Nedd4-2 regulates surface expression and may affect N-glycosylation of hyperpolarization-activated cyclic nucleotide-gated (HCN)-1 channels.
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.
1Institute of Neuroanatomy, University of Hamburg Medical Center, Martinistr. 52, 20246 Hamburg, Germany. rbender@uke.uni-hamburg.de.
Wiebke
W
Wilkars
Jessica
J
Wollberg
Evita
E
Mohr
Mieri
M
Han
Dane M
DM
Chetkovich
Robert
R
Bähring
Roland A
RA
Bender
D020816:Amino Acid Motifs; D000818:Animals; D001921:Brain / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D015536:Down-Regulation; D004594:Electrophysiology; D056827:Endosomal Sorting Complexes Required for Transport / Q000502:physiology*; D005260:Female; D005786:Gene Expression Regulation; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels / Q000378:metabolism*; D000075702:Nedd4 Ubiquitin Protein Ligases; D009865:Oocytes / Q000166:cytology; D017434:Protein Structure, Tertiary; D051381:Rats; D017208:Rats, Wistar; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000502:physiology*; D029867:Xenopus Proteins; D014982:Xenopus laevis
D056827:Endosomal Sorting Complexes Required for Transport; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; C529762:PEX5L protein, human; D018160:Receptors, Cytoplasmic and Nuclear; D029867:Xenopus Proteins; C000618479:NEDD4L protein, rat; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618482:Nedd4 protein, Xenopus; C000618483:Nedd4 protein, human; C000618484:Nedd4 protein, rat; C000618485:Nedd4L protein, human; C000618460:nedd4l protein, Xenopus; D044767:Ubiquitin-Protein Ligases
TRIP8b; ion channel; trafficking; ubiquitination