Structural and functional properties of the transporter SLC26A6 reveal mechanism of coupled anion exchange
- Department of Biochemistry, University of Zurich, Switzerland
- Department of Chemistry, Loughborough University, United Kingdom
Figures
Figure 1 with 1 supplement
Transport properties of SLC26A6.
(A) Current-Voltage relationships of HEK 293 cells expressing murine SLC26A9 and (B) human SLC26A6. Data were recorded in the whole-cell configuration in symmetric (150 mM) Cl- concentrations and …
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Figure 1—source data 1
Electrophysiology, liposomal transport assay data and western blot.
- https://cdn.elifesciences.org/articles/87178/elife-87178-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Transport data.
(A, B) Representative whole-cell recordings of HEK293 cells expressing murine SLC26A9 (A) or human SLC26A6 (B) recorded by a step protocol. (C) Proteoliposome-based ion transport assays used in this …
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Figure 1—figure supplement 1—source data 1
Extended electrophysiology and liposomal transport assay data.
- https://cdn.elifesciences.org/articles/87178/elife-87178-fig1-figsupp1-data1-v1.xlsx
Figure 2 with 3 supplements
SLC26A6 structure.
(A) Cryo-EM density of the SLC26A6 dimer at 3.28 Å and (B) ribbon representation of the model in the same orientation. Subunits are shown in unique colors and selected structural elements are …
Figure 2—figure supplement 1
Cryo-EM reconstruction of SLC26A6.
(A) Representative micrograph (of a total of 11,637) of the SLC26A6 dataset. (B) 2D class averages of SLC26A6. (C) Data-processing workflow. Particles were extracted and subjected to 2D …
Figure 2—figure supplement 2
Section of the cryo-EM density of SLC26A6.
(A) Cryo-EM density at 3.28 Å (contoured at 4σ) superimposed on indicated parts of the model. (B) Cryo-EM density surrounding the ion binding site at high (4 σ, left) and low (0.6 σ, right) contour …
Figure 2—figure supplement 3
Sequence and Topology.
(A) Sequence alignment of the human proteins SLC26A6 (isoform 3, NCBI reference sequence: NP_602298.2) and SLC26A9 (isoform b, NCBI reference sequence: NP_599152.2). Secondary structure elements of …
Figure 3 with 1 supplement
SLC26A6 TM domain.
(A) Ribbon representation of the TM unit of SLC26A6 in indicated orientations (left, view is from the outside, center and right, from within the membrane). Core and gate domains are colored in green …
Figure 3—figure supplement 1
TM domain features.
(A, B) Open book representation of the molecular surfaces of the TM (left) and its gate (center) and core domain (right) of SLC26A6 (A) and SLC26A9 (B). The coloring refers to the area contacted by …
Figure 4 with 1 supplement
Features of the substrate binding site.
(A) Ribbon representation of the core domain of SLC26A6 viewed from within the membrane from the gate domain. Asterisk indicates the location of the ion binding site, selected secondary structure …
Figure 4—figure supplement 1
Binding site comparisons.
(A, B) Superposition of secondary structure elements constituting the ion binding site of SLC26 transporters. (A) Comparison between SLC26A6 and SLC26A5 and (B) SLC26A9. (C) Positional difference …
Figure 5
Functional properties of a structure based SLC26A6 construct.
(A) SLC26 ion binding site showing surrounding residues including Arg 404. (B) Representative current trace and (C) current-voltage relationships of HEK 293 cells expressing the SLC26A6 mutant …
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Figure 5—source data 1
Transport data and Western blot of the SLC26A6 mutant R404V.
- https://cdn.elifesciences.org/articles/87178/elife-87178-fig5-data1-v1.xlsx
Figure 6
Transport mechanism.
Features of the anion binding site (left) and kinetic schemes (right) of two SLC26 paralogs with distinct functional properties. (A) The coupled antiporter SLC26A6 mediates the strict stoichiometric …
Tables
Table 1
Cryo-EM data collection, refinement, and validation statistics.
