title: Structure and stoichiometry of an accessory subunit TRIP8b interaction with hyperpolarization-activated cyclic nucleotide-gated channels.
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 single-molecule 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 nucleotide-binding 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.
authors.affiliation: Department of Physiology and Biophysics, University of Washington School of Medicine, Seattle, WA 98195, USA.
authors.forename: John R
authors.initials: JR
authors.lastname: Bankston
authors.forename: Stacey S
authors.initials: SS
authors.lastname: Camp
authors.forename: Frank
authors.initials: F
authors.lastname: DiMaio
authors.forename: Alan S
authors.initials: AS
authors.lastname: Lewis
authors.forename: Dane M
authors.initials: DM
authors.lastname: Chetkovich
authors.forename: William N
authors.initials: WN
authors.lastname: Zagotta
mesh_terms: D000818:Animals; D002850:Chromatography, Gel; D003461:Crystallography; D005454:Fluorescence Polarization; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins; D006801:Humans; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009865:Oocytes; D018408:Patch-Clamp Techniques; D015221:Potassium Channels; D011485:Protein Binding; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000378:metabolism*; D014961:X-Ray Diffraction; D014981:Xenopus
chemical_list: C578710:HCN2 protein, human; C578711:Hcn2 protein, mouse; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels; D046912:Multiprotein Complexes; D009419:Nerve Tissue Proteins; C529762:PEX5L protein, human; D015221:Potassium Channels; D018160:Receptors, Cytoplasmic and Nuclear; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins