Fig 2: Potential Model for HCN1 degradation versus 14-3-3 interaction with HCN1.A) One potential model that could explain the apparent dual nature of the S810 & S867 centered signals in the HCN1 C-terminus–the serines are required for interaction with 14-3-3 and the flanking sequences around those serines participate in HCN1 degradation by an unknown factor (X). HCN1 phosphorylation at S810 and S867 could bias protein-protein interactions at those sites towards 14-3-3 over the Factor X degradation mediator. B) HCN1 protein level is established by the balance between destabilizing forces, such as the strong degradation signal, and stabilizing forces. The role of 14-3-3 interaction with HCN1 remains unclear but it could act in combination with other proteins, such as the well-established neuron specific HCN1 binding partner TRIP8b to provide a modularized strong protection signal to balance the effects of the degradation signal.

Fig 3: The C-terminus of HCN1 contains multiple predicted 14-3-3 binding sites.Sequence alignment of HCN1 from five vertebrate species. The structured domains of HCN1 are shaded as follows: light gray, HCN domain; dark gray, transmembrane domains; and medium gray, gating ring (C-linker and Cyclic Nucleotide Binding Domain (CNBD)). The RxR ER retention motif is colored in orange and the TRIP8b binding site in cyan. 14-3-3Pred predicted 14-3-3 binding sites are underlined and the three sites that were tested in this study are further shaded in yellow.

Fig 4: 14-3-3 interacts with HCN1.A) Western blotting for HCN1 and 14-3-3 following 14-3-3 IP or control GFP IP from wild-type and HCN1 KO retina. B) Western blotting for HCN1 following pulldown with recombinant 14-3-3? or resin only control from mouse retina, whole brain, and HCN1 expressing HEK293 cells.

Fig 5: Mutating 14-3-3 interaction sites enhances HCN1 protein level in stable HEK293 cell lines.Activation voltage (A), current density (B), and maximal current density of Ih (C), recorded from HEK293 cell lines expressing mRuby3-HCN1 (black) or mRuby3-HCN1?3, 14-3-3 (grey). D) Representative western blot showing total HCN1 expression and surface HCN1 expression isolated by surface biotinylation. Western blotting against the endogenous NKA and GAPDH as membrane and cytosolic protein controls. E) Relative amount of total and surface mRuby3-HCN1?3, 14-3-3 compared to mRuby3-HCN1 normalized to NKA. Error bars represent standard error of mean for electrophysiology data (A, B, C) and standard deviation for western blot data (E). T-test p < 0.05 (*); p < 0.01 (**); p<0.001 (***); p < 0.0001 (****).