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Structure of the human cation–chloride cotransport KCC1 in an outward-open state

Journal Article · · Proceedings of the National Academy of Sciences of the United States of America
Cation–chloride cotransporters (CCCs) catalyze electroneutral symport of Cl-with Na+and/or K+across membranes. CCCs are fundamental in cell volume homeostasis, transepithelia ion movement, maintenance of intracellular Cl-concentration, and neuronal excitability. Here, we present a cryoelectron microscopy structure of human K+–Cl-cotransporter (KCC)1 bound with the VU0463271 inhibitor in an outward-open state. In contrast to many other amino acid–polyamine–organocation transporter cousins, our first outward-open CCC structure reveals that opening the KCC1 extracellular ion permeation path does not involve hinge-bending motions of the transmembrane (TM) 1 and TM6 half-helices. Instead, rocking of TM3 and TM8, together with displacements of TM4, TM9, and a conserved intracellular loop 1 helix, underlie alternate opening and closing of extracellular and cytoplasmic vestibules. We show that KCC1 intriguingly exists in one of two distinct dimeric states via different intersubunit interfaces. Our studies provide a blueprint for understanding the mechanisms of CCCs and their inhibition by small molecule compounds.
Research Organization:
Pacific Northwest National Laboratory (PNNL), Richland, WA (United States). Environmental Molecular Sciences Laboratory (EMSL); University of Utah School of Medicine, Salt Lake City, UT (United States)
Sponsoring Organization:
National Institutes of Health (NIH); USDOE
Grant/Contract Number:
AC05-76RL01830
OSTI ID:
1982396
Journal Information:
Proceedings of the National Academy of Sciences of the United States of America, Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Issue: 27 Vol. 119; ISSN 0027-8424
Publisher:
National Academy of SciencesCopyright Statement
Country of Publication:
United States
Language:
English

References (79)

