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Structural basis for modulation and agonist specificity of HCN pacemaker channels

Abstract

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.

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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.

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Acknowledgements

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.

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Correspondence to William N. Zagotta or Eric Gouaux.

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Zagotta, W., Olivier, N., Black, K. et al. Structural basis for modulation and agonist specificity of HCN pacemaker channels. Nature 425, 200–205 (2003). https://doi.org/10.1038/nature01922

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