title: 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.
authors.affiliation: Department of Developmental Physiology, National Institute for Physiological Sciences, Okazaki 444-8585, Japan.
authors.forename: Miho
authors.initials: M
authors.lastname: Watanabe
authors.affiliation: Department of Developmental Physiology, National Institute for Physiological Sciences, Okazaki 444-8585, Japan; Core Research for the Evolutionary Science and Technology, Japan Science and Technology Corporation, Saitama 333-0012, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Wake
authors.affiliation: Department of Physiology, School of Medical Sciences, University of New South Wales, Sydney, New South Wales 2052, Australia.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Moorhouse
authors.affiliation: Department of Developmental Physiology, National Institute for Physiological Sciences, Okazaki 444-8585, Japan; Core Research for the Evolutionary Science and Technology, Japan Science and Technology Corporation, Saitama 333-0012, Japan; Department of Physiological Sciences, The Graduate University for Advanced Studies (SOKENDAI), Hayama 240-0193, Japan. Electronic address: nabekura@nips.ac.jp.
authors.forename: Junichi
authors.initials: J
authors.lastname: Nabekura
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D005260:Female; D019833:Genistein; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D010766:Phosphorylation; D011247:Pregnancy; D051381:Rats; D017208:Rats, Wistar; D027981:Symporters / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*; D005680:gamma-Aminobutyric Acid / Q000378:metabolism; D000096922:K Cl- Cotransporters
chemical_list: D019833:Genistein; D027981:Symporters; D014443:Tyrosine; D005680:gamma-Aminobutyric Acid; D000096922:K Cl- Cotransporters
references.citation: Trends Neurosci. 2003 Apr;26(4):199-206
references.pmid: 12689771
references.citation: Biochemistry. 2000 Apr 4;39(13):3718-24
references.pmid: 10736171
references.citation: Brain Res. 2000 May 2;864(1):134-7
references.pmid: 10793196
references.citation: Neurosci Res. 2008 Dec;62(4):270-7
references.pmid: 18840481
references.citation: J Neurosci. 2006 Oct 11;26(41):10407-19
references.pmid: 17035525
references.citation: J Cell Biol. 2004 Dec 20;167(6):1205-15
references.pmid: 15596541
references.citation: Nat Rev Neurosci. 2002 Sep;3(9):728-39
references.pmid: 12209121
references.citation: J Neurosci. 2002 Jun 1;22(11):4412-7
references.pmid: 12040048
references.citation: Nat Rev Mol Cell Biol. 2000 Oct;1(1):31-9
references.pmid: 11413487
references.citation: J Biol Chem. 2001 Nov 9;276(45):41870-8
references.pmid: 11551954
references.citation: Nature. 1999 Jan 21;397(6716):251-5
references.pmid: 9930699
references.citation: J Neurosci. 2007 Feb 14;27(7):1642-50
references.pmid: 17301172
references.citation: Endocrinology. 2004 May;145(5):2375-83
references.pmid: 14736732
references.citation: J Biol Chem. 2003 Jul 4;278(27):24302-7
references.pmid: 12704198
references.citation: J Neurosci. 2001 Nov 1;21(21):8339-47
references.pmid: 11606621
references.citation: J Biol Chem. 2004 Jun 4;279(23):24685-91
references.pmid: 15073181
references.citation: J Neurosci. 1999 Apr 15;19(8):2843-51
references.pmid: 10191302
references.citation: Cell Physiol Biochem. 2008;22(1-4):31-44
references.pmid: 18769030
references.citation: Trends Pharmacol Sci. 2004 Jan;25(1):16-21
references.pmid: 14723974
references.citation: J Neurosci. 1996 Jul 1;16(13):4283-92
references.pmid: 8753889
references.citation: J Neurophysiol. 2000 Jul;84(1):281-8
references.pmid: 10899203
references.citation: Neurochem Int. 2005 May;46(6):489-99
references.pmid: 15769551
references.citation: Eur J Neurosci. 2005 Feb;21(4):899-904
references.pmid: 15787696
references.citation: Neuron. 1990 Jul;5(1):1-10
references.pmid: 2196069
references.citation: J Neurophysiol. 2003 Mar;89(3):1353-62
references.pmid: 12612004
references.citation: Arch Med Res. 2001 Nov-Dec;32(6):486-98
references.pmid: 11750724
references.citation: J Neurochem. 2006 Jan;96(2):598-608
references.pmid: 16336223
references.citation: Nat Neurosci. 2004 Jul;7(7):711-8
references.pmid: 15195093
references.citation: Neuron. 2005 Dec 8;48(5):773-86
references.pmid: 16337915
references.citation: J Biol Chem. 2007 Oct 12;282(41):29777-84
references.pmid: 17693402
references.citation: J Am Soc Nephrol. 2003 Dec;14(12):3039-46
references.pmid: 14638903
references.citation: Proc Natl Acad Sci U S A. 2005 Nov 15;102(46):16783-8
references.pmid: 16275911
references.citation: J Biol Chem. 2002 Feb 15;277(7):4945-50
references.pmid: 11733521
references.citation: Proc Natl Acad Sci U S A. 1992 May 1;89(9):4149-53
references.pmid: 1570341
references.citation: Am J Physiol Cell Physiol. 2006 Jan;290(1):C134-42
references.pmid: 15930150
references.citation: Brain Res Mol Brain Res. 2002 Jun 30;103(1-2):91-105
references.pmid: 12106695
references.citation: FEBS Lett. 2004 Apr 23;564(1-2):131-5
references.pmid: 15094054
references.citation: J Neurosci. 2003 Apr 15;23(8):3262-71
references.pmid: 12716933
references.citation: J Biol Chem. 1999 Apr 30;274(18):12656-64
references.pmid: 10212246
references.citation: J Neurosci. 2006 Aug 23;26(34):8748-57
references.pmid: 16928863