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- EMDB-45081: Phosphorylated human NKCC1_K289NA492E in complex with furosemide -

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Basic information

Entry
Database: EMDB / ID: EMD-45081
TitlePhosphorylated human NKCC1_K289NA492E in complex with furosemide
Map dataPhosphorylated human NKCC1_K289NA492E in complex with furosemide
Sample
  • Complex: Phosphorylated NKCC1_K289NA492E in complex with furosemide
    • Protein or peptide: Solute carrier family 12 member 2
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: 5-(AMINOSULFONYL)-4-CHLORO-2-[(2-FURYLMETHYL)AMINO]BENZOIC ACID
  • Ligand: POTASSIUM ION
  • Ligand: CHLORIDE ION
  • Ligand: SODIUM ION
  • Ligand: MAGNESIUM ION
KeywordsSodium potassium chloride cotransporter / phosphorylation / outward-open state / furosemide / 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 / ammonium transmembrane transport / negative regulation of vascular wound healing / 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 / basal plasma membrane / cytoplasmic vesicle 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 / SLC12A transporter family / Amino acid permease/ SLC12A domain / 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: 2.7 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

Supplemental images

emd_45081.png

Downloads & links

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Map

FileDownload / File: emd_45081.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPhosphorylated human NKCC1_K289NA492E in complex with furosemide
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.83 Å/pix.
x 360 pix.
= 297.216 Å		0.83 Å/pix.
x 360 pix.
= 297.216 Å		0.83 Å/pix.
x 360 pix.
= 297.216 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8256 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.18
Minimum - Maximum-0.43376985 - 1.0456535
Average (Standard dev.)0.0005536064 (±0.024893336)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 297.216 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Additional Map

Fileemd_45081_additional_1.map
AnnotationAdditional Map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: Half Map B

Fileemd_45081_half_map_1.map
AnnotationHalf Map B
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map A

Fileemd_45081_half_map_2.map
AnnotationHalf Map A
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Phosphorylated NKCC1_K289NA492E in complex with furosemide

EntireName: Phosphorylated NKCC1_K289NA492E in complex with furosemide
Components
  • Complex: Phosphorylated NKCC1_K289NA492E in complex with furosemide
    • Protein or peptide: Solute carrier family 12 member 2
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: 5-(AMINOSULFONYL)-4-CHLORO-2-[(2-FURYLMETHYL)AMINO]BENZOIC ACID
  • Ligand: POTASSIUM ION
  • Ligand: CHLORIDE ION
  • Ligand: SODIUM ION
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Phosphorylated NKCC1_K289NA492E in complex with furosemide

SupramoleculeName: Phosphorylated NKCC1_K289NA492E in complex with furosemide
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: 131.866328 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 H (TPO)AAQLGEK LLRPSLAELH DELEKEPFED GFANGEESTP TRDAVVTYTA ESKGVVKFGW INGVLVRCML NIWGVMLF I RLSWIVGQAG IGLSVLVIMM ATVVTTITGL STSAIATNGF VRGGGAYYLI SRSLGPEFGG AIGLIFAFAN AVAVAMYVV GFAETVVELL KEHSILMIDE INDIRIIGAI TVVILLGISV AGMEWEAKAQ IVLLVILLLA IGDFVIGTFI PLESKKPKGF FGYKSEIFN ENFGPDFREE ETFFSVFEIF FPAATGILAG ANISGDLADP QSAIPKGTLL AILITTLVYV GIAVSVGSCV V RDATGNVN DTIVTELTNC TSAACKLNFD FSSCESSPCS YGLMNNFQVM SMVSGFTPLI SAGIFSATLS SALASLVSAP KI FQALCKD NIYPAFQMFA KGYGKNNEPL RGYILTFLIA LGFILIAELN VIAPIISNFF LASYALINFS VFHASLAKSP GWR PAFKYY NMWISLLGAI LCCIVMFVIN WWAALLTYVI VLGLYIYVTY KKPDVNWGSS TQALTYLNAL QHSIRLSGVE DHVK NFRPQ CLVMTGAPNS RPALLHLVHD FTKNVGLMIC GHVHMGPRRQ AMKEMSIDQA KYQRWLIKNK MKAFYAPVHA DDLRE GAQY LMQAAGLGRM KPNTLVLGFK KDWLQADMRD VDMYINLFHD AFDIQYGVVV IRLKEGLDIS HLQGQEELLS SQEKSP GTK DVVVSVEYSK KSDLDTSKPL SEKPITHKVE EEDGKTATQP LLKKESKGPI VPLNVADQKL LEASTQFQKK QGKNTID VW WLFDDGGLTL LIPYLLTTKK KWKDCKIRVF IGGKINRIDH DRRAMATLLS KFRIDFSDIM VLGDINTKPK KENIIAFE E IIEPYRLHED DKEQDIADKM KEDEPWRITD NELELYKTKT YRQIRLNELL KEHSSTANII VMSLPVARKG AVSSALYMA WLEALSKDLP PILLVRGNHQ SVLTFYS

UniProtKB: Solute carrier family 12 member 2

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Macromolecule #2: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 2 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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Macromolecule #3: 5-(AMINOSULFONYL)-4-CHLORO-2-[(2-FURYLMETHYL)AMINO]BENZOIC ACID

MacromoleculeName: 5-(AMINOSULFONYL)-4-CHLORO-2-[(2-FURYLMETHYL)AMINO]BENZOIC ACID
type: ligand / ID: 3 / Number of copies: 2 / Formula: FUN
Molecular weightTheoretical: 330.744 Da
Chemical component information

ChemComp-FUN:
5-(AMINOSULFONYL)-4-CHLORO-2-[(2-FURYLMETHYL)AMINO]BENZOIC ACID

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Macromolecule #4: POTASSIUM ION

MacromoleculeName: POTASSIUM ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: K
Molecular weightTheoretical: 39.098 Da

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Macromolecule #5: CHLORIDE ION

MacromoleculeName: CHLORIDE ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: CL
Molecular weightTheoretical: 35.453 Da

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Macromolecule #6: SODIUM ION

MacromoleculeName: SODIUM ION / type: ligand / ID: 6 / Number of copies: 2
Molecular weightTheoretical: 22.99 Da

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Macromolecule #7: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 7 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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 MORGAGNI
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 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: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 8051818
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION

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