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
2011
Rare 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
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
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 transportComparison of Na-K-Cl Cotransporters NKCC1, NKCC2, AND THE HEK CELL Na-K-Cl COTRANSPORTER*
Isenring P, Jacoby S, Payne J, Forbush B. Comparison of Na-K-Cl Cotransporters NKCC1, NKCC2, AND THE HEK CELL Na-K-Cl COTRANSPORTER*. Journal Of Biological Chemistry 1998, 273: 11295-11301. PMID: 9556622, DOI: 10.1074/jbc.273.18.11295.Peer-Reviewed Original ResearchConceptsNa-K-Cl cotransporterHEK-293 cellsNa-K-Cl cotransporter NKCC1Epithelial cellsBasolateral Cl- exitHEK-293Reverse transcriptase-polymerase chain reactionNa-K-ClRenal epithelial cellsEpithelial Cl- transportResting level of activityMaintenance of cell volumeSecretory epithelial cellsInhibitor bumetanideCotransporter NKCC1NKCC2 activityCl- exitNKCC1Cl- transportNKCC2CotransporterVolume responseNa affinityResting levelBumetanide
1997
Ion and Bumetanide Binding by the Na-K-Cl Cotransporter
Isenring P, Forbush B. Ion and Bumetanide Binding by the Na-K-Cl Cotransporter. Journal Of Biological Chemistry 1997, 272: 24556-24562. PMID: 9305921, DOI: 10.1074/jbc.272.39.24556.Peer-Reviewed Original ResearchConceptsNa-K-Cl cotransporterNa-K-ClBumetanide-sensitive 86Rb influxCation-chloride cotransporter familyApparent affinityHEK-293 cellsInhibitor bumetanideCell surface deliveryElectrolyte secretionTime course of activationBumetanidePolarized epitheliaBumetanide bindingCotransporterCl- mediumCotransported ionsHEK-293SNKCC1Hydrophobic central domainSurface deliveryCourse of activationCentral domainTime courseCytoplasmic N-Chimera approach
1994
Molecular cloning and functional expression of the bumetanide-sensitive Na-K-Cl cotransporter.
Xu J, Lytle C, Zhu T, Payne J, Benz E, Forbush B. Molecular cloning and functional expression of the bumetanide-sensitive Na-K-Cl cotransporter. Proceedings Of The National Academy Of Sciences Of The United States Of America 1994, 91: 2201-2205. PMID: 8134373, PMCID: PMC43338, DOI: 10.1073/pnas.91.6.2201.Peer-Reviewed Original ResearchConceptsNa-K-Cl cotransporterBumetanide-sensitive Na-K-Cl cotransporterNa-K-ClNa-K-Cl cotransport activityNa-K-Cl cotransport proteinThiazide-sensitive Na-Cl cotransporterCoupled movement of NaUncharacterized genesShort stretches of homologyRegulation of ionic balanceStretches of homologyC. elegans cDNAHEK-293 cellsHuman HEK-293 cellsPutative transmembrane domainsBumetanide sensitivityCotransport activityCotransporter proteinMovement of NaUrinary bladderAbsorptive epitheliaNorthern blot analysisPhosphoacceptor residuesCDNA sequenceCoding region