| hSLC26A6 in GDN(EMDB-17085) (PDB 8OPQ) | ||
|---|---|---|
| Data collection and processing | Data Set 1 | Data Set 2 |
| Magnification | 130,000 | 130,000 |
| Voltage (kV) | 300 | 300 |
| Electron exposure (e–/Å2) | 61 | 67 |
| Defocus range (μm) | −0.8 to −2.4 | −1 to −2.4 |
| Pixel size (Å)* | 0.3255 (0.651) | 0.3255 (0.651) |
| Symmetry imposed | C2 | |
| Initial particle images (no.) | 1,749,907 | |
| Final particle images (no.) | 93,169 | |
| Map resolution (Å) FSC threshold 0.143 | 3.28 | |
| Map resolution range (Å) | 2.9–4.1 | |
| Refinement | ||
| Model resolution (Å) 0.5 FSC Threshold | 3.3 | |
| Model resolution range (Å) | 1.6–3.3 | |
| Map sharpening b-factor (Å2) | 121.9 | |
| Model composition Non-hydrogen atoms Protein residues Ligands | 10,006 1296 2 | |
| B factors (Å2) Protein Ligands | 58.64 30 | |
| R.m.s. deviations Bond lengths (Å) Bond angles (°) | 0.004 0.907 | |
| Validation MolProbity score Clashscore Poor rotamers (%) | 1.71 8.25 0 | |
| Ramachandran plot Favored (%) Allowed (%) Disallowed (%) | 96.18 3.82 0.0 | |
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1. *Values in parentheses indicate the pixel size in super-resolution.
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Chemical compound, drug | HyClone HyCell TransFx-H medium | Cytiva | SH30939.02 | |
| Chemical compound, drug | 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) | Avanti Polar Lipids | 850757 C | |
| Chemical compound, drug | 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) | Avanti Polar Lipids | 840457 C | |
| Chemical compound, drug | 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) | Avanti Polar Lipids | 850457 C | |
| Chemical compound, drug | Cholesterol | Sigma | C8667 | |
| Chemical compound, drug | Triton X-100 | Sigma | T9284 | |
| Chemical compound, drug | Pepstatin A | Axon lab | A2205.0100 | |
| Chemical compound, drug | Phenylmethylsulfonyl fluoride (PMSF) | Sigma | PMSF-RO | |
| Chemical compound, drug | Leupeptin | AppliChem | A2183,0100 | |
| Chemical compound, drug | Benzamidine | Sigma | B6506 | |
| Chemical compound, drug | Chloroform | Fluka | 25690 | |
| Chemical compound, drug | Glyco-diosgenin (GDN) | Anatrace | GDN101 | |
| Chemical compound, drug | Diethyl ether | Sigma | 296082 | |
| Chemical compound, drug | DNase I | AppliChem | A3778 | |
| Chemical compound, drug | Glycerin, Glycerol 86% | Roth | 4043.3 | |
| Chemical compound, drug | HCl | Merck Millipore | 1.00319.1000 | |
| Chemical compound, drug | HEPES | Sigma | H3375 | |
| Chemical compound, drug | [Eu.L1]+ | Loughborough University (Dr SJ Butler) | N/A | |
| Chemical compound, drug | ACMA | Thermofischer Scientific | A1324 | |
| Chemical compound, drug | CCCP | Sigma | C2759 | |
| Chemical compound, drug | N,N′-Dimethyl-9,9′-biacridinium-dinitrat (Lucigenin) | Sigma | M8010 | |
| Chemical compound, drug | Phosphate buffered saline | Sigma | D8537 | |
| Chemical compound, drug | Strep-Tactin Superflow high capacity resin | IBA | 2-1208-010 | |
| Chemical compound, drug | D-desthiobiotin | Sigma | D1411 | |
| Chemical compound, drug | Kolliphor P188 | Sigma | K4894 | |
| Chemical compound, drug | L-glutamine | Sigma | G7513 | |
| Chemical compound, drug | Penicillin-streptomycin | Sigma | P0781 | |
| Chemical compound, drug | Fetal bovine serum | Sigma | F7524 | |
| Chemical compound, drug | Polyethylenimine 25 K MW, linear | Polysciences | 23966–1 | |
| Chemical compound, drug | 40 kDa linear PEI MAX | Polysciences | 24765–1 | |
| Chemical compound, drug | Valproic acid | Sigma | P4543 | |
| Chemical compound, drug | Calcium Chloride | Sigma | 223506 | |
| Chemical compound, drug | Magnesium Chloride | Fluka | 63,065 | |
| Chemical compound, drug | Potassium chloride | Sigma | 746346 | |
| Chemical compound, drug | Sodium chloride | Sigma | 71380 | |
| Chemical compound, drug | Terrific broth | Sigma | T9179 | |
