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

ABSTRACT 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.—Wilkars, W., Wollberg, J., Mohr, E., Han, M., Chetkovich, D. M., Bähring, R., Bender, R. A Nedd4‐2 regulates surface expression and may affect N ‐glycosylation of hyperpolarization‐activated cyclic nucleotide‐gated (HCN)‐1 channels. FASEB J . 28, 2177–2190 (2014). www.fasebj.org