).

    Article  CAS  PubMed Central  Google Scholar 

  20. Wang, J., Chen, S., Nolan, M.F. & Siegelbaum, S.A. Activity-dependent regulation of HCN pacemaker channels by cyclic AMP: signaling through dynamic allosteric coupling. Neuron 36, 451–461 (2002).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  22. Craven, K.B. & Zagotta, W.N. CNG and HCN channels: two peas, one pod. Annu. Rev. Physiol. 68, 375–401 (2006).

    Article  CAS  Google Scholar 

  23. Kaupp, U.B. & Seifert, R. Molecular diversity of pacemaker ion channels. Annu. Rev. Physiol. 63, 235–257 (2001).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  25. Altomare, C. et al. Integrated allosteric model of voltage gating in HCN channels. J. Gen. Physiol. 117, 519–532 (2001).

    Article  CAS  PubMed Central  Google Scholar 

  26. Bruening-Wright, A., Elinder, F. & Larsson, H.P. Kinetic relationship between the voltage sensor and the activation gate in spHCN channels. J. Gen. Physiol. 130, 71–81 (2007).

    Article  CAS  PubMed Central  Google Scholar 

  27. Männikkö, R., Pandey, S., Larsson, H.P. & Elinder, F. Hysteresis in the voltage dependence of HCN channels: conversion between two modes affects pacemaker properties. J. Gen. Physiol. 125, 305–326 (2005).

    Article  PubMed Central  Google Scholar 

  28. Elinder, F., Männikkö, R., Pandey, S. & Larsson, H.P. Mode shifts in the voltage gating of the mouse and human HCN2 and HCN4 channels. J. Physiol. (Lond.) 575, 417–431 (2006).

    Article  CAS  Google Scholar 

  29. Craven, K.B. & Zagotta, W.N. Salt bridges and gating in the COOH-terminal region of HCN2 and CNGA1 channels. J. Gen. Physiol. 124, 663–677 (2004).

    Article  CAS  PubMed Central  Google Scholar 

  30. Shin, K.S., Maertens, C., Proenza, C., Rothberg, B.S. & Yellen, G. Inactivation in HCN channels results from reclosure of the activation gate: desensitization to voltage. Neuron 41, 737–744 (2004).

    Article  CAS  Google Scholar 

  31. Ulens, C. & Siegelbaum, S.A. Regulation of hyperpolarization-activated HCN channels by cAMP through a gating switch in binding domain symmetry. Neuron 40, 959–970 (2003).

    Article  CAS  Google Scholar 

  32. Chen, S., Wang, J., Zhou, L., George, M.S. & Siegelbaum, S.A. Voltage sensor movement and cAMP binding allosterically regulate an inherently voltage-independent closed-open transition in HCN channels. J. Gen. Physiol. 129, 175–188 (2007).

    Article  CAS  PubMed Central  Google Scholar 

  33. Kusch, J. et al. Interdependence of receptor activation and ligand binding in HCN2 pacemaker channels. Neuron 67, 75–85 (2010).

    Article  CAS  Google Scholar 

  34. Weiss, J.N. The Hill equation revisited: uses and misuses. FASEB J. 11, 835–841 (1997).

    Article  CAS  Google Scholar 

  35. Colquhoun, D. Binding, gating, affinity and efficacy: the interpretation of structure-activity relationships for agonists and of the effects of mutating receptors. Br. J. Pharmacol. 125, 924–947 (1998).

    Article  CAS  Google Scholar 

  36. Yifrach, O. Hill coefficient for estimating the magnitude of cooperativity on gating transitions of voltage-dependent ion channels. Biophys. J. 87, 822–830 (2004).

    Article  CAS  PubMed Central  Google Scholar 

  37. Monod, J., Wyman, J. & Changeux, J.P. On the nature of allosteric transitions: a plausible model. J. Mol. Biol. 12, 88–118 (1965).

    Article  CAS  Google Scholar 

  38. Goulding, E.H., Tibbs, G.R. & Siegelbaum, S.A. Molecular mechanism of cyclic-nucleotide-gated channel activation. Nature 372, 369–374 (1994).

    Article  CAS  Google Scholar 

  39. Biskup, C. et al. Relating ligand binding to activation gating in CNGA2 channels. Nature 446, 440–443 (2007).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  41. 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).

