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

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

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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.

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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.

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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.

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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

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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.

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Correspondence to Klaus Benndorf.

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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

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