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.ver the past two decades, it has become apparent that in living tissues ion channels do not function in isolation, but instead exist as macromolecular complexes that include auxiliary subunits, signaling molecules, scaffolding proteins, and cytoskeletal proteins (1-3). These accessory proteins associate with channels and alter their gating, expression levels, localization, and response to signals such as phosphorylation or internal calcium. Until recently, little was known about which proteins may associate with and modulate hyperpolarization-activated cyclic nucleotide-gated (HCN) channels under physiological conditions. HCN channels are members of the superfamily of voltage-gated channels that contain four subunits around a centrally located pore. Unlike most of the members of the family, however, HCN channels open in response to hyperpolarizing voltages (4, 5). In addition to their unique voltage dependence, activation of HCN channels is facilitated by direct binding of cAMP to a cyclic nucleotide-binding domain (CNBD) in the carboxyl-terminal region of each subunit. The CNBD is connected to the pore region via a gating ring formed from the C-linker of each subunit. The binding of cAMP to the CNBD causes a conformational change in the C-linker and potentiation of the pore opening, which leads to a speeding of channel activation and a depolarizing shift in the voltage ...