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
2010
Phosphorylation 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
Sites 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
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
Activation 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
1996
Regulatory phosphorylation of the secretory Na-K-Cl cotransporter: modulation by cytoplasmic Cl
Lytle C, Forbush B. Regulatory phosphorylation of the secretory Na-K-Cl cotransporter: modulation by cytoplasmic Cl. American Journal Of Physiology 1996, 270: c437-c448. PMID: 8779905, DOI: 10.1152/ajpcell.1996.270.2.c437.Peer-Reviewed Original ResearchConceptsNa-K-Cl cotransporterNa-K-ClCotransport activityNa-K-Cl cotransport proteinResponse to cell shrinkageIncreased cotransport activityExtracellular K concentrationBlocking activityRefractory to forskolinPhosphatase inhibitor calyculin AInhibitor calyculin AShark rectal glandCotransporter proteinCytoplasmic Cl-CotransporterCell ClSecretory stimuliCell shrinkageCalyculin APhosphorylationRate of entryRegulatory phosphorylationPhosphorylation stateSecretory tubulesRectal gland
1994
Kinetic heterogeneity of phosphoenzyme of Na,K-ATPase modeled by unmixed lipid phases. Competence of the phosphointermediate.
Klodos I, Post R, Forbush B. Kinetic heterogeneity of phosphoenzyme of Na,K-ATPase modeled by unmixed lipid phases. Competence of the phosphointermediate. Journal Of Biological Chemistry 1994, 269: 1734-1743. PMID: 8294422, DOI: 10.1016/s0021-9258(17)42089-8.Peer-Reviewed Original Research
1992
The Na-K-Cl cotransport protein of shark rectal gland. II. Regulation by direct phosphorylation.
Lytle C, Forbush B. The Na-K-Cl cotransport protein of shark rectal gland. II. Regulation by direct phosphorylation. Journal Of Biological Chemistry 1992, 267: 25438-25443. PMID: 1334094, DOI: 10.1016/s0021-9258(19)74060-5.Peer-Reviewed Original ResearchMeSH KeywordsAmino Acid SequenceAnimalsCarrier ProteinsChromatography, High Pressure LiquidCyclic AMPDogfishHypertonic SolutionsIsoquinolinesKineticsMembrane ProteinsMolecular Sequence DataPeptide FragmentsPhosphorylationProtein Kinase InhibitorsRectumSebaceous GlandsSodium-Potassium-Chloride SymportersSulfanilamidesThionucleotidesConceptsNa-K-Cl cotransport proteinNa-K-ClCotransporter proteinShark rectal glandCell shrinkageCAMP-dependent secretagoguesRectal gland tubulesOsmotically induced changesCotransporter phosphorylationCotransport activityKinase inhibitorsProtein kinase inhibitorsCotransporterPhosphorylation stateRectal glandActive stateH-8K-252aTubulesGlandPhosphorylationCDNA analysisCellsIncreased 5Protein phosphorylation