2022
Structural basis for inhibition of the Cation-chloride cotransporter NKCC1 by the diuretic drug bumetanide
Zhao Y, Roy K, Vidossich P, Cancedda L, De Vivo M, Forbush B, Cao E. Structural basis for inhibition of the Cation-chloride cotransporter NKCC1 by the diuretic drug bumetanide. Nature Communications 2022, 13: 2747. PMID: 35585053, PMCID: PMC9117670, DOI: 10.1038/s41467-022-30407-3.Peer-Reviewed Original ResearchConceptsTranslocation pathwayElectron cryo-microscopy structureC-terminal domainIon translocation pathwayCation-chloride cotransporters NKCC1Transmembrane domainCotransporter NKCC1C-terminal domain interactionsStructural basisDomain interactionsRenal salt reabsorptionDomain associationConformational changesFunctional studiesIon translocationElectroneutral symportCell membraneNKCC1PathwayNKCC2DomainSalt reabsorptionTransmembraneTranslocationTransporters
2021
The structural basis of function and regulation of neuronal cotransporters NKCC1 and KCC2
Zhang S, Zhou J, Zhang Y, Liu T, Friedel P, Zhuo W, Somasekharan S, Roy K, Zhang L, Liu Y, Meng X, Deng H, Zeng W, Li G, Forbush B, Yang M. The structural basis of function and regulation of neuronal cotransporters NKCC1 and KCC2. Communications Biology 2021, 4: 226. PMID: 33597714, PMCID: PMC7889885, DOI: 10.1038/s42003-021-01750-w.Peer-Reviewed Original ResearchConceptsCryo-electron microscopy structureHuman NKCC1Microscopy structureEssential residuesFunctional characterizationKCC transportersPlasma membraneStructural basisTransepithelial saltTransport activityMechanistic understandingTransportersStructural studiesCritical roleCotransporter NKCC1Computational analysisIon transportWater transportNeuronal excitabilityNKCC1PhosphorylationCell volumeNKCCKCC2Residues
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 ResearchMeSH KeywordsCysteineDNA, ComplementaryEndoplasmic ReticulumEpitopesHEK293 CellsHumansRecombinant Fusion ProteinsSolute Carrier Family 12, Member 2ConceptsNa-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 ResearchMeSH KeywordsBinding SitesBumetanideDiureticsHEK293 CellsHumansIon TransportMesylatesMutagenesisMutation, MissenseProtein Structure, SecondarySodium-Potassium-Chloride SymportersSolute Carrier Family 12, Member 2Sulfhydryl ReagentsConceptsTransmembrane 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 cells
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 expression
2006
Expression 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
2004
WNK4 regulates apical and basolateral Cl– flux in extrarenal epithelia
Kahle KT, Gimenez I, Hassan H, Wilson FH, Wong RD, Forbush B, Aronson PS, Lifton RP. WNK4 regulates apical and basolateral Cl– flux in extrarenal epithelia. Proceedings Of The National Academy Of Sciences Of The United States Of America 2004, 101: 2064-2069. PMID: 14769928, PMCID: PMC357052, DOI: 10.1073/pnas.0308434100.Peer-Reviewed Original ResearchMeSH KeywordsAnimalsBlotting, WesternCarrier ProteinsCell PolarityChloridesEpitheliumHumansImmunohistochemistryIon TransportKidneyMembrane ProteinsMembrane Transport ProteinsMiceOocytesProtein Serine-Threonine KinasesRNA, MessengerSodium-Potassium-Chloride SymportersSolute Carrier Family 12, Member 2Sulfate TransportersXenopus laevisConceptsCl fluxBlood-brain barrierUnrelated ion channelsActivity of mediatorsWNK4 mRNABile ductPancreatic ductExtrarenal expressionExtrarenal tissuesCl(-) handlingPseudohypoaldosteronism type IIChannel ROMKNaCl reabsorptionSerine-threonine kinase WNK4Specialized endotheliumExchanger SLC26A6NaCl cotransporterWNK4 effectsColonic cryptsEpitheliumVariable inhibitionSweat ductsTight junctionsKidneyElectrolyte flux
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 expressionNKCC1CellsActivationBindingCotransporterMutants
2002
A 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 timeActivation of the Na-K-Cl Cotransporter NKCC1 Detected with a Phospho-specific Antibody*
Flemmer AW, Giménez I, Dowd BF, Darman RB, Forbush B. Activation of the Na-K-Cl Cotransporter NKCC1 Detected with a Phospho-specific Antibody*. Journal Of Biological Chemistry 2002, 277: 37551-37558. PMID: 12145305, DOI: 10.1074/jbc.m206294200.Peer-Reviewed Original ResearchMeSH KeywordsAmino Acid SequenceAnimalsAntibody SpecificityBinding SitesColonDipeptidesEpinephrineHumansIon TransportIsoproterenolKineticsMolecular Sequence DataMutagenesis, Site-DirectedPhosphatesPhosphopeptidesRatsRecombinant ProteinsSodium-Potassium-Chloride SymportersSolute Carrier Family 12, Member 2ThreonineTracheaConceptsPhospho-specific antibodiesNa-K-Cl cotransporter NKCC1Divergent vertebrate speciesBasolateral membraneShark rectal glandPhosphorylation of NKCC1Regulatory lociHEK-293 cellsHigh conservationThreonine residuesPhosphorylation experimentsVertebrate speciesRegulatory domainCotransporter NKCC1NKCC1 regulationN-terminusRectal glandCell membranePhosphorylationImmunofluorescence analysisAgonist stimulationActivation stateGland tubulesRectal gland tubulesEpithelial cells