Cyclic dinucleotides bind the C-linker of HCN4 to control channel cAMP responsiveness.
cAMP mediates autonomic regulation of heart rate by means of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which underlie the pacemaker current If. cAMP binding to the C-terminal cyclic nucleotide binding domain enhances HCN open probability through a conformational change that reaches the pore via the C-linker. Using structural and functional analysis, we identified a binding pocket in the C-linker of HCN4. Cyclic dinucleotides, an emerging class of second messengers in mammals, bind the C-linker pocket (CLP) and antagonize cAMP regulation of the channel. Accordingly, cyclic dinucleotides prevent cAMP regulation of If in sinoatrial node myocytes, reducing heart rate by 30%. Occupancy of the CLP hence constitutes an efficient mechanism to hinder β-adrenergic stimulation on If. Our results highlight the regulative role of the C-linker and identify a potential drug target in HCN4. Furthermore, these data extend the signaling scope of cyclic dinucleotides in mammals beyond their first reported role in innate immune system.
1] Department of Biosciences, University of Milan, Milan, Italy. [2].
Marco
M
Lolicato
1] Department of Biosciences, University of Milan, Milan, Italy. [2].
Annalisa
A
Bucchi
Department of Biosciences, University of Milan, Milan, Italy.
Cristina
C
Arrigoni
Department of Biosciences, University of Milan, Milan, Italy.
Stefano
S
Zucca
Department of Biosciences, University of Milan, Milan, Italy.
Marco
M
Nardini
Department of Biology, Technische Universität Darmstadt, Darmstadt, Germany.
Indra
I
Schroeder
School of Chemistry, University of Leeds, Leeds, UK.
Katie
K
Simmons
Department of Biosciences, University of Milan, Milan, Italy.
Marco
M
Aquila
Department of Biosciences, University of Milan, Milan, Italy.
Dario
D
DiFrancesco
1] Department of Biosciences, University of Milan, Milan, Italy. [2] Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Milan, Italy.
Martino
0000000292535170
M
Bolognesi
BIOLOG Life Science Institute, Forschungslabor und Biochemica-Vertrieb GmbH D-28199, Bremen, Germany.
Frank
F
Schwede
BIOLOG Life Science Institute, Forschungslabor und Biochemica-Vertrieb GmbH D-28199, Bremen, Germany.
Dmitry
D
Kashin
School of Chemistry, University of Leeds, Leeds, UK.
Colin W G
CW
Fishwick
School of Chemistry, University of Leeds, Leeds, UK.
A Peter
AP
Johnson
Department of Biology, Technische Universität Darmstadt, Darmstadt, Germany.
Gerhard
G
Thiel
1] Department of Biosciences, University of Milan, Milan, Italy. [2] Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Milan, Italy.
Anna
A
Moroni
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D018360:Crystallography, X-Ray; D000242:Cyclic AMP / Q000378:metabolism*; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D015226:Dinucleoside Phosphates / Q000737:chemistry / Q000378:metabolism*; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels / Q000235:genetics / Q000378:metabolism*; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D008024:Ligands; D051379:Mice; D008810:Mice, Inbred C57BL; D062105:Molecular Docking Simulation; D015394:Molecular Structure; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism*; D012849:Sinoatrial Node / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology; D014162:Transfection
D015226:Dinucleoside Phosphates; C119587:HCN4 protein, human; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D008024:Ligands; D009124:Muscle Proteins; D015221:Potassium Channels; D054852:Small Molecule Libraries; C528998:cyclic diadenosine phosphate; C062025:bis(3',5')-cyclic diguanylic acid; D000242:Cyclic AMP; D006152:Cyclic GMP