title: A mechanism for the auto-inhibition of hyperpolarization-activated cyclic nucleotide-gated (HCN) channel opening and its relief by cAMP.
abstract: Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels control neuronal and cardiac electrical rhythmicity. There are four homologous isoforms (HCN1-4) sharing a common multidomain architecture that includes an N-terminal transmembrane tetrameric ion channel followed by a cytoplasmic "C-linker," which connects a more distal cAMP-binding domain (CBD) to the inner pore. Channel opening is primarily stimulated by transmembrane elements that sense membrane hyperpolarization, although cAMP reduces the voltage required for HCN activation by promoting tetramerization of the intracellular C-linker, which in turn relieves auto-inhibition of the inner pore gate. Although binding of cAMP has been proposed to relieve auto-inhibition by affecting the structure of the C-linker and CBD, the nature and extent of these cAMP-dependent changes remain limitedly explored. Here, we used NMR to probe the changes caused by the binding of cAMP and of cCMP, a partial agonist, to the apo-CBD of HCN4. Our data indicate that the CBD exists in a dynamic two-state equilibrium, whose position as gauged by NMR chemical shifts correlates with the V½ voltage measured through electrophysiology. In the absence of cAMP, the most populated CBD state leads to steric clashes with the activated or "tetrameric" C-linker, which becomes energetically unfavored. The steric clashes of the apo tetramer are eliminated either by cAMP binding, which selects for a CBD state devoid of steric clashes with the tetrameric C-linker and facilitates channel opening, or by a transition of apo-HCN to monomers or dimer of dimers, in which the C-linker becomes less structured, and channel opening is not facilitated.
authors.affiliation: From the Departments of Chemistry and Chemical Biology and.
authors.forename: Madoka
authors.initials: M
authors.lastname: Akimoto
authors.affiliation: the Biomolecular NMR and Munich Center for Integrated Protein Science, Department of Chemie, Technische Universität München, Lichtenbergstrasse 4, 85747 Garching, Germany.
authors.forename: Zaiyong
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario L8S 4M1, Canada.
authors.forename: Stephen
authors.initials: S
authors.lastname: Boulton
authors.affiliation: From the Departments of Chemistry and Chemical Biology and.
authors.forename: Rajeevan
authors.initials: R
authors.lastname: Selvaratnam
authors.affiliation: From the Departments of Chemistry and Chemical Biology and.
authors.forename: Bryan
authors.initials: B
authors.lastname: VanSchouwen
authors.affiliation: From the Departments of Chemistry and Chemical Biology and Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario L8S 4M1, Canada.
authors.forename: Melanie
authors.initials: M
authors.lastname: Gloyd
authors.affiliation: the Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada, and.
authors.forename: Eric A
authors.initials: EA
authors.lastname: Accili
authors.affiliation: the Biomolecular NMR and Munich Center for Integrated Protein Science, Department of Chemie, Technische Universität München, Lichtenbergstrasse 4, 85747 Garching, Germany, the Institute of Structural Biology, Helmholtz Zentrum München, 85764 Neuherberg, Germany.
authors.forename: Oliver F
authors.initials: OF
authors.lastname: Lange
authors.affiliation: From the Departments of Chemistry and Chemical Biology and Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario L8S 4M1, Canada, melacin@mcmaster.ca.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Melacini
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D000242:Cyclic AMP / Q000378:metabolism*; D003563:Cyclic CMP / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015640:Ion Channel Gating; D008564:Membrane Potentials; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D015221:Potassium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: C578710:HCN2 protein, human; C119587:HCN4 protein, human; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D009124:Muscle Proteins; D015221:Potassium Channels; D011994:Recombinant Proteins; D003563:Cyclic CMP; D000242:Cyclic AMP
keywords: Allosteric Regulation; Allostery; Cyclic AMP (cAMP); HCN; Intrinsically Disordered Proteins (IDPs); Nuclear Magnetic Resonance (NMR); Protein Conformation; Signaling; cAMP-binding Domain (CBD); cCMP