Clustering of Neuronal K+-Cl− Cotransporters in Lipid Rafts by Tyrosine Phosphorylation

Abstract

] and hyperpolarizing GABAergic synaptic potentials. Depolarizing ␥-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. In addition to changes in the expression levels of KCC2 protein, the function of KCC2 can be more dynamically and rapidly modulated by the availability of transport substrates and by various forms of kinase activity. Cl Ϫ extrusion is quantitatively regulated by the K ϩ driving force across the membrane (8). Protein kinase C can down-regulate both KCC2 function (9) and surface expression (10). Staurosporine, a broad spectrum kinase inhibitor, produces a rapid up-regulation of KCC2 function in immature neurons (11). Brain-type creatinine kinase binding to KCC2 may also regulate its function (12). Finally, WNK3, by interacting with Ste20-related proline-alanine-rich kinase, prevents the cell swelling-induced activation of KCC2 in Xenopus oocytes (13,14).KCC2 contains one tyrosine protein kinase phosphorylation consensus site (Tyr-1087) within the long carboxyl terminus in the intracellular region (15). Tyr-1087 is not present in KCC1, another family of KCCs, suggesting that direct tyrosine phosphorylation may uniquely regulate KCC2. The receptor tyrosine kinase, IGF-1, and the soluble tyrosine kinase, Src kinase, activate KCC2 during maturation of hippocampal neurons (16). Oxidative stress decreases the tyrosine phosphorylation of KCC2 and reduces KCC2 function (17). However, just how tyrosine phosphorylation regulates KCC2 function under more physiological conditions is unclear, al...

Neto2 is a KCC2 interacting protein required for neuronal Cl − regulation in hippocampal neurons

“…We determined biochemically that Neto2 preferentially interacts with oligomerized KCC2. KCC2 oligomerization, which depends on tyrosine phosphorylation of the C terminus (29), correlates with the maturation of inhibitory neurotransmission in the CNS (28), and is believed to play a major role in the change from the inactive to the active state of this transporter (5,30). Next, we demonstrated electrophysiologically that Neto2-null neurons were unable to extrude a Cl − load, indicating that the function of the remaining neuronal KCC2 in Neto2-null is severely compromised.…”

“…Efficient KCC2-mediated Cl − extrusion occurs when KCC2 exists in the oligomerized state (28)(29)(30). We, therefore, tested whether Neto2 could associate with the active oligomeric fraction of KCC2.…”

Taurine Inhibits K+-Cl− Cotransporter KCC2 to Regulate Embryonic Cl− Homeostasis via With-no-lysine (WNK) Protein Kinase Signaling Pathway

“…Ser-940 phosphorylation regulates KCC2 function by modulating cell surface KCC2 expression (56). Tyr-1087 phosphorylation affects oligomerization, which plays a pivotal role in KCC2 activity without affecting cell surface expression (20,55). Rinehart et al (54) indicated that Thr-906 and Thr-1007 phosphorylation does not affect cell surface KCC2 expression.…”

“…KCC2 gene up-regulation is essential for Cl Ϫ homeostasis during development, and phosphorylation of KCC2 is another important factor (5,12,15,18,55,56). Ser-940 phosphorylation regulates KCC2 function by modulating cell surface KCC2 expression (56).…”

A Novel Regulatory Locus of Phosphorylation in the C Terminus of the Potassium Chloride Cotransporter KCC2 That Interferes with N-Ethylmaleimide or Staurosporine-mediated Activation*♦

“…On the cellular level, location in membrane rafts was shown to modify KCC2 transport activity (23,24). On the molecular level, interaction partners such as the ATPase subunit ␣2 (25), CIP1 (26), Neto2 (27), and several protein kinases, including brain-specific creatine kinase (28), SPAK (29), OSR1 (30), and WNKs (31) were identified.…”