Nedd4-2 haploinsufficiency in mice causes increased seizure susceptibility and impaired Kir4.1 ubiquitination

Neural precursor cell expressed developmentally down-regulated gene 4-like (NEDD4-2) encodes a ubiquitin E3 ligase that is involved in epileptogenesis with mechanisms needing further investigation. We constructed a novel Nedd4-2^+/- mouse model with half level of both Nedd4-2 long and short isoforms in the brain. Nedd4-2 haploinsufficiency caused increased susceptibility and severity of pentylenetetrazole (PTZ)-induced seizures. Of the 3379 proteins identified by the hippocampal proteomic analysis, 55 were considered altered in Nedd4-2^+/- mice compared with wild-type control, among which the inwardly rectifying K⁺ channel Kir4.1 was up-regulated by 1.83-fold. Kir4.1 was subsequently confirmed to be less ubiquitinated in response to comprised Nedd4-2 in mouse brains and C6 cells. Kir4.1 associated with Nedd4-2 through the threonine³¹²-proline motif in the intracellular domain by target mutagenesis. Adaptor protein 14-3-3 facilitated Nedd4-2-mediated ubiquitination of Kir4.1. Our data consolidate the detailed molecular mechanism of Nedd4-2-mediated Kir4.1 ubiquitination, and provide a possible relationship between increased seizure susceptibility and impaired Kir4.1 ubiquitination in the brain.