Structural basis for modulation and agonist specificity of HCN pacemaker channels

William N. Zagotta; Nelson B. Olivier; Kevin D. Black; Edgar C. Young; Rich Olson; Eric Gouaux

doi:10.1038/nature01922

Structural basis for modulation and agonist specificity of HCN pacemaker channels

William N. Zagotta, Nelson B. Olivier, Kevin D. Black, Edgar C. Young, Rich Olson, Eric Gouaux

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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 integration. These nonselective cation channels, underlying the I_f, I_h and I_q currents of heart and nerve cells, are activated by membrane hyperpolarization and modulated by the binding of cyclic nucleotides such as cAMP and cGMP. The cAMP-mediated enhancement of channel activity is largely responsible for the increase in heart rate caused by β-adrenergic agonists. 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.

Original language	English (US)
Pages (from-to)	200-205
Number of pages	6
Journal	Nature
Volume	425
Issue number	6954
DOIs	https://doi.org/10.1038/nature01922
State	Published - Sep 11 2003
Externally published	Yes

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@article{e1d9eaa418c9464187f4233fa3b6ea2f,

title = "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 integration. 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 cGMP. The cAMP-mediated enhancement of channel activity is largely responsible for the increase in heart rate caused by β-adrenergic agonists. 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.",

author = "Zagotta, {William N.} and Olivier, {Nelson B.} and Black, {Kevin D.} and Young, {Edgar C.} and Rich Olson and Eric Gouaux",

note = "Funding Information: Acknowledgements We thank L. Jing, Y. Hao and Y. He for assistance; W. Zhang for HEK293 cells and discussions on expression cloning; D. McClay and R. Fehon for use of cell culture and imaging facilities; J. Schroeder, J. Kwak, J. Harper and A. VanDogen for vectors; J. Schroeder, T. Sun and R. Venters for discussions; and J. Siedow, X. Dong, W. Durrant, P. Benfey, W. Zhang and R. Fehon for comments on the manuscript. R.T. was supported in part by a Hargitt Fellowship. This work was supported by start-up funds from Duke University and by a grant from the NSF to Z.-M.P. Funding Information: 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.",

year = "2003",

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day = "11",

doi = "10.1038/nature01922",

language = "English (US)",

volume = "425",

pages = "200--205",

journal = "Nature",

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T1 - Structural basis for modulation and agonist specificity of HCN pacemaker channels

AU - Zagotta, William N.

AU - Olivier, Nelson B.

AU - Black, Kevin D.

AU - Young, Edgar C.

AU - Olson, Rich

AU - Gouaux, Eric

N1 - Funding Information: Acknowledgements We thank L. Jing, Y. Hao and Y. He for assistance; W. Zhang for HEK293 cells and discussions on expression cloning; D. McClay and R. Fehon for use of cell culture and imaging facilities; J. Schroeder, J. Kwak, J. Harper and A. VanDogen for vectors; J. Schroeder, T. Sun and R. Venters for discussions; and J. Siedow, X. Dong, W. Durrant, P. Benfey, W. Zhang and R. Fehon for comments on the manuscript. R.T. was supported in part by a Hargitt Fellowship. This work was supported by start-up funds from Duke University and by a grant from the NSF to Z.-M.P. Funding Information: 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.

PY - 2003/9/11

Y1 - 2003/9/11

N2 - 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 integration. 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 cGMP. The cAMP-mediated enhancement of channel activity is largely responsible for the increase in heart rate caused by β-adrenergic agonists. 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.

AB - 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 integration. 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 cGMP. The cAMP-mediated enhancement of channel activity is largely responsible for the increase in heart rate caused by β-adrenergic agonists. 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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DO - 10.1038/nature01922

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VL - 425

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JF - Nature

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Structural basis for modulation and agonist specificity of HCN pacemaker channels

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