title: TRIP8b regulates HCN1 channel trafficking and gating through two distinct C-terminal interaction sites.
abstract: Hyperpolarization-activated cyclic nucleotide-regulated (HCN) channels in the brain associate with their auxiliary subunit TRIP8b (also known as PEX5R), a cytoplasmic protein expressed as a family of alternatively spliced isoforms. Recent in vitro and in vivo studies have shown that association of TRIP8b with HCN subunits both inhibits channel opening and alters channel membrane trafficking, with some splice variants increasing and others decreasing channel surface expression. Here, we address the structural bases of the regulatory interactions between mouse TRIP8b and HCN1. We find that HCN1 and TRIP8b interact at two distinct sites: an upstream site where the C-linker/cyclic nucleotide-binding domain of HCN1 interacts with an 80 aa domain in the conserved central core of TRIP8b; and a downstream site where the C-terminal SNL (Ser-Asn-Leu) tripeptide of the channel interacts with the tetratricopeptide repeat domain of TRIP8b. These two interaction sites play distinct functional roles in the effects of TRIP8b on HCN1 trafficking and gating. Binding at the upstream site is both necessary and sufficient for TRIP8b to inhibit channel opening. It is also sufficient to mediate the trafficking effects of those TRIP8b isoforms that downregulate channel surface expression, in combination with the trafficking motifs present in the N-terminal region of TRIP8b. In contrast, binding at the downstream interaction site serves to stabilize the C-terminal domain of TRIP8b, allowing for optimal interaction between HCN1 and TRIP8b as well as for proper assembly of the molecular complexes that mediate the effects of TRIP8b on HCN1 channel trafficking.
authors.affiliation: Department of Neuroscience and Pharmacology, Howard Hughes Medical Institute, Columbia University, New York, New York 10032, USA.
authors.forename: Bina
authors.initials: B
authors.lastname: Santoro
authors.forename: Lei
authors.initials: L
authors.lastname: Hu
authors.forename: Haiying
authors.initials: H
authors.lastname: Liu
authors.forename: Andrea
authors.initials: A
authors.lastname: Saponaro
authors.forename: Phillip
authors.initials: P
authors.lastname: Pian
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Piskorowski
authors.forename: Anna
authors.initials: A
authors.lastname: Moroni
authors.forename: Steven A
authors.initials: SA
authors.lastname: Siegelbaum
mesh_terms: D017398:Alternative Splicing / Q000502:physiology; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D015153:Blotting, Western; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000378:metabolism*; D004594:Electrophysiology; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D047468:Immunoprecipitation; D015640:Ion Channel Gating / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D000074401:Peroxins; D015221:Potassium Channels / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology
chemical_list: D054815:Cyclic Nucleotide-Gated Cation Channels; C578666:Hcn1 protein, mouse; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D008565:Membrane Proteins; D000074401:Peroxins; C501732:Pex5l protein, mouse; D015221:Potassium Channels