A di-arginine ER retention signal regulates trafficking of HCN1 channels from the early secretory pathway to the plasma membrane

Abstract

Hyperpolarization-activated cyclic nucleotide-gated 1 (HCN1) channels carry Ih, which contributes to neuronal excitability and signal transmission in the nervous system. Controlling the trafficking of HCN1 is an important aspect of its regulation, yet the details of this process are poorly understood. Here, we investigated how the C-terminus of HCN1 regulates trafficking by testing for its ability to redirect the localization of a non-targeted reporter in transgenic Xenopus laevis photoreceptors. We found that HCN1 contains an ER localization signal and through a series of deletion constructs, identified the responsible di-arginine ER retention signal. This signal is located in the intrinsically disordered region of the C-terminus of HCN1. To test the function of the ER retention signal in intact channels, we expressed wild type and mutant HCN1 in HEK293 cells and found this signal negatively regulates surface expression of HCN1. In summary, we report a new mode of regulating HCN1 trafficking: through the use of a di-arginine ER retention signal that monitors processing of the channel in the early secretory pathway.

Fig. 1

Overview of HCN1 structure and experimental design. a Schematic representation of an HCN1 homotetramer (left). Each monomer (cyan) consists of six transmembrane domains with cytoplasmic N- and C-termini (right). The cyan oval in the C-terminus indicates the relative position of the CNBD. b Disorder analysis of HCN1 using meta protein disorder prediction system. Residues above the threshold (gray dashed line) are predicted to be intrinsically disordered. c A cartoon of the reporter (left) consisting of a single pass transmembrane domain (red), GFP (green) and a palmitoylated peptide (red) to which the C-terminus (cyan) of Xenopus HCN1 (or portions thereof) was attached. d A summary of the design and targeting behavior of all constructs expressed in transgenic Xenopus photoreceptors. The linear arrangement of sequence motifs in the HCN1 C-terminus is displayed on top. The range of HCN1 amino acids attached to the reporter in each construct is listed in the first column. The gray shaded area is required for ER localization. TMD transmembrane domain, CNBD cyclic nucleotide-binding domain, QP glutamine- and proline-rich region, OS outer segment, ER endoplasmic reticulum

Fig. 2

The C-terminus of HCN1 directs localization to the ER. a Transgenic Xenopus rod photoreceptors expressing the reporter alone or b in fusion to the cytoplasmic C-terminus (aa 373–839) of Xenopus HCN1. The transgenically expressed protein (b, green) colocalizes with calnexin (c, red) in the inner segment (d, transmitted light). OS outer segments, IS inner segments, N (blue) nuclei, ST synaptic terminals. Scale bars 5 μm

Fig. 3

The C-linker and CNBD are not required for ER localization. Transgenic Xenopus rod photoreceptors expressing the indicated Reporter-HCN1 fragments detailed in Fig. 1d. OS outer segments, IS inner segments, N (blue) nuclei, ST synaptic terminals. Scale bars 5 μm

Fig. 4

Identification of a di-arginine ER retention signal. a–d Transgenic Xenopus rod photoreceptors expressing the indicated Reporter-HCN1 fragments detailed in Fig. 1d. OS outer segments, IS inner segments, N (blue) nuclei, ST synaptic terminals. Scale bars 5 μm. e ClustalW sequence alignment of X. tropicalis HCN1 (aa 576–635) to HCN1 from various animal species. Identical residues shaded in black, partially conserved residues in gray, with the di-arginine motif outlined in red [asterisks mark arginines forming the RxR (red) or RxRxR (blue) motif]. Accession numbers are: X. tropicalis, XP002933077; X. laevis a, b deduced from genomic scaffold v7.1 52441 and 337825. C. mydas, XP_007052900; A. sinensis, XP_006017356; C. livia, XP_005500951; F. peregrinus, XP_005242028; G. gallus, XP429145; B. mutus, ELR46479; R. norvegicus, W9JKB0; M. musculus, O88704; H. sapiens, O60741

Fig. 5

The di-arginine motif influences plasma membrane localization of HCN1. Immunostaining of HEK293 cells expressing the following proteins: a wild-type HA-tagged HCN1 and GFP; b HA-tagged HCN1 with a mutated ER retention signal (RxR to AxA) and GFP; c wild-type HA-HCN1 and GFP-TRIP8b; d GFP alone. HA tag (red), GFP (green), Nuclei (blue), and scale bars 10 μm

Fig. 6

Mutating the di-arginine motif enhances the surface expression. Biotinylation assays of HEK293 cells transfected as described in (a, Fig. 5). a After biotinylation of transfected cells, surface proteins were pulled down using NeutrAvidin beads. The level of HCN1 and TRIP8b in the total and surface pools was detected by Western blotting. NKA and GAPDH were used as controls for membrane and cytosolic proteins, respectively. b Densitometry of Western blots represented in (a) was used to calculate the surface to total ratio of HCN1 after normalization to the loading control NKA. Asterisks indicate statistical significance with p < 0.01 (t test)