    Article  CAS  Google Scholar 

  42. Liu, D.T., Tibbs, G.R., Paoletti, P. & Siegelbaum, S.A. Constraining ligand-binding site stoichiometry suggests that a cyclic nucleotide-gated channel is composed of two functional dimers. Neuron 21, 235–248 (1998).

    Article  CAS  Google Scholar 

  43. Burzomato, V., Beato, M., Groot-Kormelink, P.J., Colquhoun, D. & Sivilotti, L.G. Single-channel behavior of heteromeric alpha1beta glycine receptors: an attempt to detect a conformational change before the channel opens. J. Neurosci. 24, 10924–10940 (2004).

    Article  CAS  Google Scholar 

  44. Nache, V. et al. Activation of olfactory-type cyclic nucleotide-gated channels is highly cooperative. J. Physiol. (Lond.) 569, 91–102 (2005).

    Article  CAS  Google Scholar 

  45. Jonas, P. High-speed solution switching using piezo-based micropositioning stages. in Single-Channel Recording (eds. Sakmann, B. & Neher, E.) 231–243 (Plenum Press, New York, 1995).

  46. Zheng, J. & Zagotta, W.N. Gating rearrangements in cyclic nucleotide-gated channels revealed by patch-clamp fluorometry. Neuron 28, 369–374 (2000).

    Article  CAS  Google Scholar 

  47. Zheng, J. & Zagotta, W.N. Patch-clamp fluorometry recording of conformational rearrangements of ion channels. Sci. STKE 2003, PL7 (2003).

    PubMed  Google Scholar 

  48. Wu, S. et al. State-dependent cAMP binding to functioning HCN channels studied by patch-clamp fluorometry. Biophys. J. 100, 1226–1232 (2011).

    Article  CAS  PubMed Central  Google Scholar 

Download references

Acknowledgements

We are indebted to U.B. Kaupp for providing the cDNA and to F. Lehmann (Dyomics GmbH, Jena, Germany) for providing the dyes. We would also like to thank G. Ditze, G. Sammler, F. Horn, M. Händel, K. Schoknecht, S. Bernhardt, A. Kolchmeier and B. Tietsch for excellent technical assistance. This work was supported by grants from the Deutsche Forschungsgemeinschaft (to K.B.) and from the Friedrich-Schiller-University (to J.K.). F.S. acknowledges support from the Bremen Innovation Agency.

Author information

Author notes

  1. Jana Kusch and Susanne Thon: These authors contributed equally to this work.

Authors and Affiliations

  1. Institut für Physiologie II, Universitätsklinikum Jena, Friedrich-Schiller-Universität Jena, Jena, Germany

    Jana Kusch, Susanne Thon, Christoph Biskup, Vasilica Nache, Thomas Zimmer & Klaus Benndorf

  2. Fachhochschule Schmalkalden, Fakultät Elektrotechnik, Blechhammer, Schmalkalden, Germany

    Eckhard Schulz

  3. Arbeitsgruppe Biomolekulare Photonik, Universitätsklinikum Jena, Friedrich-Schiller-Universität Jena, Germany

    Christoph Biskup

  4. Abteilung Molekulare Neurosensorik, Center of Advanced European Studies and Research, Bonn, Germany

    Reinhard Seifert

  5. BIOLOG Life Science Institute, Bremen, Germany

    Frank Schwede

Authors
  1. Jana Kusch
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Susanne Thon
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Eckhard Schulz
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Christoph Biskup
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Vasilica Nache
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Thomas Zimmer
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Reinhard Seifert
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Frank Schwede
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Klaus Benndorf
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.K. and S.T. designed and carried out the electrophysiological and fluorometrical measurements, analyzed the experimental data (apart from the global fit) and prepared the figures. J.K. also contributed to the manuscript preparation. E.S. performed the mathematical analysis of the experimental data. C.B. developed the software for analyzing the fluorometrical data. V.N. and R.S. contributed to the experimental design. T.Z. provided molecular biological support. F.S. synthesized the fluorescently labeled cAMP. K.B. planned the project, designed experiments and wrote the manuscript.

Corresponding author

Correspondence to Klaus Benndorf.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Methods and Supplementary Results (PDF 211 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kusch, J., Thon, S., Schulz, E. et al. How subunits cooperate in cAMP-induced activation of homotetrameric HCN2 channels. Nat Chem Biol 8, 162–169 (2012). https://doi.org/10.1038/nchembio.747

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.747

This article is cited by

Search

Advanced search

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