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Structural basis for inhibition of the Cation-chloride cotransporter $$\mathrm{NKCC1}$$ by the diuretic drug bumetanide

Journal Article · · Nature Communications
 [1];  [2];  [3];  [3];  [3];  [2];  [4]
  1. University of Utah, Salt Lake City, UT (United States). School of Medicine; OSTI
  2. Yale University, New Haven, CT (United States). School of Medicine
  3. Istituto Italiano di Tecnologia, Genova (Italy)
  4. University of Utah, Salt Lake City, UT (United States). School of Medicine
+ Show Author Affiliations
Cation-chloride cotransporters (CCCs) NKCC1 and NKCC2 catalyze electroneutral symport of 1 Na+, 1K+, and 2 Cl- across cell membranes. NKCC1 mediates trans-epithelial Cl- secretion and regulates excitability of some neurons and NKCC2 is critical to renal salt reabsorption. Both transporters are inhibited by the so-called loop diuretics including bumetanide, and these drugs are a mainstay for treating edema and hypertension. Here, our single-particle electron cryo-microscopy structures supported by functional studies reveal an outward-facing conformation of NKCC1, showing bumetanide wedged into a pocket in the extracellular ion translocation pathway. Based on these and the previously published inward-facing structures, we define the translocation pathway and the conformational changes necessary for ion translocation. We also identify an NKCC1 dimer with separated transmembrane domains and extensive transmembrane and C-terminal domain interactions. We further define an N-terminal phosphoregulatory domain that interacts with the C-terminal domain, suggesting a mechanism whereby (de)phosphorylation regulates NKCC1 by tuning the strength of this domain association.
Research Organization:
University of Utah, Salt Lake City, UT (United States); Yale University, New Haven, CT (United States)
Sponsoring Organization:
NY State Assembly; National Institutes of Health (NIH); Pew Charitable Foundation; Simons Foundation; USDOE Office of Science (SC), Biological and Environmental Research (BER)
Grant/Contract Number:
AC05-76RL01830
OSTI ID:
1903925
Journal Information:
Nature Communications, Journal Name: Nature Communications Journal Issue: 1 Vol. 13; ISSN 2041-1723
Publisher:
Nature Publishing GroupCopyright Statement
Country of Publication:
United States
Language:
English

References (78)

