Mechanism for the inhibition of the cAMP dependence of HCN ion channels by the auxiliary subunit TRIP8b

J Biol Chem. 2017 Oct 27;292(43):17794-17803. doi: 10.1074/jbc.M117.800722. Epub 2017 Sep 1.


TRIP8b, an accessory subunit of hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels, alters both the cell surface expression and cyclic nucleotide dependence of these channels. However, the mechanism by which TRIP8b exerts these dual effects is still poorly understood. In addition to binding to the carboxyl-terminal tripeptide of HCN channels, TRIP8b also binds directly to the cyclic nucleotide-binding domain (CNBD). That interaction, which requires a small central portion of TRIP8b termed TRIP8bcore, is both necessary and sufficient for reducing the cAMP-dependent regulation of HCN channels. Here, using fluorescence anisotropy, we report that TRIP8b binding to the CNBD of HCN2 channels decreases the apparent affinity of cAMP for the CNBD. We explored two possible mechanisms for this inhibition. A noncompetitive mechanism in which TRIP8b inhibits the conformational change of the CNBD associated with cAMP regulation and a competitive mechanism in which TRIP8b and cAMP compete for the same binding site. To test these two mechanisms, we used a combination of fluorescence anisotropy, biolayer interferometry, and double electron-electron resonance spectroscopy. Fitting these models to our fluorescence anisotropy binding data revealed that, surprisingly, the TRIP8b-dependent reduction of cAMP binding to the CNBD can largely be explained by partial competition between TRIP8b and cAMP. On the basis of these findings, we propose that TRIP8b competes with a portion of the cAMP-binding site or distorts the binding site by making interactions with the binding pocket, thus acting predominantly as a competitive antagonist that inhibits the cyclic-nucleotide dependence of HCN channels.

Keywords: cyclic nucleotide; electron paramagnetic resonance (EPR); fluorescence anisotropy; inhibition mechanism; ion channel; protein-protein interaction.

MeSH terms

  • Animals
  • Binding Sites
  • Cyclic AMP* / chemistry
  • Cyclic AMP* / genetics
  • Cyclic AMP* / metabolism
  • Humans
  • Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels*
  • Potassium Channels*
  • Protein Domains
  • Receptors, Cytoplasmic and Nuclear* / chemistry
  • Receptors, Cytoplasmic and Nuclear* / genetics
  • Receptors, Cytoplasmic and Nuclear* / metabolism
  • Xenopus laevis


  • HCN2 protein, human
  • Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels
  • PEX5L protein, human
  • Potassium Channels
  • Receptors, Cytoplasmic and Nuclear
  • Cyclic AMP

Associated data

  • PDB/1Q43