A variant of KCC 2 from patients with febrile seizures impairs neuronal Cl extrusion and dendritic spine formation journal March 2014
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
Physiology and pathophysiology of SLC12A1/2 transporters journal October 2013
Thiazide Diuretics in Chronic Kidney Disease journal March 2015
Loop Diuretics in the Treatment of Hypertension journal March 2016
Numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes journal March 1977
Ototoxicity of Loop Diuretics journal October 1993
Genetic disruption of KCC cotransporters in a mouse model of thalassemia intermedia journal March 2020
Further optimization of the K-Cl cotransporter KCC2 antagonist ML077: Development of a highly selective and more potent in vitro probe journal July 2012
Sites of Regulated Phosphorylation that Control K-Cl Cotransporter Activity journal August 2009
Reactivation of Dormant Relay Pathways in Injured Spinal Cord by KCC2 Manipulations journal September 2018
SPFP: Speed without compromise—A mixed precision model for GPU accelerated molecular dynamics simulations journal February 2013
Automated electron microscope tomography using robust prediction of specimen movements journal October 2005
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
K-Cl cotransporters, cell volume homeostasis, and neurological disease journal August 2015
Cation-chloride cotransporters NKCC1 and KCC2 as potential targets for novel antiepileptic and antiepileptogenic treatments journal June 2013
Chloride Dysregulation, Seizures, and Cerebral Edema: A Relationship with Therapeutic Potential journal May 2017
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
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
Glide:  A New Approach for Rapid, Accurate Docking and Scoring. 1. Method and Assessment of Docking Accuracy journal March 2004
Extra Precision Glide:  Docking and Scoring Incorporating a Model of Hydrophobic Enclosure for Protein−Ligand Complexes journal October 2006
Determination of Alkali and Halide Monovalent Ion Parameters for Use in Explicitly Solvated Biomolecular Simulations journal July 2008
The mechanism of sodium and substrate release from the binding pocket of vSGLT journal December 2010
X-ray structures of LeuT in substrate-free outward-open and apo inward-open states journal January 2012
X-ray structures and mechanism of the human serotonin transporter journal April 2016
Mutations in SLC12A5 in epilepsy of infancy with migrating focal seizures journal September 2015
Roles of the cation–chloride cotransporters in neurological disease journal September 2008
The K–Cl cotransporter KCC3 is mutant in a severe peripheral neuropathy associated with agenesis of the corpus callosum journal October 2002
Chloride extrusion enhancers as novel therapeutics for neurological diseases journal October 2013
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
Serotonin transporter–ibogaine complexes illuminate mechanisms of inhibition and transport journal April 2019
Structure and mechanism of the cation–chloride cotransporter NKCC1 journal July 2019
The structural basis of function and regulation of neuronal cotransporters NKCC1 and KCC2 journal February 2021
Rhodol-based thallium sensors for cellular imaging of potassium channel activity journal January 2018
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
Small-molecule screen identifies inhibitors of the neuronal K-Cl cotransporter KCC2 journal March 2009
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
The Residues Determining Differences in Ion Affinities among the Alternative Splice Variants F, A, and B of the Mammalian Renal Na-K-Cl Cotransporter (NKCC2) journal March 2007
The KCC3 cotransporter as a therapeutic target for peripheral neuropathy journal January 2017
HMSN/ACC truncation mutations disrupt brain-type creatine kinase-dependant activation of K+/Cl− co-transporter 3 journal June 2008
Stock-based detection of protein oligomeric states in jsPISA journal April 2015
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
A de novo missense mutation inSLC12A5found in a compound heterozygote patient with epilepsy of infancy with migrating focal seizures journal September 2017
Brain-type creatine kinase activates neuron-specific K+-Cl-co-transporter KCC2 journal January 2006
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
A Competitive Inhibitor Traps LeuT in an Open-to-Out Conformation journal December 2008
Clustering by fast search and find of density peaks journal June 2014
Cryo-EM structures of the human cation-chloride cotransporter KCC1 journal October 2019
De novo variants in SLC12A6 cause sporadic early-onset progressive sensorimotor neuropathy journal August 2019
Shared Molecular Mechanisms of Membrane Transporters journal June 2016
Common Folds and Transport Mechanisms of Secondary Active Transporters journal May 2013
Physiology of SLC12 transporters: lessons from inherited human genetic mutations and genetically engineered mouse knockouts journal April 2013
Erythrocyte K-Cl cotransport: properties and regulation journal November 1992
Rare mutations in the human Na-K-Cl cotransporter (NKCC2) associated with lower blood pressure exhibit impaired processing and transport function journal April 2011
Molecular Physiology and Pathophysiology of Electroneutral Cation-Chloride Cotransporters journal April 2005
Sodium-Potassium-Chloride Cotransport journal January 2000
K-Cl Cotransport: Properties and Molecular Mechanism journal January 2000
Activation of the erythroid K-Cl cotransporter Kcc1 enhances sickle cell disease pathology in a humanized mouse model journal December 2015
Novel truncating and missense mutations of the KCC3 gene associated with Andermann syndrome journal April 2006
A Sensitive Membrane-Targeted Biosensor for Monitoring Changes in Intracellular Chloride in Neuronal Processes journal April 2012
Phospho‐regulation, nucleotide binding and ion access control in potassium‐chloride cotransporters journal May 2021
Genetically encoded impairment of neuronal KCC 2 cotransporter function in human idiopathic generalized epilepsy journal June 2014
Bumetanide A Review of its Pharmacodynamic and Pharmacokinetic Properties and Therapeutic Use journal November 1984
Regulatory domain or CpG site variation in SLC12A5, encoding the chloride transporter KCC2, in human autism and schizophrenia journal October 2015
Identification of KCC2 Mutations in Human Epilepsy Suggests Strategies for Therapeutic Transporter Modulation journal November 2019
Cryo-EM structure of the potassium-chloride cotransporter KCC4 in lipid nanodiscs journal April 2020