Bumetanide-The Way to Its Chemical Structure journal November 1981
MolProbity: More and better reference data for improved all-atom structure validation: PROTEIN SCIENCE.ORG journal November 2017
An overview of the Amber biomolecular simulation package: Amber biomolecular simulation package
  • Salomon-Ferrer, Romelia; Case, David A.; Walker, Ross C.
  • Wiley Interdisciplinary Reviews: Computational Molecular Science, Vol. 3, Issue 2 https://doi.org/10.1002/wcms.1121
journal September 2012
Loop Diuretics in the Treatment of Hypertension journal March 2016
Cellular mechanism of action of loop diuretics: Implications for drug effectiveness and adverse effects journal January 1986
Numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes journal March 1977
Green fluorescent protein-based halide indicators with improved chloride and iodide affinities journal June 2001
[3H]bumetanide binding to membranes isolated from dog kidney outer medulla. Relationship to the Na,K,Cl co-transport system. journal October 1983
[3H]bumetanide binding to duck red cells. Correlation with inhibition of (Na + K + 2Cl) co-transport. journal June 1986
Ototoxicity of Loop Diuretics journal October 1993
Cation–chloride co-transporters in neuronal communication, development and trauma journal April 2003
Discovery of a Small Molecule Drug Candidate for Selective NKCC1 Inhibition in Brain Disorders journal August 2020
Loop diuretics are most appropriate for hypertension treatment in chronic kidney disease journal April 2016
Ototoxic effects and mechanisms of loop diuretics journal December 2016
RELION: Implementation of a Bayesian approach to cryo-EM structure determination journal December 2012
CTFFIND4: Fast and accurate defocus estimation from electron micrographs journal November 2015
Chloride transporters and GABA polarity in developmental, neurological and psychiatric conditions journal July 2018
Chloride Dysregulation, Seizures, and Cerebral Edema: A Relationship with Therapeutic Potential journal May 2017
The search for NKCC1-selective drugs for the treatment of epilepsy: Structure–function relationship of bumetanide and various bumetanide derivatives in inhibiting the human cation-chloride cotransporter NKCC1A journal June 2016
The search for brain-permeant NKCC1 inhibitors for the treatment of seizures: Pharmacokinetic-pharmacodynamic modelling of NKCC1 inhibition by azosemide, torasemide, and bumetanide in mouse brain journal January 2021
PACKMOL-Memgen: A Simple-To-Use, Generalized Workflow for Membrane-Protein–Lipid-Bilayer System Building journal May 2019
ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB journal July 2015
Design, Synthesis, In Vitro and In Vivo Characterization of Selective NKCC1 Inhibitors for the Treatment of Core Symptoms in Down Syndrome journal June 2021
PROPKA3: Consistent Treatment of Internal and Surface Residues in Empirical p K a Predictions journal December 2010
Lipid14: The Amber Lipid Force Field journal January 2014
Aminobenzoic acid diuretics. 5. 3-Amino-4-arylmethyl-5-sulfamylbenzoic acid derivatives and related compounds journal October 1973
Determination of Alkali and Halide Monovalent Ion Parameters for Use in Explicitly Solvated Biomolecular Simulations journal July 2008
Deafness and imbalance associated with inactivation of the secretory Na-K-2Cl co-transporter journal June 1999
Comparison of loop diuretics in patients with chronic renal insufficiency journal October 1987
Mutations in kelch-like 3 and cullin 3 cause hypertension and electrolyte abnormalities journal January 2012
NKCC1 transporter facilitates seizures in the developing brain journal October 2005
cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination journal February 2017
MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy journal February 2017
Screening and large-scale expression of membrane proteins in mammalian cells for structural studies journal October 2014
Cryo-EM structures of the full-length human KCC2 and KCC3 cation-chloride cotransporters journal November 2020
Structure of the human cation–chloride cotransporter NKCC1 determined by single-particle electron cryo-microscopy journal February 2020
Structure and mechanism of the cation–chloride cotransporter NKCC1 journal July 2019
Positive-unlabeled convolutional neural networks for particle picking in cryo-electron micrographs journal October 2019
Azosemide is more potent than bumetanide and various other loop diuretics to inhibit the sodium-potassium-chloride-cotransporter human variants hNKCC1A and hNKCC1B journal June 2018
The structural basis of function and regulation of neuronal cotransporters NKCC1 and KCC2 journal February 2021
Comparison of simple potential functions for simulating liquid water journal July 1983
Particle mesh Ewald: An N ⋅log( N ) method for Ewald sums in large systems journal June 1993
Mice Lacking the Basolateral Na-K-2Cl Cotransporter Have Impaired Epithelial Chloride Secretion and Are Profoundly Deaf journal September 1999
Clustering of Neuronal K+-Cl− Cotransporters in Lipid Rafts by Tyrosine Phosphorylation journal October 2009
Regulatory Activation Is Accompanied by Movement in the C Terminus of the Na-K-Cl Cotransporter (NKCC1) journal January 2012
Loop Diuretic and Ion-binding Residues Revealed by Scanning Mutagenesis of Transmembrane Helix 3 (TM3) of Na-K-Cl Cotransporter (NKCC1) journal May 2012
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 March 2014
A Regulatory Locus of Phosphorylation in the N Terminus of the Na-K-Cl Cotransporter, NKCC1 journal October 2002
Activation of the Na-K-Cl Cotransporter NKCC1 Detected with a Phospho-specific Antibody journal October 2002
Identification of a Functionally Important Conformation-sensitive Region of the Secretory Na+-K+-2Cl− Cotransporter (NKCC1) journal April 2003
Stock-based detection of protein oligomeric states in jsPISA journal April 2015
Bumetanide to Treat Parkinson Disease journal January 2016
PHENIX: a comprehensive Python-based system for macromolecular structure solution journal January 2010
Features and development of Coot journal March 2010
A Bayesian approach to beam-induced motion correction in cryo-EM single-particle analysis journal January 2019
Bumetanide reduces seizure frequency in patients with temporal lobe epilepsy journal October 2012
Thiazide and Loop Diuretics journal July 2011
Cl−uptake promoting depolarizing GABA actions in immature rat neocortical neurones is mediated by NKCC1 journal June 2004
How LeuT shapes our understanding of the mechanisms of sodium-coupled neurotransmitter transporters: Role of LeuT in understanding neurotransmitter transporters journal August 2013
Structures and an activation mechanism of human potassium-chloride cotransporters journal December 2020
Human Hypertension Caused by Mutations in WNK Kinases journal August 2001
Cryo-EM structures of the human cation-chloride cotransporter KCC1 journal October 2019
The WNK-SPAK/OSR1 pathway: Master regulator of cation-chloride cotransporters journal July 2014
Shared Molecular Mechanisms of Membrane Transporters journal June 2016
On the substrate recognition and negative regulation of SPAK, a kinase modulating Na + -K + -2Cl − cotransport activity journal September 2010
Na-K-Cl cotransport in the shark rectal gland. I. Regulation in the intact perfused gland journal April 1992
A model of Na-K-2Cl cotransport based on ordered ion binding and glide symmetry journal February 1998
Loop diuretics: from the Na-K-2Cl transporter to clinical use journal January 2003
Regulation of the renal NaCl cotransporter by the WNK/SPAK pathway: lessons learned from genetically altered animals journal January 2019
Decreased Seizure Activity in a Human Neonate Treated With Bumetanide, an Inhibitor of the Na+-K+-2Cl- Cotransporter NKCC1 journal April 2009
A Sensitive Membrane-Targeted Biosensor for Monitoring Changes in Intracellular Chloride in Neuronal Processes journal April 2012
Functional Expression of Human NKCC1 from a Synthetic Cassette-Based cDNA: Introduction of Extracellular Epitope Tags and Removal of Cysteines journal December 2013
Oligomerization of KCC2 Correlates with Development of Inhibitory Neurotransmission journal October 2006
Phospho‐regulation, nucleotide binding and ion access control in potassium‐chloride cotransporters journal May 2021
How Do Loop Diuretics Act? journal January 1991
Off-Label Use of Bumetanide for Brain Disorders: An Overview journal April 2019
Loop Diuretics in Clinical Practice journal January 2015
Cryo-EM structure of the potassium-chloride cotransporter KCC4 in lipid nanodiscs journal April 2020

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Related Subjects

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
59 BASIC BIOLOGICAL SCIENCES
cryoelectron microscopy
ion channels
membrane proteins
permeation and transport