2014
Molecular Motions Involved in Na-K-Cl Cotransporter-mediated Ion Transport and Transporter Activation Revealed by Internal Cross-linking between Transmembrane Domains 10 and 11/12*
Monette MY, Somasekharan S, Forbush B. Molecular Motions Involved in Na-K-Cl Cotransporter-mediated Ion Transport and Transporter Activation Revealed by Internal Cross-linking between Transmembrane Domains 10 and 11/12*. Journal Of Biological Chemistry 2014, 289: 7569-7579. PMID: 24451383, PMCID: PMC3953270, DOI: 10.1074/jbc.m113.542258.Peer-Reviewed Original ResearchMeSH KeywordsAmino Acid SequenceBinding SitesCell LineChloridesCopperCross-Linking ReagentsDisulfidesHomeostasisHumansIon TransportIonsKineticsMicroscopy, ConfocalMicroscopy, FluorescenceModels, MolecularMolecular Sequence DataMutationPhenanthrolinesPhosphorylationProtein Structure, TertiaryRubidium RadioisotopesSequence Homology, Amino AcidSodium-Potassium-Chloride SymportersSolute Carrier Family 12, Member 2ConceptsTransmembrane domain 10Domain 10Copper phenanthrolineLarge C terminusNa-K-Cl cotransporterRegulatory domainCysteine pairsC-terminusN-terminusDephosphorylation rateTransporter activationDisulfide formationIon transportHomology modelNKCC activationInhibition of transportTransport functionLow micromolar concentrationsSame transporterCross-link formationActivation statePhosphorylationTerminusTransportersOcclusion step
2013
Functional Expression of Human NKCC1 from a Synthetic Cassette-Based cDNA: Introduction of Extracellular Epitope Tags and Removal of Cysteines
Somasekharan S, Monette MY, Forbush B. Functional Expression of Human NKCC1 from a Synthetic Cassette-Based cDNA: Introduction of Extracellular Epitope Tags and Removal of Cysteines. PLOS ONE 2013, 8: e82060. PMID: 24339991, PMCID: PMC3855340, DOI: 10.1371/journal.pone.0082060.Peer-Reviewed Original ResearchConceptsNa-K-Cl cotransporterEpitope tagYFP tagPlasma membraneMost animal cellsRemoval of cysteineOpen reading frameSilent restriction sitesHuman NKCC1Regulation of NKCC1Na-K-Cl cotransport activityHEK-293 cellsCellular homeostasisBiosynthetic machinerySynthetic cassetteAnimal cellsNovel cDNACysteine mutantsReading frameSynthetic cDNACysteine mutationsEndoplasmic reticulumFunctional expressionCotransport activityCDNA
2012
Loop Diuretic and Ion-binding Residues Revealed by Scanning Mutagenesis of Transmembrane Helix 3 (TM3) of Na-K-Cl Cotransporter (NKCC1)*
Somasekharan S, Tanis J, Forbush B. Loop Diuretic and Ion-binding Residues Revealed by Scanning Mutagenesis of Transmembrane Helix 3 (TM3) of Na-K-Cl Cotransporter (NKCC1)*. Journal Of Biological Chemistry 2012, 287: 17308-17317. PMID: 22437837, PMCID: PMC3366785, DOI: 10.1074/jbc.m112.356014.Peer-Reviewed Original ResearchConceptsTransmembrane helix 3Na-K-Cl cotransporterTranslocation pathwayHelix 3Homology modelTryptophan-scanning mutagenesisMutation of residuesStructural homology modelEpithelial salt transportExtracellular gateCellular chloride homeostasisScanning mutagenesisOpen conformationIntracellular endPore residuesFunctional roleIon translocationTranslocation rateResiduesMutagenesisCentral roleChloride homeostasisMutationsPathwayLarge effect
2011
Regulatory Activation Is Accompanied by Movement in the C Terminus of the Na-K-Cl Cotransporter (NKCC1)*
