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- EMDB-10704: Structure of Human Potassium Chloride Transporter KCC3b (S45D/T94... -

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Basic information

Entry
Database: EMDB / ID: EMD-10704
TitleStructure of Human Potassium Chloride Transporter KCC3b (S45D/T940D/T997D) in KCl
Map data
Sample
  • Complex: Structure of Human Potassium Chloride Transporter KCC3b (S45D/T940D/T997D) in KCl
    • Protein or peptide: Solute carrier family 12 member 6
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Function / homology
Function and homology information


Defective SLC12A6 causes agenesis of the corpus callosum, with peripheral neuropathy (ACCPN) / ammonium import across plasma membrane / potassium ion transmembrane transporter activity / potassium:chloride symporter activity / Cation-coupled Chloride cotransporters / chloride ion homeostasis / cellular hypotonic response / ammonium transmembrane transporter activity / cellular hypotonic salinity response / potassium ion homeostasis ...Defective SLC12A6 causes agenesis of the corpus callosum, with peripheral neuropathy (ACCPN) / ammonium import across plasma membrane / potassium ion transmembrane transporter activity / potassium:chloride symporter activity / Cation-coupled Chloride cotransporters / chloride ion homeostasis / cellular hypotonic response / ammonium transmembrane transporter activity / cellular hypotonic salinity response / potassium ion homeostasis / cell volume homeostasis / potassium ion import across plasma membrane / monoatomic ion transport / potassium ion transmembrane transport / chloride transmembrane transport / cellular response to glucose stimulus / chemical synaptic transmission / basolateral plasma membrane / angiogenesis / axon / synapse / protein kinase binding / membrane / metal ion binding / plasma membrane
Similarity search - Function
K/Cl co-transporter / SLC12A transporter family / SLC12A transporter, C-terminal / Solute carrier family 12 / Amino acid permease/ SLC12A domain / Amino acid permease
Similarity search - Domain/homology
Solute carrier family 12 member 6
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.08 Å
AuthorsChi G / Man H / Ebenhoch R / Reggiano G / Pike ACW / Wang D / McKinley G / Mukhopadhyay SMM / MacLean EM / Chalk R ...Chi G / Man H / Ebenhoch R / Reggiano G / Pike ACW / Wang D / McKinley G / Mukhopadhyay SMM / MacLean EM / Chalk R / Moreau C / Snee M / Bohstedt T / Singh NK / Abrusci P / Arrowsmith CH / Bountra C / Edwards AM / Marsden BD / Burgess-Brown NA / DiMaio F / Duerr KL
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
European Commission115766 United Kingdom
CitationJournal: EMBO J / Year: 2021
Title: Phospho-regulation, nucleotide binding and ion access control in potassium-chloride cotransporters.
Authors: Gamma Chi / Rebecca Ebenhoch / Henry Man / Haiping Tang / Laurence E Tremblay / Gabriella Reggiano / Xingyu Qiu / Tina Bohstedt / Idlir Liko / Fernando G Almeida / Alexandre P Garneau / Dong ...Authors: Gamma Chi / Rebecca Ebenhoch / Henry Man / Haiping Tang / Laurence E Tremblay / Gabriella Reggiano / Xingyu Qiu / Tina Bohstedt / Idlir Liko / Fernando G Almeida / Alexandre P Garneau / Dong Wang / Gavin McKinley / Christophe P Moreau / Kiran D Bountra / Patrizia Abrusci / Shubhashish M M Mukhopadhyay / Alejandra Fernandez-Cid / Samira Slimani / Julie L Lavoie / Nicola A Burgess-Brown / Ben Tehan / Frank DiMaio / Ali Jazayeri / Paul Isenring / Carol V Robinson / Katharina L Dürr /
Abstract: Potassium-coupled chloride transporters (KCCs) play crucial roles in regulating cell volume and intracellular chloride concentration. They are characteristically inhibited under isotonic conditions ...Potassium-coupled chloride transporters (KCCs) play crucial roles in regulating cell volume and intracellular chloride concentration. They are characteristically inhibited under isotonic conditions via phospho-regulatory sites located within the cytoplasmic termini. Decreased inhibitory phosphorylation in response to hypotonic cell swelling stimulates transport activity, and dysfunction of this regulatory process has been associated with various human diseases. Here, we present cryo-EM structures of human KCC3b and KCC1, revealing structural determinants for phospho-regulation in both N- and C-termini. We show that phospho-mimetic KCC3b is arrested in an inward-facing state in which intracellular ion access is blocked by extensive contacts with the N-terminus. In another mutant with increased isotonic transport activity, KCC1Δ19, this interdomain interaction is absent, likely due to a unique phospho-regulatory site in the KCC1 N-terminus. Furthermore, we map additional phosphorylation sites as well as a previously unknown ATP/ADP-binding pocket in the large C-terminal domain and show enhanced thermal stabilization of other CCCs by adenine nucleotides. These findings provide fundamentally new insights into the complex regulation of KCCs and may unlock innovative strategies for drug development.
History
DepositionFeb 25, 2020-
Header (metadata) releaseJul 15, 2020-
Map releaseJul 15, 2020-
UpdateJul 28, 2021-
Current statusJul 28, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.012
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.012
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6y5v
  • Surface level: 0.012
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10704.png

