Structure and stoichiometry of an accessory subunit TRIP8b interaction with hyperpolarization-activated cyclic nucleotide-gated channels

John R. Bankston, Stacey S. Camp, Frank DiMaio, Alan S. Lewis, Dane M. Chetkovich, William N. Zagotta*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

37 Scopus citations

Abstract

Ion channels operate in intact tissues as part of large macromolecular complexes that can include cytoskeletal proteins, scaffolding proteins, signaling molecules, and a litany of other molecules. The proteins that make up these complexes can influence the trafficking, localization, and biophysical properties of the channel. TRIP8b (tetratricopetide repeat-containing Rab8b-interacting protein) is a recently discovered accessory subunit of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels that contributes to the substantial dendritic localization of HCN channels in many types of neurons. TRIP8b interacts with the carboxyl-terminal region of HCN channels and regulates their cell-surface expression level and cyclic nucleotide dependence. Here we examine the molecular determinants of TRIP8b binding to HCN2 channels. Using a singlemolecule fluorescence bleaching method, we found that TRIP8b and HCN2 form an obligate 4:4 complex in intact channels. Fluorescence- detection size-exclusion chromatography and fluorescence anisotropy allowed us to confirm that two different domains in the carboxyl-terminal portion of TRIP8b-the tetratricopepide repeat region and the TRIP8b conserved region-interact with two different regions of the HCN carboxyl-terminal region: the carboxyl- terminal three amino acids (SNL) and the cyclic nucleotidebinding domain, respectively. And finally, using X-ray crystallography, we determined the atomic structure of the tetratricopepide region of TRIP8b in complex with a peptide of the carboxy-terminus of HCN2. Together, these experiments begin to uncover the mechanism for TRIP8b binding and regulation of HCN channels.

Original languageEnglish (US)
Pages (from-to)7899-7904
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume109
Issue number20
DOIs
StatePublished - May 15 2012

Keywords

  • Beta subunit
  • Protein-protein interaction
  • Single molecule

ASJC Scopus subject areas

  • General

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