Monette MY, Forbush B. Regulatory Activation Is Accompanied by Movement in the C Terminus of the Na-K-Cl Cotransporter (NKCC1)*. Journal Of Biological Chemistry 2011, 287: 2210-2220. PMID: 22121194, PMCID: PMC3265899, DOI: 10.1074/jbc.m111.309211.Peer-Reviewed Original ResearchConceptsC-terminusFluorescence resonance energy transferNa-K-Cl cotransporterFRET decreasesSame C-terminusMost vertebrate cellsKey structural roleEmbryonic kidney cell lineYellow fluorescent proteinHuman embryonic kidney cell lineRegulation of NKCC1Vertebrate cellsKidney cell linePlasma membraneNKCC1 regulationN-terminusFluorescent proteinStructural roleRegulatory activationTransporter activationConformational changesTerminusTransport activityResonance energy transferHEK cellsRare mutations in the human Na-K-Cl cotransporter (NKCC2) associated with lower blood pressure exhibit impaired processing and transport function
Monette MY, Rinehart J, Lifton RP, Forbush B. Rare mutations in the human Na-K-Cl cotransporter (NKCC2) associated with lower blood pressure exhibit impaired processing and transport function. American Journal Of Physiology. Renal Physiology 2011, 300: f840-f847. PMID: 21209010, PMCID: PMC3074999, DOI: 10.1152/ajprenal.00552.2010.Peer-Reviewed Original ResearchConceptsHEK-293 cellsNa-K-Cl cotransporterTransport functionMajor salt transport pathwayPlasma membrane localizationHEK cellsLow transport activitySequence conservationMembrane localizationProtein functionHeterologous expressionXenopus laevis oocytesImportant residuesMutantsRenal salt reabsorptionMolecular mechanismsIndependent mutationsConstitutive activityTransport activityBlood pressureFunctional consequencesImpaired transport functionSuch mutationsProcessing defectsLaevis oocytes
2008
Na-K-Cl Cotransporter-1 in the Mechanism of Ammonia-induced Astrocyte Swelling*
Jayakumar A, Liu M, Moriyama M, Ramakrishnan R, Forbush B, Reddy P, Norenberg M. Na-K-Cl Cotransporter-1 in the Mechanism of Ammonia-induced Astrocyte Swelling*. Journal Of Biological Chemistry 2008, 283: 33874-33882. PMID: 18849345, PMCID: PMC2590687, DOI: 10.1074/jbc.m804016200.Peer-Reviewed Original ResearchConceptsNKCC activityNa-K-ClAstrocyte swellingCell swellingNa-K-Cl cotransporter-1Na-K-Cl cotransporterComplication of acute liver failureN-nitro-L-arginine methyl esterNitric oxide synthase inhibitionActivation of NKCC1NKCC1 protein expressionCultured astrocytesInduce oxidative/nitrosative stressAcute liver failureLoss of ion homeostasisSwelling of astrocytesPhosphorylated NKCC1NKCC1 expressionLiver failureNKCC1Synthase inhibitionBrain herniationBrain edemaIntracranial pressureProtein expressionExon Loss Accounts for Differential Sorting of Na-K-Cl Cotransporters in Polarized Epithelial Cells
Carmosino M, Giménez I, Caplan M, Forbush B. Exon Loss Accounts for Differential Sorting of Na-K-Cl Cotransporters in Polarized Epithelial Cells. Molecular Biology Of The Cell 2008, 19: 4341-4351. PMID: 18667527, PMCID: PMC2555935, DOI: 10.1091/mbc.e08-05-0478.Peer-Reviewed Original ResearchConceptsDileucine motifNa-K-Cl cotransporterRenal Na-K-Cl cotransporterPolarized epithelial cellsAmino acid stretchApical proteinsApical sortingEvolutionary lossRenal epithelial cell lineGene structurePhylogenetic analysisDifferential sortingDirect traffickingEpithelial cell lineAdditional exonC-terminusMammalian kidneyApical membraneExonsNovel mechanismNKCC2 geneCell linesBasolateral membraneMotifEpithelial cells
2007
Intramolecular and Intermolecular Fluorescence Resonance Energy Transfer in Fluorescent Protein-tagged Na-K-Cl Cotransporter (NKCC1) SENSITIVITY TO REGULATORY CONFORMATIONAL CHANGE AND CELL VOLUME* * This work was supported by National Institutes of Health Grant DK47661. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The on-line version of this article (available at http://www.jbc.org) contains supplemental Methods 1 and Table 1.
