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- PDB-9c0h: Phosphorylated human NKCC1 in complex with bumetanide -

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Basic information

Entry
Database: PDB / ID: 9c0h
TitlePhosphorylated human NKCC1 in complex with bumetanide
ComponentsSolute carrier family 12 member 2
KeywordsTRANSPORT PROTEIN / Phosphorylation / sodium potassium chloride cotransporter / outward-open / bumetanide
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 / transepithelial chloride transport / Cation-coupled Chloride cotransporters / potassium ion transmembrane transporter activity / 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 potassium ion / T cell chemotaxis / cellular response to chemokine / potassium ion homeostasis / intracellular sodium ion homeostasis / hyperosmotic response / sodium ion import across plasma membrane / intracellular potassium ion homeostasis / cell volume homeostasis / regulation of spontaneous synaptic transmission / gamma-aminobutyric acid signaling pathway / maintenance of blood-brain barrier / potassium ion import across plasma membrane / lateral plasma membrane / transport across blood-brain barrier / monoatomic ion transport / sodium ion transmembrane transport / cytoplasmic vesicle membrane / chloride transmembrane transport / basal plasma membrane / 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
3-(butylamino)-4-phenoxy-5-sulfamoylbenzoic acid / : / Solute carrier family 12 member 2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsZhao, Y.X. / Cao, E.H.
Funding support United States, 1items
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, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 12, 2025Provider: repository / Type: Initial release
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Revision 1.1Feb 19, 2025Group: Data collection / Database references / Category: citation / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Solute carrier family 12 member 2
B: Solute carrier family 12 member 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)264,41110
Polymers263,4872
Non-polymers9248
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Solute carrier family 12 member 2 / Basolateral Na-K-Cl symporter / Bumetanide-sensitive sodium-(potassium)-chloride cotransporter 2


Mass: 131743.391 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC12A2, NKCC1 / Production host: Homo sapiens (human) / References: UniProt: P55011
#2: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: K
#3: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl
#4: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Na
#5: Chemical ChemComp-82U / 3-(butylamino)-4-phenoxy-5-sulfamoylbenzoic acid


Mass: 364.416 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C17H20N2O5S / Comment: medication*YM
Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Phosphorylated human NKCC1 in complex with bumetanide / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: FEI TECNAI 20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3500 nm / Nominal defocus min: 700 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1694969 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00413430
ELECTRON MICROSCOPYf_angle_d0.62318266
ELECTRON MICROSCOPYf_dihedral_angle_d7.7691922
ELECTRON MICROSCOPYf_chiral_restr0.0422108
ELECTRON MICROSCOPYf_plane_restr0.0052268

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