Early changes in KCC2 phosphorylation in response to neuronal stress result in functional downregulation.
The K+ Cl- cotransporter KCC2 plays an important role in chloride homeostasis and in neuronal responses mediated by ionotropic GABA and glycine receptors. The expression levels of KCC2 in neurons determine whether neurotransmitter responses are inhibitory or excitatory. KCC2 expression is decreased in developing neurons, as well as in response to various models of neuronal injury and epilepsy. We investigated whether there is also direct modulation of KCC2 activity by changes in phosphorylation during such neuronal stressors. We examined tyrosine phosphorylation of KCC2 in rat hippocampal neurons under different conditions of in vitro neuronal stress and the functional consequences of changes in tyrosine phosphorylation. Oxidative stress (H2O2) and the induction of seizure activity (BDNF) and hyperexcitability (0 Mg2+) resulted in a rapid dephosphorylation of KCC2 that preceded the decreases in KCC2 protein or mRNA expression. Dephosphorylation of KCC2 is correlated with a reduction of transport activity and a decrease in [Cl-]i, as well as a reduction in KCC2 surface expression. Manipulation of KCC2 tyrosine phosphorylation resulted in altered neuronal viability in response to in vitro oxidative stress. During continued neuronal stress, a second phase of functional KCC2 downregulation occurs that corresponds to decreases in KCC2 protein expression levels. We propose that neuronal stress induces a rapid loss of tyrosine phosphorylation of KCC2 that results in translocation of the protein and functional loss of transport activity. Additional understanding of the mechanisms involved may provide means for manipulating the extent of irreversible injury resulting from different neuronal stressors.
Wake
Hiroaki
H
Division of Homeostatic Development, National Institute of Physiological Sciences, Okazaki 444-8585, Japan.
Watanabe
Miho
M
Moorhouse
Andrew J
AJ
Kanematsu
Takashi
T
Horibe
Shoko
S
Matsukawa
Noriyuki
N
Asai
Kiyofumi
K
Ojika
Kosei
K
Hirata
Masato
M
Nabekura
Junichi
J
D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D004307:Dose-Response Relationship, Radiation; D015536:Down-Regulation / Q000502:physiology*; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004558:Electric Stimulation / Q000379:methods; D004622:Embryo, Mammalian; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D018682:GABA Agents / Q000494:pharmacology; D006624:Hippocampus / Q000166:cytology; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D009474:Neurons / Q000187:drug effects* / Q000502:physiology*; D018408:Patch-Clamp Techniques / Q000379:methods; D010766:Phosphorylation / Q000187:drug effects; D011247:Pregnancy; D051381:Rats; D017208:Rats, Wistar; D027981:Symporters / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism; D000096922:K Cl- Cotransporters
D002614:Chelating Agents; D004533:Egtazic Acid; D004791:Enzyme Inhibitors; D018682:GABA Agents; D006861:Hydrogen Peroxide; D027981:Symporters; D014443:Tyrosine; D000096922:K Cl- Cotransporters; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester