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- EMDB-45084: Phosphorylated human NKCC1 in complex with bumetanide -

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Basic information

Entry
Database: EMDB / ID: EMD-45084
TitlePhosphorylated human NKCC1 in complex with bumetanide
Map dataAdditional Map
Sample
  • Complex: Phosphorylated human NKCC1 in complex with bumetanide
    • Protein or peptide: Solute carrier family 12 member 2
  • Ligand: POTASSIUM ION
  • Ligand: CHLORIDE ION
  • Ligand: SODIUM ION
  • Ligand: 3-(butylamino)-4-phenoxy-5-sulfamoylbenzoic acid
KeywordsPhosphorylation / sodium potassium chloride cotransporter / outward-open / bumetanide / TRANSPORT PROTEIN
Function / homology
Function and homology information


positive regulation of cell volume / positive regulation of aspartate secretion / transepithelial ammonium transport / regulation of matrix metallopeptidase secretion / cell body membrane / metal ion transmembrane transporter activity / inorganic anion import across plasma membrane / inorganic cation import across plasma membrane / chloride:monoatomic cation symporter activity / sodium:potassium:chloride symporter activity ...positive regulation of cell volume / positive regulation of aspartate secretion / transepithelial ammonium transport / regulation of matrix metallopeptidase secretion / cell body membrane / metal ion transmembrane transporter activity / inorganic anion import across plasma membrane / inorganic cation import across plasma membrane / chloride:monoatomic cation symporter activity / sodium:potassium:chloride symporter activity / Cation-coupled Chloride cotransporters / potassium ion transmembrane transporter activity / transepithelial chloride transport / intracellular chloride ion homeostasis / negative regulation of vascular wound healing / ammonium transmembrane transport / sodium ion homeostasis / ammonium channel activity / chloride ion homeostasis / cell projection membrane / cellular response to chemokine / T cell chemotaxis / potassium ion homeostasis / intracellular sodium ion homeostasis / sodium ion import across plasma membrane / cell volume homeostasis / cellular response to potassium ion / hyperosmotic response / regulation of spontaneous synaptic transmission / gamma-aminobutyric acid signaling pathway / maintenance of blood-brain barrier / potassium ion import across plasma membrane / intracellular potassium ion homeostasis / lateral plasma membrane / transport across blood-brain barrier / monoatomic ion transport / sodium ion transmembrane transport / cytoplasmic vesicle membrane / basal plasma membrane / chloride transmembrane transport / cell periphery / cell projection / Hsp90 protein binding / extracellular vesicle / protein-folding chaperone binding / cell body / basolateral plasma membrane / neuron projection / apical plasma membrane / neuronal cell body / protein kinase binding / extracellular exosome / membrane / plasma membrane
Similarity search - Function
Solute carrier family 12 member 1/2 / Solute carrier family 12 member 2 / Amino acid permease, N-terminal / Amino acid permease N-terminal / SLC12A transporter, C-terminal / Solute carrier family 12 / Amino acid permease/ SLC12A domain / SLC12A transporter family / Amino acid permease
Similarity search - Domain/homology
Solute carrier family 12 member 2
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsZhao YX / Cao EH
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)5R01DK128592 United States
CitationJournal: EMBO J / Year: 2025
Title: Structural basis for human NKCC1 inhibition by loop diuretic drugs.
Authors: Yongxiang Zhao / Pietro Vidossich / Biff Forbush / Junfeng Ma / Jesse Rinehart / Marco De Vivo / Erhu Cao /
Abstract: Na-K-Cl cotransporters functions as an anion importers, regulating trans-epithelial chloride secretion, cell volume, and renal salt reabsorption. Loop diuretics, including furosemide, bumetanide, and ...Na-K-Cl cotransporters functions as an anion importers, regulating trans-epithelial chloride secretion, cell volume, and renal salt reabsorption. Loop diuretics, including furosemide, bumetanide, and torsemide, antagonize both NKCC1 and NKCC2, and are first-line medicines for the treatment of edema and hypertension. NKCC1 activation by the molecular crowding sensing WNK kinases is critical if cells are to combat shrinkage during hypertonic stress; however, how phosphorylation accelerates NKCC1 ion transport remains unclear. Here, we present co-structures of phospho-activated NKCC1 bound with furosemide, bumetanide, or torsemide showing that furosemide and bumetanide utilize a carboxyl group to coordinate and co-occlude a K, whereas torsemide encroaches and expels the K from the site. We also found that an amino-terminal segment of NKCC1, once phosphorylated, interacts with the carboxyl-terminal domain, and together, they engage with intracellular ion exit and appear to be poised to facilitate rapid ion translocation. Together, these findings enhance our understanding of NKCC-mediated epithelial ion transport and the molecular mechanisms of its inhibition by loop diuretics.
History
DepositionMay 25, 2024-
Header (metadata) releaseFeb 12, 2025-
Map releaseFeb 12, 2025-
UpdateMay 14, 2025-
Current statusMay 14, 2025Processing site: RCSB / Status: Released

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Structure visualization

Downloads & links

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Map

FileDownload / File: emd_45084.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAdditional Map
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.33
Minimum - Maximum-0.6770213 - 1.4058706
Average (Standard dev.)0.001306137 (±0.031726956)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 317.99997 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Phosphorylated human NKCC1 in complex with bumetanide

EntireName: Phosphorylated human NKCC1 in complex with bumetanide
Components
  • Complex: Phosphorylated human NKCC1 in complex with bumetanide
    • Protein or peptide: Solute carrier family 12 member 2
  • Ligand: POTASSIUM ION
  • Ligand: CHLORIDE ION
  • Ligand: SODIUM ION
  • Ligand: 3-(butylamino)-4-phenoxy-5-sulfamoylbenzoic acid

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Supramolecule #1: Phosphorylated human NKCC1 in complex with bumetanide

SupramoleculeName: Phosphorylated human NKCC1 in complex with bumetanide / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Solute carrier family 12 member 2

MacromoleculeName: Solute carrier family 12 member 2 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 131.743391 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MEPRPTAPSS GAPGLAGVGE TPSAAALAAA RVELPGTAVP SVPEDAAPAS RDGGGVRDEG PAAAGDGLGR PLGPTPSQSR FQVDLVSEN AGRAAAAAAA AAAAAAAAGA GAGAKQTPAD GEASGESEPA KGSEEAKGRF RVNFVDPAAS SSAEDSLSDA A GVGVDGPN ...String:
MEPRPTAPSS GAPGLAGVGE TPSAAALAAA RVELPGTAVP SVPEDAAPAS RDGGGVRDEG PAAAGDGLGR PLGPTPSQSR FQVDLVSEN AGRAAAAAAA AAAAAAAAGA GAGAKQTPAD GEASGESEPA KGSEEAKGRF RVNFVDPAAS SSAEDSLSDA A GVGVDGPN VSFQNGGDTV LSEGSSLHSG GGGGSGHHQH YYYDTHTNTY YLR(TPO)FGHN(TPO)M DAVPRIDHYR HTAA QLGEK LLRPSLAELH DELEKEPFED GFANGEESTP TRDAVVTYTA ESKGVVKFGW IKGVLVRCML NIWGVMLFIR LSWIV GQAG IGLSVLVIMM ATVVTTITGL STSAIATNGF VRGGGAYYLI SRSLGPEFGG AIGLIFAFAN AVAVAMYVVG FAETVV ELL KEHSILMIDE INDIRIIGAI TVVILLGISV AGMEWEAKAQ IVLLVILLLA IGDFVIGTFI PLESKKPKGF FGYKSEI FN ENFGPDFREE ETFFSVFAIF FPAATGILAG ANISGDLADP QSAIPKGTLL AILITTLVYV GIAVSVGSCV VRDATGNV N DTIVTELTNC TSAACKLNFD FSSCESSPCS YGLMNNFQVM SMVSGFTPLI SAGIFSATLS SALASLVSAP KIFQALCKD NIYPAFQMFA KGYGKNNEPL RGYILTFLIA LGFILIAELN VIAPIISNFF LASYALINFS VFHASLAKSP GWRPAFKYYN MWISLLGAI LCCIVMFVIN WWAALLTYVI VLGLYIYVTY KKPDVNWGSS TQALTYLNAL QHSIRLSGVE DHVKNFRPQC L VMTGAPNS RPALLHLVHD FTKNVGLMIC GHVHMGPRRQ AMKEMSIDQA KYQRWLIKNK MKAFYAPVHA DDLREGAQYL MQ AAGLGRM KPNTLVLGFK KDWLQADMRD VDMYINLFHD AFDIQYGVVV IRLKEGLDIS HLQGQEELLS SQEKSPGTKD VVV SVEYSK KSDLDTSKPL SEKPITHKVE EEDGKTATQP LLKKESKGPI VPLNVADQKL LEASTQFQKK QGKNTIDVWW LFDD GGLTL LIPYLLTTKK KWKDCKIRVF IGGKINRIDH DRRAMATLLS KFRIDFSDIM VLGDINTKPK KENIIAFEEI IEPYR LHED DKEQDIADKM KEDEPWRITD NELELYKTKT YRQIRLNELL KEHSSTANII VMSLPVARKG AVSSALYMAW LEALSK DLP PILLVRGNHQ SVLTFYS

UniProtKB: Solute carrier family 12 member 2

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Macromolecule #2: POTASSIUM ION

MacromoleculeName: POTASSIUM ION / type: ligand / ID: 2 / Number of copies: 2 / Formula: K
Molecular weightTheoretical: 39.098 Da

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Macromolecule #3: CHLORIDE ION

MacromoleculeName: CHLORIDE ION / type: ligand / ID: 3 / Number of copies: 2 / Formula: CL
Molecular weightTheoretical: 35.453 Da

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Macromolecule #4: SODIUM ION

MacromoleculeName: SODIUM ION / type: ligand / ID: 4 / Number of copies: 2
Molecular weightTheoretical: 22.99 Da

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Macromolecule #5: 3-(butylamino)-4-phenoxy-5-sulfamoylbenzoic acid

MacromoleculeName: 3-(butylamino)-4-phenoxy-5-sulfamoylbenzoic acid / type: ligand / ID: 5 / Number of copies: 2 / Formula: 82U
Molecular weightTheoretical: 364.416 Da
Chemical component information

ChemComp-82U:
3-(butylamino)-4-phenoxy-5-sulfamoylbenzoic acid / medication*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TECNAI 20
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.5 µm / Nominal defocus min: 0.7000000000000001 µm

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 1694969
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION

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