Downloads & links

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Map

FileDownload / File: emd_10704.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.012 / Movie #1: 0.012
Minimum - Maximum-0.05140601 - 0.07878588
Average (Standard dev.)2.7610193e-05 (±0.0025908886)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 249.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.830.830.83
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z249.000249.000249.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0510.0790.000

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Supplemental data

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Mask #1

Fileemd_10704_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_10704_half_map_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_10704_half_map_2.map
Projections & Slices
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Sample components

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Entire : Structure of Human Potassium Chloride Transporter KCC3b (S45D/T94...

EntireName: Structure of Human Potassium Chloride Transporter KCC3b (S45D/T940D/T997D) in KCl
Components
  • Complex: Structure of Human Potassium Chloride Transporter KCC3b (S45D/T940D/T997D) in KCl
    • Protein or peptide: Solute carrier family 12 member 6
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Structure of Human Potassium Chloride Transporter KCC3b (S45D/T94...

SupramoleculeName: Structure of Human Potassium Chloride Transporter KCC3b (S45D/T940D/T997D) in KCl
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
Molecular weightTheoretical: 240 KDa

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Macromolecule #1: Solute carrier family 12 member 6

MacromoleculeName: Solute carrier family 12 member 6 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 122.344422 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MPHFTVTKVE DPEEGAAASI SQEPSLADIK ARIQDSDEPD LSQNDITGEH SQLLDDGHKK ARNAYLNNSN YEEGDEYFDK NLALFEEEM DTRPKVSSLL NRMANYTNLT QGAKEHEEAE NITEGKKKPT KTPQMGTFMG VYLPCLQNIF GVILFLRLTW V VGTAGVLQ ...String:
MPHFTVTKVE DPEEGAAASI SQEPSLADIK ARIQDSDEPD LSQNDITGEH SQLLDDGHKK ARNAYLNNSN YEEGDEYFDK NLALFEEEM DTRPKVSSLL NRMANYTNLT QGAKEHEEAE NITEGKKKPT KTPQMGTFMG VYLPCLQNIF GVILFLRLTW V VGTAGVLQ AFAIVLICCC CTMLTAISMS AIATNGVVPA GGSYFMISRA LGPEFGGAVG LCFYLGTTFA AAMYILGAIE IF LVYIVPR AAIFHSDDAL KESAAMLNNM RVYGTAFLVL MVLVVFIGVR YVNKFASLFL ACVIVSILAI YAGAIKSSFA PPH FPVCML GNRTLSSRHI DVCSKTKEIN NMTVPSKLWG FFCNSSQFFN ATCDEYFVHN NVTSIQGIPG LASGIITENL WSNY LPKGE IIEKPSAKSS DVLGSLNHEY VLVDITTSFT LLVGIFFPSV TGIMAGSNRS GDLKDAQKSI PIGTILAILT TSFVY LSNV VLFGACIEGV VLRDKFGDAV KGNLVVGTLS WPSPWVIVIG SFFSTCGAGL QSLTGAPRLL QAIAKDNIIP FLRVFG HSK ANGEPTWALL LTAAIAELGI LIASLDLVAP ILSMFFLMCY LFVNLACALQ TLLRTPNWRP RFRYYHWALS FMGMSIC LA LMFISSWYYA IVAMVIAGMI YKYIEYQGAE KEWGDGIRGL SLSAARFALL RLEEGPPHTK NWRPQLLVLL KLDEDLHV K HPRLLTFASQ LKAGKGLTIV GSVIVGNFLE NYGEALAAEQ TIKHLMEAEK VKGFCQLVVA AKLREGISHL IQSCGLGGM KHNTVVMGWP NGWRQSEDAR AWKTFIGTVR VTTAAHLALL VAKNISFFPS NVEQFSEGNI DVWWIVHDGG MLMLLPFLLK QHKVWRKCS IRIFTVAQLE DNSIQMKKDL ATFLYHLRIE AEVEVVEMHD SDISAYTYER DLMMEQRSQM LRHMRLSKTE R DREAQLVK DRNSMLRLTS IGSDEDEETE TYQEKVHMDW TKDKYMASRG QKAKSMEGFQ DLLNMRPDQS NVRRMHTAVK LN EVIVNKS HEAKLVLLNM PGPPRNPEGD ENYMEFLEVL TEGLERVLLV RGGGSEVITI YSA

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Macromolecule #3: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 3 / Number of copies: 2 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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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

Concentration6 mg/mL
BufferpH: 7.5
GridModel: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.08 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 293233
FSC plot (resolution estimation)

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