Pedersen M, Carmosino M, Forbush B. Intramolecular and Intermolecular Fluorescence Resonance Energy Transfer in Fluorescent Protein-tagged Na-K-Cl Cotransporter (NKCC1) SENSITIVITY TO REGULATORY CONFORMATIONAL CHANGE AND CELL VOLUME* * This work was supported by National Institutes of Health Grant DK47661. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The on-line version of this article (available at http://www.jbc.org) contains supplemental Methods 1 and Table 1. Journal Of Biological Chemistry 2007, 283: 2663-2674. PMID: 18045874, DOI: 10.1074/jbc.m708194200.Peer-Reviewed Original ResearchMeSH KeywordsAnimalsBacterial ProteinsCell LineCell SizeChloridesFluorescence Resonance Energy TransferGreen Fluorescent ProteinsHumansLuminescent ProteinsModels, MolecularPhosphorylationProtein ConformationRecombinant Fusion ProteinsSharksSodium-Potassium-Chloride SymportersSolute Carrier Family 12, Member 1TransfectionConceptsFluorescence resonance energy transferRegulatory domainC-terminusLevel of FRETN-terminusFluorescent proteinFRET changesResonance energy transferRegulatory phosphorylation eventsRegulatory conformational changesFluorescent protein tagsExtreme N-terminusEmbryonic kidney cell lineYellow fluorescent proteinHuman embryonic kidney cell lineN-terminal residuesPhosphorylation eventsU.S.C. Section 1734Na-K-Cl cotransporterMembrane domainsProtein tagsKidney cell lineIntermolecular fluorescence resonance energy transferYFP fluorescenceCosts of publication
2006
The Residues Determining Differences in Ion Affinities among the Alternative Splice Variants F, A, and B of the Mammalian Renal Na-K-Cl Cotransporter (NKCC2)*
Giménez I, Forbush B. The Residues Determining Differences in Ion Affinities among the Alternative Splice Variants F, A, and B of the Mammalian Renal Na-K-Cl Cotransporter (NKCC2)*. Journal Of Biological Chemistry 2006, 282: 6540-6547. PMID: 17186942, DOI: 10.1074/jbc.m610780200.Peer-Reviewed Original ResearchExpression of the basolateral Na–K–Cl cotransporter during mouse nephrogenesis and embryonic development
Heuvel G, Payne J, Igarashi P, Forbush B. Expression of the basolateral Na–K–Cl cotransporter during mouse nephrogenesis and embryonic development. Gene Expression Patterns 2006, 6: 1000-1006. PMID: 16814616, DOI: 10.1016/j.modgep.2006.04.004.Peer-Reviewed Original ResearchConceptsIn situ hybridizationNa-K-Cl cotransporterDorsal root ganglionBranching ureteric budEndocapillary cellsPeripheral nervous systemRoot ganglionTooth budsNasal epitheliumMouse nephrogenesisChoroid plexusSubmandibular glandMature nephronsUreteric budNorthern blot analysisSLC12A2Nervous systemMesenchymal cellsNKCC1Blot analysisMetanephroiNephric structuresGlomeruliMiceEmbryonic development
2003
PASK (Proline-Alanine-rich STE20-related Kinase), a Regulatory Kinase of the Na-K-Cl Cotransporter (NKCC1)*
Dowd BF, Forbush B. PASK (Proline-Alanine-rich STE20-related Kinase), a Regulatory Kinase of the Na-K-Cl Cotransporter (NKCC1)*. Journal Of Biological Chemistry 2003, 278: 27347-27353. PMID: 12740379, DOI: 10.1074/jbc.m301899200.Peer-Reviewed Original ResearchMeSH KeywordsAnimalsBlotting, WesternCell LineDNA, ComplementaryDose-Response Relationship, DrugGenes, DominantGenetic VectorsHumansMarine ToxinsOxazolesOxidative StressPhosphorylationPrecipitin TestsProtein BindingProtein Serine-Threonine KinasesProtein Structure, TertiaryRatsRubidiumSharksSodium-Potassium-Chloride SymportersSolute Carrier Family 12, Member 2ThreonineTime FactorsConceptsNa-K-Cl cotransporterN-terminal regulatory domainPhosphorylation-dependent activationHEK cellsInhibitor calyculin ANKCC1 activityRegulatory domainCoimmunoprecipitation assaysRegulatory kinasesActivation of NKCC1Calyculin ARegulated eventNKCC1 activationPhosphorylationKinaseSharksCotransporter activityOverexpressionCotransporter expressionNKCC1CellsActivationBindingCotransporterMutantsShort-term Stimulation of the Renal Na-K-Cl Cotransporter (NKCC2) by Vasopressin Involves Phosphorylation and Membrane Translocation of the Protein*
