The Neuron-specific K-Cl Cotransporter, KCC2

Williams, Jeffery; Sharp, James W.; Kumari, Vijaya G.; Wilson, Martin; Payne, John A.

doi:10.1074/jbc.274.18.12656

Cited by 211 publications

(74 citation statements)

References 39 publications

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“…The maintenance of such low levels of intracellular Cl Ϫ ions is responsible for hyperpolarizing Cl Ϫ currents upon activation of GABA A and Gly receptors, which are responsible for fast synaptic inhibition in the adult central nervous system (2)(3)(4)(5). Molecular studies have demonstrated that KCC2 is a member of a CCC superfamily and that these transporters are composed of 12-transmembrane domains with N-and C-terminal cytoplasmic domains (2,6,7). KCC2 is expressed exclusively in neurons throughout the adult brain.…”

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Direct Protein Kinase C-dependent Phosphorylation Regulates the Cell Surface Stability and Activity of the Potassium Chloride Cotransporter KCC2

Lee

Walker

Williams

et al. 2007

Journal of Biological Chemistry

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264

326

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The potassium chloride cotransporter KCC2 plays a major role in the maintenance of transmembrane chloride potential in mature neurons; thus KCC2 activity is critical for hyperpolarizing membrane currents generated upon the activation of ␥-aminobutyric acid type A and glycine (Gly) receptors that underlie fast synaptic inhibition in the adult central nervous system. However, to date an understanding of the cellular mechanism that neurons use to modulate the functional expression of KCC2 remains rudimentary. Using Escherichia coli expression coupled with in vitro kinase assays, we first established that protein kinase C (PKC) can directly phosphorylate serine 940 (Ser 940) within the C-terminal cytoplasmic domain of KCC2. We further demonstrated that Ser 940 is the major site for PKC-dependent phosphorylation for full-length KCC2 molecules when expressed in HEK-293 cells. Phosphorylation of Ser 940 increased the cell surface stability of KCC2 in this system by decreasing its rate of internalization from the plasma membrane. Coincident phosphorylation of Ser 940 increased the rate of ion transport by KCC2. It was further evident that phosphorylation of endogenous KCC2 in cultured hippocampal neurons is regulated by PKC-dependent activity. Moreover, in keeping with our recombinant studies, enhancing PKC-dependent phosphorylation increased the targeting of KCC2 to the neuronal cell surface. Our studies thus suggest that PKC-dependent phosphorylation of KCC2 may play a central role in modulating both the functional expression of this critical transporter in the brain and the strength of synaptic inhibition. Cation-chloride cotransporters (CCC)3 regulate Cl Ϫ homeostasis in cells and the generation of transmembrane chloride gradients (1). Adult mammalian neurons maintain low intracellular Cl Ϫ concentrations, which arise principally from the activity of the potassium chloride cotransporter-2 (KCC2). The maintenance of such low levels of intracellular Cl Ϫ ions is responsible for hyperpolarizing Cl Ϫ currents upon activation of GABA A and Gly receptors, which are responsible for fast synaptic inhibition in the adult central nervous system (2-5). Molecular studies have demonstrated that KCC2 is a member of a CCC superfamily and that these transporters are composed of 12-transmembrane domains with N-and C-terminal cytoplasmic domains (2, 6, 7). KCC2 is expressed exclusively in neurons throughout the adult brain. Developmentally KCC2 is first detected around 10 days in vitro in cultured rat neurons, which is coincident with the emergence of hyperpolarizing GABA A receptor-mediated Cl Ϫ currents (4, 8). Gene knock-out of KCC2 has revealed that ablating the expression of this protein results in early postnatal death. Neurons derived from these animals exhibit compromised GABA A receptor-mediated synaptic inhibition (9).Under pathological conditions such as epilepsy or ischemic brain injury, deficits in the expression of KCC2 are evident together with decreased efficacy of GABAergic inhibition and with the emergence of depola...

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confidence: 99%

Direct Protein Kinase C-dependent Phosphorylation Regulates the Cell Surface Stability and Activity of the Potassium Chloride Cotransporter KCC2

Lee

Walker

Williams

et al. 2007

Journal of Biological Chemistry

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264

326

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“…HEK293 cells, and Xenopus oocytes. In this study, we therefore examined the role and mechanisms of tyrosine phosphorylation in the regulation of KCC2 function in cultured hippocampal neurons and in GT1-7 cells, a brain-derived cell line that possesses many neuronal characteristics but does not express endogenous KCC2 (18,19) (also, see "Experimental Procedures"). The present study proposes that tyrosine phosphorylation of KCC2 results in clustering within lipid rafts via interactions within the carboxyl terminus of KCC2 and that this clustering results in efficient extrusion of Cl Ϫ .…”

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

Watanabe

Wake

Moorhouse

et al. 2009

Journal of Biological Chemistry

118

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] 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...

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“…Third, KCC2 has perhaps the most clearly defined physiological role of the four KCCs. KCC2 is thus a neuron-specific gene (22,23), with a crucial role in determining intracellular chloride activity ([Cl Ϫ ] i ) and the neuronal response to GABA and glycine (24,25). Early in postnatal life there is robust neuronal expression of the Na ϩ -K ϩ -2Cl Ϫ cotransporter NKCC1 (26), with only minimal expression of KCC2.…”

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confidence: 99%