Nedd4-2 regulates surface expression and may affect N-glycosylation of hyperpolarization-activated cyclic nucleotide-gated (HCN)-1 channels
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Nedd4-2 regulates surface expression and may affect N-glycosylation of hyperpolarization-activated cyclic nucleotide-gated (HCN)-1 channels. / Wilkars, Wiebke; Wollberg, Jessica; Mohr, Evita; Han, Mieri; Chetkovich, Dane M; Bähring, Robert; Bender, Roland A.
in: FASEB J, Jahrgang 28, Nr. 5, 01.05.2014, S. 2177-90.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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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 -