Giménez I, Forbush B. Short-term Stimulation of the Renal Na-K-Cl Cotransporter (NKCC2) by Vasopressin Involves Phosphorylation and Membrane Translocation of the Protein*. Journal Of Biological Chemistry 2003, 278: 26946-26951. PMID: 12732642, DOI: 10.1074/jbc.m303435200.Peer-Reviewed Original ResearchConceptsNa-K-Cl cotransporterRenal Na-K-Cl cotransporterCell membrane compartmentsRegulatory threonineMembrane compartmentsAntidiuretic hormone vasopressinPhosphospecific antibodiesMembrane translocationAmino terminusCytoplasmic vesiclesNKCC2 phosphorylationShort-term activationApical membranePhosphorylationTerm activationHormone vasopressinShort-term stimulationThick ascending limbProteinTranslocationCotransporterNKCC2ActivationAscending limbMorphometric analysis
2002
Functional comparison of renal Na-K-Cl cotransporters between distant species
Gagnon E, Forbush B, Caron L, Isenring P. Functional comparison of renal Na-K-Cl cotransporters between distant species. American Journal Of Physiology - Cell Physiology 2002, 284: c365-c370. PMID: 12388059, DOI: 10.1152/ajpcell.00262.2002.Peer-Reviewed Original ResearchConceptsRenal Na-K-Cl cotransporterNa-K-Cl cotransporterSplice variantsSecond transmembrane domainDistant vertebratesDistant speciesTransmembrane domainAlternative splicingXenopus laevis oocytesTransport activityCl(-) affinityRenal NKCC2Functional comparisonLaevis oocytesSpeciesSharksSimilar affinityIon dependenceA Regulatory Locus of Phosphorylation in the N Terminus of the Na-K-Cl Cotransporter, NKCC1*
Darman RB, Forbush B. A Regulatory Locus of Phosphorylation in the N Terminus of the Na-K-Cl Cotransporter, NKCC1*. Journal Of Biological Chemistry 2002, 277: 37542-37550. PMID: 12145304, DOI: 10.1074/jbc.m206293200.Peer-Reviewed Original ResearchMeSH KeywordsAmino Acid SequenceAnimalsBinding SitesCell LineDogfishHumansIon TransportKineticsModels, MolecularPeptide FragmentsPhosphopeptidesPhosphorylationProtein ConformationRecombinant ProteinsSalt GlandSodium-Potassium-Chloride SymportersSolute Carrier Family 12, Member 2Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationTransfectionTrypsinConceptsProtein phosphatase 1 bindsN-terminusSer/ThrPhosphorylation-dependent mechanismN-terminal domainStrong consensus siteNa-K-Cl cotransporter NKCC1Matrix-assisted laser desorption ionization timePhosphoregulatory mechanismsPhosphoacceptor sitesRegulatory lociHEK-293 cellsNa-K-Cl cotransporterLaser desorption ionization timeCalyculin AConsensus sitesPhosphorylation stoichiometryDesorption ionization timeAmino acidsTryptic fragmentsProteinGland tubulesRectal gland tubulesFlight mass spectrometryIonization time
2001
Ion transport and ligand binding by the Na–K–Cl cotransporter, structure–function studies
Isenring P, Forbush B. Ion transport and ligand binding by the Na–K–Cl cotransporter, structure–function studies. Comparative Biochemistry And Physiology Part A Molecular & Integrative Physiology 2001, 130: 487-497. PMID: 11913460, DOI: 10.1016/s1095-6433(01)00420-2.Peer-Reviewed Original ResearchConceptsNa-K-Cl cotransporterK-Cl cotransporterCation-Cl(-) cotransportersC-terminusStructure-function studiesGroups of residuesTransmembrane segmentsMutational approachAnimal cellsCentral domainVariant residuesLigand bindingIon transportDistinct carriersNa-Cl cotransporterResiduesSpecies differencesCotransporterBindingLoop diureticsAnion transportAvailable sulfhydryl groupsSulfhydryl groupsMovement of NaDifferent substratesModulation of Ion Transport by Direct Targeting of Protein Phosphatase Type 1 to the Na-K-Cl Cotransporter*
