2024
Structural bases for Na+-Cl− cotransporter inhibition by thiazide diuretic drugs and activation by kinases
Zhao Y, Schubert H, Blakely A, Forbush B, Smith M, Rinehart J, Cao E. Structural bases for Na+-Cl− cotransporter inhibition by thiazide diuretic drugs and activation by kinases. Nature Communications 2024, 15: 7006. PMID: 39143061, PMCID: PMC11324901, DOI: 10.1038/s41467-024-51381-y.Peer-Reviewed Original ResearchConceptsNa+-Cl- cotransporterFamilial hyperkalemic hypertensionRenal salt retentionThiazide diuretic drugsNa+-Cl-Cotransporter inhibitionNCC activitySalt reabsorptionDiuretic drugsBlood pressureBalanced electrolyteTreat hypertensionIon translocation pathwayIon translocationThiazideHypertensionSalt retentionOrthosteric siteCo-structureCarboxyl-terminal domainKinase cascadeEdemaChlorthalidoneCotransporterTranslocation
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
2016
Photolysis quantum yield measurements in the near-UV; a critical analysis of 1-(2-nitrophenyl)ethyl photochemistry
Corrie JE, Kaplan JH, Forbush B, Ogden DC, Trentham DR. Photolysis quantum yield measurements in the near-UV; a critical analysis of 1-(2-nitrophenyl)ethyl photochemistry. Photochemical & Photobiological Sciences 2016, 15: 604-608. PMID: 27050155, DOI: 10.1039/c5pp00440c.Peer-Reviewed Original Research
2015
Commentary on “Caged Phosphate and the Slips and Misses in Determination of Quantum Yields for Ultraviolet‐A‐Induced Photouncaging” by G. Gasser and Co‐Workers
Corrie JE, Kaplan JH, Forbush B, Ogden DC, Trentham DR. Commentary on “Caged Phosphate and the Slips and Misses in Determination of Quantum Yields for Ultraviolet‐A‐Induced Photouncaging” by G. Gasser and Co‐Workers. ChemPhysChem 2015, 16: 1861-1862. PMID: 25704071, DOI: 10.1002/cphc.201402808.Peer-Reviewed Original Research
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
2010
Drosophila glia use a conserved cotransporter mechanism to regulate extracellular volume
Leiserson WM, Forbush B, Keshishian H. Drosophila glia use a conserved cotransporter mechanism to regulate extracellular volume. Glia 2010, 59: 320-332. PMID: 21125654, PMCID: PMC3005002, DOI: 10.1002/glia.21103.Peer-Reviewed Original ResearchMeSH KeywordsAction PotentialsAnimalsAnimals, Genetically ModifiedBlood-Nerve BarrierCells, CulturedDrosophilaDrosophila ProteinsExtracellular SpaceHumansIn Vitro TechniquesLarvaMicroscopy, Electron, TransmissionModels, BiologicalMutationNeural ConductionNeurogliaNeuronsPeripheral NervesProtein Serine-Threonine KinasesSymportersTwo-Hybrid System TechniquesConceptsHuman NKCC1Yeast two hybrid assaysExtracellular solute compositionLarvae mutantDrosophila gliaNcc69Osmotic homeostasisExtracellular volumeMolecular mechanismsNervous systemOrthologsExtracellular solutesPhysiological mechanismsBlood-brain barrierBlood-nerve barrierSimilar roleAccumulation of fluidAction potential conductionGlial cellsPeripheral neuropathyNKCC1Serious health threatDetectable impactBlood barrierGliaPhosphorylation state of the Na+–K+–Cl− cotransporter (NKCC1) in the gills of Atlantic killifish (Fundulus heteroclitus) during acclimation to water of varying salinity
