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Clustering of neuronal K+-Cl- cotransporters in lipid rafts by tyrosine phosphorylation.

ABSTRACT: The neuronal K(+)-Cl(-) cotransporter (KCC2) is a membrane transport protein that extrudes Cl(-) from neurons and helps maintain low intracellular [Cl(-)] and hyperpolarizing GABAergic synaptic potentials. Depolarizing gamma-aminobutyric acid (GABA) responses in neonatal neurons and following various forms of neuronal injury are associated with reduced levels of KCC2 expression. Despite the importance for plasticity of inhibitory transmission, less is known about cellular mechanisms involved in more dynamic changes in KCC2 function. In this study, we investigated the role of tyrosine phosphorylation in KCC2 localization and function in hippocampal neurons and in cultured GT1-7 cells. Mutation to the putative tyrosine phosphorylation site within the long intracellular carboxyl terminus of KCC2(Y1087D) or application of the tyrosine kinase inhibitor genistein shifted the GABA reversal potential (E(GABA)) to more depolarized values, indicating reduced KCC2 function. This was associated with a change in the expression pattern of KCC2 from a punctate distribution to a more uniform distribution, suggesting that functional tyrosine-phosphorylated KCC2 forms clusters in restricted membrane domains. Sodium vanadate, a tyrosine phosphatase inhibitor, increased the proportion of KCC2 associated with lipid rafts membrane domains. Loss of tyrosine phosphorylation also reduced oligomerization of KCC2. A loss of the punctuate distribution and oligomerization of KCC2 and a more depolarized E(GABA) were seen when the 28-amino-acid carboxyl terminus of KCC2 was deleted. These results indicate that direct tyrosine phosphorylation of KCC2 results in membrane clusters and functional transport activity, suggesting a mechanism by which intracellular Cl(-) concentrations and GABA responses can be rapidly modulated.

SUBMITTER: Watanabe M

PROVIDER: S-EPMC2788850 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Clustering of neuronal K+-Cl- cotransporters in lipid rafts by tyrosine phosphorylation.

Watanabe Miho M Wake Hiroaki H Moorhouse Andrew J AJ Nabekura Junichi J

The Journal of biological chemistry 20090813 41

The neuronal K(+)-Cl(-) cotransporter (KCC2) is a membrane transport protein that extrudes Cl(-) from neurons and helps maintain low intracellular [Cl(-)] and hyperpolarizing GABAergic synaptic potentials. Depolarizing gamma-aminobutyric acid (GABA) responses in neonatal neurons and following various forms of neuronal injury are associated with reduced levels of KCC2 expression. Despite the importance for plasticity of inhibitory transmission, less is known about cellular mechanisms involved in ...[more]

PMID: 19679663

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Project description:UnlabelledRapid reorganization and stabilization of the actin cytoskeleton in dendritic spines enables cellular processes underlying learning, such as long-term potentiation (LTP). Dendritic spines are enriched in exceptionally short and dynamic actin filaments, but the studies so far have not revealed the molecular mechanisms underlying the high actin dynamics in dendritic spines. Here, we show that actin in dendritic spines is dynamically phosphorylated at tyrosine-53 (Y53) in rat hippocampal and cortical neurons. Our findings show that actin phosphorylation increases the turnover rate of actin filaments and promotes the short-term dynamics of dendritic spines. During neuronal maturation, actin phosphorylation peaks at the first weeks of morphogenesis, when dendritic spines form, and the amount of Y53-phosphorylated actin decreases when spines mature and stabilize. Induction of LTP transiently increases the amount of phosphorylated actin and LTP induction is deficient in neurons expressing mutant actin that mimics phosphorylation. Actin phosphorylation provides a molecular mechanism to maintain the high actin dynamics in dendritic spines during neuronal development and to induce fast reorganization of the actin cytoskeleton in synaptic plasticity. In turn, dephosphorylation of actin is required for the stabilization of actin filaments that is necessary for proper dendritic spine maturation and LTP maintenance.Significance statementDendritic spines are small protrusions from neuronal dendrites where the postsynaptic components of most excitatory synapses reside. Precise control of dendritic spine morphology and density is critical for normal brain function. Accordingly, aberrant spine morphology is linked to many neurological diseases. The actin cytoskeleton is a structural element underlying the proper morphology of dendritic spines. Therefore, defects in the regulation of the actin cytoskeleton in neurons have been implicated in neurological diseases. Here, we revealed a novel mechanism for regulating neuronal actin cytoskeleton that explains the specific organization and dynamics of actin in spines. The better we understand the regulation of the dendritic spine morphology, the better we understand what goes wrong in neurological diseases.

Dataset Information

Clustering of neuronal K+-Cl- cotransporters in lipid rafts by tyrosine phosphorylation.

Publications

Clustering of neuronal K+-Cl- cotransporters in lipid rafts by tyrosine phosphorylation.

Similar Datasets

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