| Chemical compound, drug | Mouse-anti-myc primary antibody | Sigma | M4439 | (WB 1:5000) |
| Chemical compound, drug | Peroxidase AffiniPure polyclonal Goat Anti-Mouse IgG (H+L) | Jackson ImmunoResearch | 115-035-003 RRID: AB_10015289 | (WB 1:10000) |
| Commercial assay or kit | 4–20% Mini-PROTEAN TGX Precast Protein Gels, 15-well, 15 µl | BioRad Laboratories | 4561096DC | |
| Commercial assay or kit | Amicon Ultra-4 Centrifugal Filters Ultracel 100 K, 4 ml | Merck Millipore | UFC810024 | |
| Commercial assay or kit | Borosilicate glass capillary with filament | Sutter Instrument | BF150-86-10HP | |
| Commercial assay or kit | 0.22 µm Ultrafree-MC Centrifugal Filtfer | EMD Millipore | UFC30GV | |
| Commercial assay or kit | Biobeads SM-2 adsorbents | BioRad Laboratories | 152–3920 | |
| Commercial assay or kit | Avestin Extruder kit | Sigma | Z373400 | |
| Commercial assay or kit | Pierce Cell Surface Biotinylation and Isolation Kit | Thermofischer Scientific | A44390 | |
| Commercial assay or kit | Amersham ECL Prime Western Blotting Detection Reagent | Cytiva | RPN2232 | |
| Commercial assay or kit | Polycarbonate Membranes 400 nm | Sigma | 610007 | |
| Commercial assay or kit | Polycarbonate Membranes 50 nm | Sigma | 610003 | |
| Commercial assay or kit | 96-well black walled microplates | Thermofischer Scientific | M33089 | |
| Commercial assay or kit | 384-well black microplate flat-bottom | Greiner | 781076 | |
| Commercial assay or kit | Quantifoil R1.2/1.3 Au 200 Mesh | Electron Microscopy Sciences | Q2100AR1.3 | |
| Commercial assay or kit | Superose 6 10/300 GL | Cytiva | 17517501 | |
| Other | BioQuantum Energy Filter | Gatan | N/A | |
| Other | HPL6 | Maximator | N/A | |
| Other | K3 Direct Detector | Gatan | N/A | |
| Other | Titan Krios G3i | ThermoFisher Scientific | N/A | |
| Other | Viber Fusion FX7 imaging system | Witec | N/A | |
| Other | TECAN M1000 Infinite | TECAN | N/A | |
| Other | TECAN SPARK | TECAN | N/A | |
| Other | Vitrobot Mark IV | ThermoFisher Scientific | N/A | |
| Cell line (human) | HEK293S GnTi- & HEK293T | ATCC | CRL-3022 & CRL-1573 | |
| Recombinant DNA reagent | Mammalian expression vector with C-terminal 3 C cleavage site, venus fluorescent tag, myc tag, SBP tag. | Dutzler laboratory | N/A | |
| Recombinant DNA reagent | Mus musculus SLC26A9 ORF shuttle clone | BioScience | GenBank BC160193 | |
| Recombinant DNA reagent | Homo sapiens SLC26A6 cDNA clone | BioScience | GenBank BC017697 | |
| Recombinant protein | HRV 3 C protease | Expressed (pET_3 C) and purified in Dutzler laboratory | N/A | |
| Software, algorithm | ASTRA7.2 | Wyatt Technology | RRID:SCR_016255 https://www.wyatt.com/products/software/astra.html | |
| Software, algorithm | ChimeraX 1.4 | Pettersen et al., 2021 | RRID:SCR_015872 https://www.rbvi.ucsf.edu/chimerax/ | |
| Software, algorithm | Biorender | https://app.biorender.com/biorender-templates | ||
| Software, algorithm | Coot v.0.9.4 | Emsley et al., 2010 | RRID:SCR_014222 https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/ | |
| Software, algorithm | cryoSPARC v3.2.0–4.0 | Structura Biotechnology Inc. | RRID:SCR_016501 https://cryosparc.com/ | |
| Software, algorithm | DINO | RRID:SCR_013497 http://www.dino3d.org | ||
| Software, algorithm | EPU2.9 | Thermo Fisher Scientific | N/A | |
| Software, algorithm | Phenix | Liebschner et al., 2019 | RRID:SCR_014224 https://www.phenix-online.org/ | |
| Software, algorithm | DeepEMhancer | Sanchez-Garcia et al., 2021 | https://github.com/rsanchezgarc/deepEMhancer | |
| Software, algorithm | JALVIEW | Waterhouse et al., 2009 | ||
| Software, algorithm | Muscle | Edgar, 2004 | ||
| Software, algorithm | Axon Clampex 10.6 | Molecular Devices | N/A | |
| Software, algorithm | Axon Clampfit 10.6 | Molecular Devices | N/A | |
| Software, algorithm | Prism 9 | GraphPad | N/A | |
| Strain, strain background (E Coli) | E. coli MC1061 | Thermo Fisher Scientific | C66303 |