Flemmer AW, Monette MY, Djurisic M, Dowd B, Darman R, Gimenez I, Forbush B. Phosphorylation state of the Na+–K+–Cl− cotransporter (NKCC1) in the gills of Atlantic killifish (Fundulus heteroclitus) during acclimation to water of varying salinity. Journal Of Experimental Biology 2010, 213: 1558-1566. PMID: 20400641, PMCID: PMC2856500, DOI: 10.1242/jeb.039644.Peer-Reviewed Original ResearchConceptsSalinity acclimationAtlantic killifishNKCC1 phosphorylationCAMP-protein kinase A (PKA) pathwayEnvironmental salinityFW fishRole of phosphorylationPhospho-specific antibodiesLong-term acclimationKinase A PathwayFreshwaterCAMP-PKA pathwayTransfer of fishPhosphorylation stateKillifish gillEuryhaline teleostNKCC1 proteinKillifishAcclimationRegulatory rolePhosphorylationA PathwayRich kinaseFishGills
2009
The Potassium Chloride Cotransporter KCC-2 Coordinates Development of Inhibitory Neurotransmission and Synapse Structure in Caenorhabditis elegans
Tanis JE, Bellemer A, Moresco JJ, Forbush B, Koelle MR. The Potassium Chloride Cotransporter KCC-2 Coordinates Development of Inhibitory Neurotransmission and Synapse Structure in Caenorhabditis elegans. Journal Of Neuroscience 2009, 29: 9943-9954. PMID: 19675228, PMCID: PMC2737711, DOI: 10.1523/jneurosci.1989-09.2009.Peer-Reviewed Original ResearchMeSH KeywordsAnimalsCaenorhabditis elegansCaenorhabditis elegans ProteinsChloridesFurosemideHypotonic SolutionsMotor NeuronsMusclesMutationReceptors, G-Protein-CoupledSequence HomologySexual Behavior, AnimalSodium Potassium Chloride Symporter InhibitorsSymportersSynapsesSynaptic TransmissionSynaptic VesiclesUp-RegulationConceptsEgg-laying behaviorChloride channelsC. elegans behaviorGenetic screenHSN neuronsMature neural circuitsChloride gradientFunctional analysisInhibitory neurotransmissionSynapse developmentVesicle populationsAdult mammalian brainSynaptic vesicle populationPotassium-chloride cotransporterTransport chlorideSynapse maturationPotassium-chloride cotransporter KCC2CaenorhabditisAppropriate activity levelsMammalian brainSynapse structureChloride cotransporterHypotonic conditionsLoop diuretic furosemideCoordinate developmentSites of Regulated Phosphorylation that Control K-Cl Cotransporter Activity
Rinehart J, Maksimova YD, Tanis JE, Stone KL, Hodson CA, Zhang J, Risinger M, Pan W, Wu D, Colangelo CM, Forbush B, Joiner CH, Gulcicek EE, Gallagher PG, Lifton RP. Sites of Regulated Phosphorylation that Control K-Cl Cotransporter Activity. Cell 2009, 138: 525-536. PMID: 19665974, PMCID: PMC2811214, DOI: 10.1016/j.cell.2009.05.031.Peer-Reviewed Original ResearchConceptsIntrinsic transport activityK-Cl cotransporterTransport activityCell volume regulationRegulated phosphorylationRNA interferenceAlanine substitutionsCultured cellsHomologous sitesKCC activityCl exitWNK1 expressionNeonatal mouse brainVolume regulationNeuronal functionHypotonic conditionsActive cotransportPhosphorylationIntracellular chloride concentrationCotransporter activityKCC3Human red blood cellsKCC2 activationFundamental roleMouse brain
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
2005
SGK1 activates Na+-K+-ATPase in amphibian renal epithelial cells
de la Rosa D, Gimenez I, Forbush B, Canessa CM. SGK1 activates Na+-K+-ATPase in amphibian renal epithelial cells. American Journal Of Physiology - Cell Physiology 2005, 290: c492-c498. PMID: 16192298, DOI: 10.1152/ajpcell.00556.2004.Peer-Reviewed Original ResearchConceptsRenal epithelial cellsEpithelial cellsEffects of aldosteroneCell linesActivation of ENaCGlucocorticoid-induced kinase 1Epithelial cell lineRenal epithelial cell lineAldosteroneSGK1 expressionSame cell lineSubunit abundanceSGK1Channel activityTotal proteinImportant regulatorKinase 1Tetracycline-inducible promoterActivationCellsApical membraneATPase activityPrevious studiesATPase functionChronic