Darman R, Flemmer A, Forbush B. Modulation of Ion Transport by Direct Targeting of Protein Phosphatase Type 1 to the Na-K-Cl Cotransporter*. Journal Of Biological Chemistry 2001, 276: 34359-34362. PMID: 11466303, DOI: 10.1074/jbc.c100368200.Peer-Reviewed Original ResearchConceptsProtein phosphatase 1Substrate proteinsNa-K-Cl cotransporterProtein phosphatase type 1Phosphatase type 1Intracellular chloride regulationPhosphatase specificityRegulatory phosphorylationPhosphatase 1Catalytic subunitMotif bindsSubunit bindsN-terminusPP1cMajor proteinsDirect bindingDirect interactionChloride regulationProteinGeneral mechanismDirect targetingMutantsMotifSubunitsBinds
2000
The Na-K-Cl Cotransporter of Secretory Epithelia
Haas M, Forbush B. The Na-K-Cl Cotransporter of Secretory Epithelia. Annual Review Of Physiology 2000, 62: 515-534. PMID: 10845101, DOI: 10.1146/annurev.physiol.62.1.515.Peer-Reviewed Original ResearchConceptsNa-K-ClNa-K-Cl cotransporterRegulation of NKCC1Secretory epitheliaNa-K-Cl cotransporter isoformsK-Cl cotransporter isoformsApical Cl- channelsNa-Cl cotransporterNon-epithelial cellsNKCC1 regulationCl- channelsBasolateral membraneCotransporter proteinFluid secretionNKCC isoformsNKCC1Ascending limbHenle's loopEpithelial cellsMacula densaIon transport proteinsTransport NaEpitheliaCotransporterCell volume
1999
Functional interaction of the K-Cl cotransporter (KCC1) with the Na-K-Cl cotransporter in HEK-293 cells
Gillen C, Forbush B. Functional interaction of the K-Cl cotransporter (KCC1) with the Na-K-Cl cotransporter in HEK-293 cells. American Journal Of Physiology 1999, 276: c328-c336. PMID: 9950760, DOI: 10.1152/ajpcell.1999.276.2.c328.Peer-Reviewed Original ResearchConceptsNa-K-Cl cotransporterNa-K-Cl cotransport activityNa-K-ClK-Cl cotransportHEK-293 cellsCotransport activityK-ClHEK-293Activation of Na-K-Cl cotransportCell swellingK-Cl cotransport activityHypotonic cell swellingIntracellular Cl- concentrationFunctional interactionsKCC1External KCotransporterIon-transporting epitheliaCell linesN-ethylmaleimidePhysiological rangeCellsInvestigate functional interactionsCross-talkPretreatment
1998
Mutagenic Mapping of the Na-K-Cl Cotransporter for Domains Involved in Ion Transport and Bumetanide Binding
Isenring P, Jacoby S, Chang J, Forbush B. Mutagenic Mapping of the Na-K-Cl Cotransporter for Domains Involved in Ion Transport and Bumetanide Binding. The Journal Of General Physiology 1998, 112: 549-558. PMID: 9806964, PMCID: PMC2229443, DOI: 10.1085/jgp.112.5.549.Peer-Reviewed Original ResearchMeSH KeywordsAnimalsBinding SitesBumetanideCarrier ProteinsCells, CulturedChloridesDiureticsHumansKidneyKineticsMutagenesis, Site-DirectedOligonucleotide ProbesPotassiumProtein Structure, TertiaryRecombinant Fusion ProteinsRubidium RadioisotopesSharksSodiumSodium-Potassium-Chloride SymportersSpecies SpecificityThe role of transmembrane domain 2 in cation transport by the Na–K–Cl cotransporter
Isenring P, Jacoby S, Forbush B. The role of transmembrane domain 2 in cation transport by the Na–K–Cl cotransporter. Proceedings Of The National Academy Of Sciences Of The United States Of America 1998, 95: 7179-7184. PMID: 9618559, PMCID: PMC22778, DOI: 10.1073/pnas.95.12.7179.Peer-Reviewed Original ResearchConceptsNa-K-Cl cotransporterBumetanide-sensitive 86Rb influxTransmembrane domain 2Na-Cl cotransporterBumetanide bindingNa-K-ClHEK-293 cellsHuman cotransportersDomain 2Third transmembrane domainCotransporterNa affinityHEK-293Stable transfectionNKCC2BumetanideCation transportTransmembrane domain 3Transmembrane domainDomain 3Alternative splicingIon transport