ABSTRACT
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 Nedd4-2
SUMMARY 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 ABBREVIATION
- Nedd4
- Neuronal precursor expressed developmentally downregulated 4
- Ubi
- ubiquitin
- Kir4.1
- inwardly rectifying K+ channel 4.1
- KO
- knockout
- Cx43
- connexin43
- Cx30
- connexin30
- E1
- ubiquitin activating enzyme
- E2
- ubiquitin conjugating enzyme
- E3
- ubiquitin ligase
- HECT
- homologous to the E6AP carboxyl terminus
- N4-2
- Nedd4-2
- N4-1
- Nedd4-1
- LFP
- local field potential
- N4-1/2 bDKO
- brain-specific conditional Nedd4-1/Nedd4-2 knockout
- N4-2 bKO
- brain-specific Nedd4-2 knockout
- SM
- synaptic plasma membrane fraction
- iTRAQ
- isobaric tags for relative and absolute quantitation
- N4-1 bKO
- brain-specific Nedd4-1 single knockout
- CTL
- control
- Kir4.1-HA
- HA-tagged Kir4.1
- Cx43-HA
- HA-tagged connexin43
- K63-Ub-chains
- K63-linked polyubiquitin chain
- K48-Ub-chains
- K48-linked polyubiquitin chain
- ERAD
- endoplasmic reticulum-associated degradation
- WT
- wild type
- C/S
- catalytically inactive point mutant
- AM
- acetomethoxy
- FRAP
- fluorescence recovery after photobleaching
- tdTom
- tdTomato
- N4-2 AstKO
- astrocyte-specific Nedd4-2 knockout
- PV
- parvalbumin
- ACSF
- artificial cerebrospinal fluid