PT - JOURNAL ARTICLE AU - Bekir Altas AU - Anes Ju AU - Hugo Cruces Solís AU - Samir Karaca AU - Jan Winchenbach AU - Öyküm Kaplan AU - Manuela Schwark AU - Georg L. Wieser AU - Viduth K. Chaugule AU - Irina Majoul AU - Mohamed A. Hassan AU - Rashi Goel AU - Sonja M. Wojcik AU - Andrea Pichler AU - Miso Mitkovski AU - Livia de Hoz AU - Henning Urlaub AU - Olaf Jahn AU - Gesine Saher AU - JeongSeop Rhee AU - Hiroshi Kawabe TI - Physiological and pathophysiological homeostasis of astroglial channel proteins by Nedd4-2 AID - 10.1101/683441 DP - 2019 Jan 01 TA - bioRxiv PG - 683441 4099 - http://biorxiv.org/content/early/2019/07/04/683441.short 4100 - http://biorxiv.org/content/early/2019/07/04/683441.full AB - Nedd4-2 is an E3 ubiquitin ligase, missense mutation of which is related to familial epilepsy, indicating its critical role in regulating neuronal network activity. However, Nedd4-2 substrates involved in neuronal network function have yet to be identified. Using mouse lines lacking Nedd4-1 and Nedd4-2, we identified astrocytic channel proteins inwardly rectifying K+ channel 4.1 (Kir4.1) and Connexin43 as Nedd4-2 substrates. We found that the expression of Kir4.1 and Connexin43 is increased upon conditional deletion of Nedd4-2 in astrocytes, leading to an elevation of astrocytic membrane ion permeability and gap junction activity, with a consequent reduction of γ-oscillatory neuronal network activity. Interestingly, our biochemical data demonstrate that missense mutations found in familial epileptic patients produce gain-of-function of Nedd4-2 gene product. Our data reveal a process of coordinated astrocytic ion channel proteostasis that controls astrocyte function and astrocyte-dependent neuronal network activity, and elucidate a potential mechanism by which aberrant Nedd4-2 function leads to epilepsy.CONDENSED TITLE Ubiquitination of Astrocytic Channels by Nedd4-2SUMMARY Ubiquitination is a key regulatory machinery for protein expression. Our present work provides evidence that astrocytic ion channel proteostasis coordinated by an E3 ubiquitin ligase Nedd4-2 is of particular importance for the maintenance of neuronal network activity.LIST OF ABBREVIATIONNedd4Neuronal precursor expressed developmentally downregulated 4UbiubiquitinKir4.1inwardly rectifying K+ channel 4.1KOknockoutCx43connexin43Cx30connexin30E1ubiquitin activating enzymeE2ubiquitin conjugating enzymeE3ubiquitin ligaseHECThomologous to the E6AP carboxyl terminusN4-2Nedd4-2N4-1Nedd4-1LFPlocal field potentialN4-1/2 bDKObrain-specific conditional Nedd4-1/Nedd4-2 knockoutN4-2 bKObrain-specific Nedd4-2 knockoutSMsynaptic plasma membrane fractioniTRAQisobaric tags for relative and absolute quantitationN4-1 bKObrain-specific Nedd4-1 single knockoutCTLcontrolKir4.1-HAHA-tagged Kir4.1Cx43-HAHA-tagged connexin43K63-Ub-chainsK63-linked polyubiquitin chainK48-Ub-chainsK48-linked polyubiquitin chainERADendoplasmic reticulum-associated degradationWTwild typeC/Scatalytically inactive point mutantAMacetomethoxyFRAPfluorescence recovery after photobleachingtdTomtdTomatoN4-2 AstKOastrocyte-specific Nedd4-2 knockoutPVparvalbuminACSFartificial cerebrospinal fluid