Nedd4-2 regulates surface expression and may affect N-glycosylation of hyperpolarization-activated cyclic nucleotide-gated (HCN)-1 channels

TY - JOUR

T1 - Nedd4-2 regulates surface expression and may affect N-glycosylation of hyperpolarization-activated cyclic nucleotide-gated (HCN)-1 channels

AU - Wilkars, Wiebke

AU - Wollberg, Jessica

AU - Mohr, Evita

AU - Han, Mieri

AU - Chetkovich, Dane M

AU - Bähring, Robert

AU - Bender, Roland A

PY - 2014/5/1

Y1 - 2014/5/1

N2 - HCN channels are important regulators of neuronal excitability. The proper function of these channels is governed by various mechanisms, including post-translational modifications of channel subunits. Here, we provide evidence that ubiquitination via a ubiquitin ligase, neuronal precursor cell expressed developmentally downregulated (Nedd)-4-2, is involved in the regulation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. We identified a PY motif (L/PPxY), the characteristic binding motif for Nedd4-2 in the C terminus of the HCN1 subunit, and showed that HCN1 and Nedd4-2 interacted both in vivo (rat hippocampus, neocortex, and cerebellum) and in vitro [human embryonic kidney 293 (HEK293) cells], resulting in increased HCN1 ubiquitination. Elimination of the PY motif reduced, but did not abolish, Nedd4-2 binding, which further involved a stretch of ∼100 aa downstream in the HCN1 C terminus. Coexpression of Nedd4-2 and HCN1 drastically reduced the HCN1-mediated h-current amplitude (85-92%) in Xenopus laevis oocytes and reduced surface expression (34%) of HCN1 channels in HEK293 cells, thereby opposing effects of tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b)-(1a-4), an auxiliary subunit that promotes HCN1 surface expression. Regulation may further include N-glycosylation of HCN1 channels, which is significantly enhanced by TRIP8b(1a-4), but may be reduced by Nedd4-2. Taken together, our data indicate that Nedd4-2 plays an important role in the regulation of HCN1 trafficking and may compete with TRIP8b(1a-4) in this process.

AB - HCN channels are important regulators of neuronal excitability. The proper function of these channels is governed by various mechanisms, including post-translational modifications of channel subunits. Here, we provide evidence that ubiquitination via a ubiquitin ligase, neuronal precursor cell expressed developmentally downregulated (Nedd)-4-2, is involved in the regulation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. We identified a PY motif (L/PPxY), the characteristic binding motif for Nedd4-2 in the C terminus of the HCN1 subunit, and showed that HCN1 and Nedd4-2 interacted both in vivo (rat hippocampus, neocortex, and cerebellum) and in vitro [human embryonic kidney 293 (HEK293) cells], resulting in increased HCN1 ubiquitination. Elimination of the PY motif reduced, but did not abolish, Nedd4-2 binding, which further involved a stretch of ∼100 aa downstream in the HCN1 C terminus. Coexpression of Nedd4-2 and HCN1 drastically reduced the HCN1-mediated h-current amplitude (85-92%) in Xenopus laevis oocytes and reduced surface expression (34%) of HCN1 channels in HEK293 cells, thereby opposing effects of tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b)-(1a-4), an auxiliary subunit that promotes HCN1 surface expression. Regulation may further include N-glycosylation of HCN1 channels, which is significantly enhanced by TRIP8b(1a-4), but may be reduced by Nedd4-2. Taken together, our data indicate that Nedd4-2 plays an important role in the regulation of HCN1 trafficking and may compete with TRIP8b(1a-4) in this process.

KW - Amino Acid Motifs

KW - Animals

KW - Brain

KW - Cell Membrane

KW - Down-Regulation

KW - Electrophysiology

KW - Endosomal Sorting Complexes Required for Transport

KW - Female

KW - Gene Expression Regulation

KW - Glycosylation

KW - HEK293 Cells

KW - Humans

KW - Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels

KW - Oocytes

KW - Protein Structure, Tertiary

KW - Rats

KW - Rats, Wistar

KW - Receptors, Cytoplasmic and Nuclear

KW - Ubiquitin-Protein Ligases

KW - Xenopus laevis

U2 - 10.1096/fj.13-242032

DO - 10.1096/fj.13-242032

M3 - SCORING: Journal article

C2 - 24451387

VL - 28

SP - 2177

EP - 2190

JO - FASEB J

JF - FASEB J

SN - 0892-6638

IS - 5

ER -